US007871607B2
(12) United States Patent (10) Patent No.: US 7,871,607 B2 B00kbinder et al. (45) Date of Patent: *Jan. 18, 2011
(54) SOLUBLE GLYCOSAMINOGLYCANASES 5,292,509 A 3/1994 Hageman ...... 424/94.61 AND METHODS OF PREPARING AND USING 5,323,907 A 6/1994 Kalvelage ...... 206,531 SOLUBLE GLYCOSAMINOGLYCANASES 5,324,844 A 6/1994 Zalipsky ...... 548,520 5,354,566 A 10/1994 Addesso et al...... 426.9 (75) Inventors: Louis H. Bookbinder, San Diego, CA 5,436,128 A 7/1995 Harpold et al...... 435/6 (US); Anirban Kundu, San Diego, CA 5,446,090 A 8/1995 Harris ...... 525,541 (US); Gregory I. Frost, Del Mar, CA 5,591,767 A 1/1997 Mohr et al...... 514,413 (US) 5,612.460 A 3/1997 Zalipsky ...... 530.391.9 (73) Assignee: Halozyme, Inc., San Diego, CA (US) 5,631,018 A 5/1997 Zalipsky et al...... 424/450 5,639,476 A 6/1997 Oshlack et al...... 424/468 (*) Notice: Subject to any disclaimer, the term of this 5,643,575 A 7/1997 Martinez et al...... 424,194.1 patent is extended or adjusted under 35 5,665,069 A 9, 1997 Cumer et al...... 604,116 U.S.C. 154(b) by 1175 days. 5,672,662 A 9, 1997 Harris et al...... 525,408 5,674,533 A 10/1997 Santus et al...... 424,493 This patent is Subject to a terminal dis 5,714, 166 A 2, 1998 Tomalia et al...... 424/486 claimer. 5,721,348 A 2f1998 Primakoff et al. 5,723, 147 A 3/1998 Kim et al...... 424/450 (21) Appl. No.: 11/065,716 5,733,566 A 3/1998 Lewis ...... 424/426 (22) Filed: Feb. 23, 2005 (65) Prior Publication Data (Continued) US 2005/026O186A1 Nov. 24, 2005 FOREIGN PATENT DOCUMENTS (51) Int. Cl. EP O13420 T 1980 A6 IK 38/46 (2006.01) CI2N 9/26 (2006.01) (52) U.S. Cl...... 424/94.62; 435/201 (58) Field of Classification Search ...... None (Continued) See application file for complete search history. OTHER PUBLICATIONS (56) References Cited Adams et al., “The c-myc oncogene driven by immunoglobulin U.S. PATENT DOCUMENTS enhancers induces lymphoid malignancy in transgenic mice.” Nature 318:533-538 (1985). 3,536,809 A 10/1970 AppleZweig ...... 424/435 3,598,123 A 8, 1971 Zaffaroni ...... 424/435 (Continued) 3,630,200 A 12/1971 Higuchi ...... 424/427 3,710,795 A 1/1973 Higuchi et al...... 424/424 Primary Examiner Nashaat T Nashed 3,845,770 A 11, 1974 Theeuwes et al...... 424/427 (74) Attorney, Agent, or Firm K&L Gates LLP; Stephanie 3,916,899 A 11/1975 Theeuwes et al...... 424/424 Seidman RE28,819 E 5/1976 Thompson ...... 514,174 4,002,531 A 1/1977 Royer ...... 435,188 (57) ABSTRACT 4,008,719 A 2f1977 Theeuwes et al. ... 424/427 4,044,126 A 8, 1977 Cook et al...... 514,180 4,179,337 A 12, 1979 Davis et al...... 435/181 4.328,245 A 5/1982 Yu et al...... 514,530 The invention relates to the discovery of novel soluble neutral 4,358,603 A 1 1/1982 Yu ...... 560.2 active Hyaluronidase Glycoproteins (shASEGPs), methods 4,364,923 A 12/1982 Cook et al. ... 424/46 of manufacture, and their use to facilitate administration of 4.409,239 A 10, 1983 Yu ...... 514,530 other molecules or to alleviate glycosaminoglycan associated 4.410,545 A 10, 1983 Yu et al...... 514,530 pathologies. Minimally active polypeptide domains of the 4.414,209 A 11, 1983 Cook et al...... 514,180 soluble, neutral active shASEGP domains are described that 4,522,811 A 6, 1985 Eppstein et al...... 514/2 include asparagine-linked Sugar moieties required for a func 4,687,610 A 8, 1987 Vassilatos ... . 264/211.14 tional neutral active hyaluronidase domain. Included are 4,769,027 A 9, 1988 Baker et al...... 424,493 modified amino-terminal leader peptides that enhance secre 4,820,516 A 4, 1989 Sawyer et al...... 424/96.2 tion of shASEGP. The invention further comprises sialated 4,952.496 A 8, 1990 Studier et al...... 435,9141 and pegylated forms of a recombinant shASEGP to enhance 4,980,286 A 12/1990 Morgan et al...... 435/371 stability and serum pharmacokinetics over naturally occur 5,041,292 A 8/1991 Feijen ...... 424/484 ring slaughterhouse enzymes. Further described are Suitable 5,052,558 A 10, 1991 Carter ...... 206,439 formulations of a substantially purified recombinant shA 5,059,595 A 10, 1991 Le Grazie ...... 424/468 SEGP glycoprotein derived from a eukaryotic cell that gen 5,073,543 A 12, 1991 Marshall et al...... 514, 21 5, 120,548 A 6, 1992 McClelland et al. ... 424/473 erate the proper glycosylation required for its optimal activ 5,122,614 A 6/1992 Zalipsky ...... 548,520 5, 183,550 A 2f1993 Mattiessen .. 204,415 5,215,899 A 6/1993 Dattagupta ...... 435/6 32 Claims, 1 Drawing Sheet US 7,871,607 B2 Page 2
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Medications and Fluids.” San Diego, CA. Jun. 27, 2006, http://phx. 4, 2006, http://phx.corporate-ir.net/phoenix.Zhtml?c=175436 corporate-ir.net/phoenix.Zhtml?c=175436&p=irol &p=irol-newsArticle Print&ID=852102&highlight= (accessed newsArticle Print&ID=876530&highlight= (accessed Jan. 6. Jan. 6, 2009), 3 pages. 2009), 3 pages. News Release, Halozyme Therapeutics Inc., "Halozyme Therapeu News Release, Halozyme Therapeutics Inc., "Halozyme Therapeu tics Initiates Chemophase Phase I/IIa Clinical Trial for Superficial tics Begins Phase 1 Clinical Trial of Bisphosphonate Administered Bladder Cancer.” San Diego, CA, Apr. 26, 2006, http://phx.corpo With rHuPH20 Enzyme.” San Diego, CA, Dec. 10, 2008, http://phx. rate-ir.net/phoenix.Zhtml?c=175436&p=irol-newsArticle Print corporate-ir.net/phoenix.Zhtml?c=175436&p=irol &ID=847794&highlight= (accessed Jan. 6, 2009), 2 pages. newsArticle Print&ID=1234.643&highlight= (accessed Jan. 6. 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News Release, Halozyme Therapeutics Inc., "Halozyme Therapeu net/phoenix.Zhtml?c=175436&p=irol-newsArticle Print tics Begins Phase 2 Clinical Trial of Insulin With rHuPH20 in Type 1 Diabetic Patients.” San Diego, CA, Nov. 3, 2008, http://phx.corpo &ID=814561&highlight= (accessed Jan. 6, 2009), 4 pages. rate4-ir.net/phoenix.Zhtml?c=175436&p=irol-newsArticle Print News Release, Halozyme Therapeutics Inc., "Halozyme Therapeu tics Initiates Clinical Trial of Subcutaneous Morphine With &ID=1220870&highlight= (accessed Jan. 6, 2009), 2 pages. Hylenex.” San Diego, CA, Feb. 2, 2006, http://phx.corporate-ir.net/ News Release, Halozyme Therapeutics Inc., "Halozyme Therapeu phoenix.Zhtml?c=175436&p=irol-newsArticle Print&ID=811906 tics Announces That Chemophase Meets Primary Endpoint in Phase &highlight (accessed Jan. 6, 2009), 2 pages. IIIa Clinical Trial.” San Diego, CA. 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Copenhagen, Denmark, Jun. 22, 2005, http://phx.corpo Subcutaneous Absorption of a Large Molecule Protein Therapeutic.” rate-ir.net/phoenix.Zhtml?c=175436&p=irol-newsArticle Print San Diego, CA, Aug. 8, 2006. http://ph.X.corporate-ir.net/phoenix. &ID=722843&highlight= (accessed Jan. 6, 2009), 3 pages. Zhtml?c=175436&p=irol-newsArticle Print&ID=893361&high News Release, Halozyme Therapeutics Inc., "Halozyme Therapeu light (accessed Jan. 6, 2009), 2 pages. tics Announces Launch of Cumulase for In Vitro Fertilization.” San News Release, Halozyme Therapeutics Inc., "Halozyme Therapeu Diego, CA. Jun. 20, 2005, http://phx.corporate-ir.net/phoenix. tics Announces FDA 510(k) Clearance for MediCult's SynVitro Zhtml?c=175436&p=irol-newsArticle Print&ID=721999&high Cumulase Product.” San Diego, CA. May 23, 2006, http://phx.cor light (accessed Jan. 6, 2009), 2 pages. porate-ir.net/phoenix.Zhtml?c=175436&p=irol-newsArticle Print News Release, Halozyme Therapeutics Inc., "Halozyme Therapeu &ID=859761&highlight= (accessed Jan. 6, 2009), 2 pages. tics Announces FDA Acceptance of Hylenex NDA.” San Diego, CA, News Release, Halozyme Therapeutics Inc., "Halozyme Therapeu May 26, 2005, http://phx.corporate-ir.net/phoenix.Zhtml?c=175436 tics Announces 22% Increase in IVF ICSI Fertilization Rates in Eggs &p=irol-newsArticle Print&ID=714327&highlight= (accessed Treated With Cumulase Versus Bovine Extract.” San Diego, CA, May Jan. 6, 2009), 2 pages. US 7,871,607 B2 Page 10
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J Trauma in acute myocardial infarction.” Br Heart J. 45:350 (1981). de Oliveria et al., “Intravenous injection of Hyaluronidase in acute 15(11):992-998 (1975). myocardial infarction: Preliminary report of clinical and experimen Kozak et al., “Recombinant human hyaluronidase facilitates tal observations.” American Heart Journal 57(5):712-722 (1959). dexamethasone penetration into the posterior ocular segment after Dorfman, A. and M. Ott, "A turbidimetric method for the assay of sub-tenon's injection.” Association for Research in Vision and Oph hyaluronidase.” J. Biol. Chem. 172:367-375 (1948). thalmology Annual Meeting, May 1-5, 2005, Fort Lauderdale, FL. 3 Drug Shortage Bulletin: Hyaluronidase Injection-Discontinued, pub pageS. lished Jan. 18, 2005, American Society of Health-System Pharma Kundu et al., “Dispersion of the cumulus matrix with a highly puri cist, retrieved from the Internet: Sac - 2 - GAGCI'C Psal - 830 - C TGCA'G Scal ... 030. AGTACT NheI-1052 - G'CTAG C Sca. 5234. AGAC BanhI - 396 - GGATCC Baish I - 2507 - GGATC C ClaE-3960 - ATCG AT Noti - 3722 - GC'GGCC GC flui - 3693 - ACGCGT Not I-31 ls - GCGGCC GC Sac - 3389 - GAGCTC Sca-3223-AGTACT VECTOR MAP of SHASEGP VECTOR HZ24 F.G. 1 US 7,871,607 B2 1. 2 SOLUBLE GLYCOSAMINOGLYCANASES Hyaluronidases are a group of generally neutral- or acid AND METHODS OF PREPARING AND USING active enzymes found throughout the animal kingdom. SOLUBLE GLYCOSAMINOGLYCANASES Hyaluronidases vary with respect to Substrate specificity, and mechanism of action. CROSS REFERENCE TO RELATED There are three general classes of hyaluronidases: APPLICATION(S) 1. Mammalian-type hyaluronidases, (EC 3.2.1.35) which are endo-beta-N-acetylhexosaminidases with tetrasaccha Background of the Invention rides and hexasaccharides as the major end products. They have both hydrolytic and transglycosidase activities, and can 1. Field of the Invention 10 degrade hyaluronan and chondroitin Sulfates (CS), generally The present invention is generally related to Glycosami C4-S and C6-S. noglycanase enzymes, including Neutral-Active, Soluble 2. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyalu Hyaluronidase Glycoproteins (shASEGPs), and portions roman and, and to various extents, CS and DS. They are thereof, particularly Hyaluronidase domains. More specifi endo-beta-N-acetylhexosaminidases that operate by a beta cally, the invention is related to chemical modifications, phar 15 elimination reaction that yields primarily disaccharide end maceutical compositions, expression plasmids, methods for products. manufacture and therapeutic methods using the Glycosami 3. Hyaluronidases (EC 3.2.1.36) from leeches, other para noglycanases (and domains thereof and the encoding nucleic sites, and crustaceans are endo-beta-glucuronidases that gen acid molecules) for the therapeutic modification of gly erate tetrasaccharide and hexasaccharide end products cosaminoglycans in the treatment of disease and for use to through hydrolysis of the beta 1-3 linkage. increase diffusion of other molecules Such as injected mol Mammalian hyaluronidases can be further divided into two ecules in an animal. groups: neutral-active and acid-active enzymes. There are six 2. Background Information hyaluronidase-like genes in the human genome, HYAL1. Glycosaminoglycans (GAGs) are complex linear polysac HYAL2, HYAL3, HYAL4, HYALP1 and PH20/SPAM1. charides of the extracellular matrix (ECM). GAGs are char 25 HYALP1 is a pseudogene, and HYAL3 has not been shown to acterized by repeating disaccharide structures of an N-Substi possess enzyme activity toward any known Substrates. tuted hexosamine and an uronic acid (in the case of HYAL4 is a chondroitinase and exhibits little activity towards hyaluronan (HA), chondroitin sulfate (CS), chondroitin (C), hyaluronan. HYAL1 is the prototypical acid-active enzyme dermatan sulfate (DS), heparan sulfate (HS), and heparin and PH20 is the prototypical neutral-active enzyme. Acid (H)), or a galactose (in the case of keratan Sulfate (KS)). 30 active hyaluronidases, such as HYAL1 and HYAL2 generally Except for HA, all exist covalently bound to core proteins. lack catalytic activity at neutral pH (i.e. pH 7). For example, The GAGs with their core proteins are structurally referred to HYAL1 has little catalytic activity in vitro over pH 4.5 (Frost as proteoglycans (PGs). et al Anal Biochemistry, 1997). HYAL2 is an acid-active Hyaluronan (HA) is found in mammals predominantly in enzyme with a very low specific activity in vitro. connective tissues, skin, cartilage, and in synovial fluid. 35 The hyaluronidase-like enzymes can also be characterized Hyaluronan is also the main constituent of the vitreous of the by those which are generally locked to the plasma membrane eye. In connective tissue, the water of hydration associated via a glycosylphosphatidyl inositol anchor Such as human with hyaluronan creates hydrated matrices between tissues. HYAL2 and human PH20 (Danilkovitch-Miagkova, et al. Hyaluronan plays a key role in biological phenomena asso Proc Natl Acad Sci USA. 2003 Apr. 15: 100(8): 4580-5, ciated with cell motility including rapid development, regen 40 Phelps et al., Science 1988), and those which are generally eration, repair, embryogenesis, embryological development, soluble such as human HYAL1 (Frost etal, Biochem Biophys wound healing, angiogenesis, and tumorigenesis (Toole 1991 Res Commun. 1997 Jul.9; 236(1): 10-5). However, there are Cell Biol. Extracell. Matrix, Hay (ed), Plenum Press, New variations from species to species: bovine, PH20 for example York, 1384-1386; Bertrand et al. 1992 Int. J. Cancer 52: 1-6: is very loosely attached to the plasma membrane and is not Knudson et al., 1993 FASEB J. 7: 1233-1241). In addition, 45 anchored via a phospholipase sensitive anchor (Lalancette et hyaluronan levels correlate with tumor aggressiveness al, Biol Reprod. 2001 August: 65(2): 628-36.). This unique (Ozello et al. 1960 Cancer Res. 20: 600-604: Takeuchi et al. feature of bovine hyaluronidase has permitted the use of the 1976, Cancer Res. 36: 2133-2139; Kimata et al. 1983 Cancer soluble bovine testes hyaluronidase enzyme as an extract for Res.43: 1347-1354). clinical use (WydaseTM, HyalaseTM). Other PH20 species are HA is found in the extracellular matrix of many cells, 50 lipid anchored enzymes that are generally not soluble without especially in soft connective tissues. HA has been assigned the use of detergents or lipases. For example, human PH20 is various physiological functions, such as in water and plasma anchored to the plasma membrane via a GPI anchor. Attempts protein homeostasis (Laurent T C et al (1992) FASEB J 6: to make human PH20 DNA constructs that would not intro 2397-2404). HA production increases in proliferating cells duce a lipid anchor into the polypeptide resulted in either a and may play a role in mitosis. It has also been implicated in 55 catalytically inactive enzyme, or an insoluble enzyme (Arm locomotion and cell migration. HA seems to play important ing etal Eurj. Biochem. 1997 Aug. 1: 247(3): 810-4). Natu roles in cell regulation, development, and differentiation rally occurring macaque sperm hyaluronidase is found in (Laurent et al. Supra). both a soluble and membrane bound form. While the 64 kDa HA has been used in clinical medicine. Its tissue protective membrane bound form possesses enzyme activity at pH 7.0, and rheological properties have proved useful in ophthalmic 60 the 54 kDa form is only active at pH 4.0 (Cherretal, Dev Biol. Surgery (e.g. to protect the corneal endothelium during cata 1996 Apr. 10; 175(1): 142-53.). Thus, soluble forms of PH20 ract Surgery). Serum HA is diagnostic of liver disease and are often lacking enzyme activity under neutral conditions. various inflammatory conditions, such as rheumatoid arthri Chondroitinases are enzymes found throughout the animal tis. Interstitial edema caused by accumulation of HA may kingdom. These enzymes degrade glycosaminoglycans cause dysfunction in various organs (Laurent et al. Supra). 65 through an endoglycosidase reaction. Specific examples of Hyaluronan protein interactions also are involved in the known Chondroitinases include: Chondroitinase ABC (de structure of the extracellular matrix or “ground substance'. rived from Proteus vulgaris; Japanese Patent Application US 7,871,607 B2 3 4 Laid-open No 6-153947, T. Yamagata, H. Saito, O. Habuchi, nases are presently preferred for applications in which the and S. Suzuki, J. Biol. Chem.,243, 1523 (1968), S. Suzuki, H. enzyme is to be employed within the human body, including Saito, T. Yamagata, K. Anno, N. Seno, Y. Kawai, and T. many medical applications as described and illustrated Furuhashi, J. Biol. Chem. 243, 1543 (1968)); Chondroitinase herein. AC (derived from Flavobacterium heparinum; T. Yamagata, One aspect of the invention is based upon the discovery that H. Saito, O. Habuchi, and S. Suzuki, J. Biol. Chem., 243, a soluble, neutral-active hyaluronidase activity can be pro 1523 (1968)); Chondroitinase AC II (derived from Arthro duced with high yield in a mammalian expression system by bacter aurescens; K. Hiyama, and S. Okada, J. Biol. Chem. introducing nucleic acids that lack a narrow region encoding 250, 1824 (1975), K. Hiyama and S. Okada, J. Biochem. amino acids in the carboxy terminus of the human PH20 (Tokyo), 80, 1201 (1976)); Hyaluronidase ACIII (derived 10 cDNA. Additional modifications of thesHASEGP to enhance from Flavobacterium sp. Hp 102: Hirofumi Miyazono, secretion by use of non-native leader peptides are also pro Hiroshi Kikuchi, Keiichi Yoshida, Kiyoshi Morikawa, and vided. Further provided are methods to modify thesHASEGP Kiyochika Tokuyasu, Seikagaku, 61, 1023 (1989)); Chon to prolong its half life by way of masking the protein with droitinase B (derived from Flavobacterium heparinum;Y. M. polyethylene glycol (PEG) and/or post-translational modifi Michelacci and C. P. Dietrich, Biochem. Biophys. Res. Com 15 cations to native glycosylation. Previous attempts to generate mun., 56,973 (1974), Y. M. Michelacci and C. P. Dietrich, soluble neutral-active human hyaluronidase glycoproteins Biochem. J., 151, 121 (1975), Kenichi Maeyama, Akira were unsuccessful. It was concluded that truncations of the Tawada, Akiko Ueno, and Keiichi Yoshida, Seikagaku, 57. human hyaluronidase polypeptide resulted in both a loss of 1189 (1985)); Chondroitinase C (derived from Flavobacte neutral enzymatic activity, and an inability of cells to secrete rium sp. Hp 102: Hirofumi Miyazono, Hiroshi Kikuchi, Keli the recombinant protein in mammalian expression systems chi Yoshida, Kiyoshi Morikawa, and Kiyochika Tokuyasu, (Arming, etal Eur J. Biochem 1997 Aug. 1: 247 (3): 810-4). It Seikagaku, 61, 1023 (1989)); and the like. is critical to generate neutral-acting secreted shASEGP for Glycoproteins are composed of a polypeptide chain commercial production and therapeutic utility as a hyalu covalently bound to one or more carbohydrate moieties. ronidase. The invention, disclosed herein, overcomes these There are two broad categories of glycoproteins that possess 25 and other challenges. carbohydrates coupled though either N-glycosidic or O-gly The invention further comprises a catalytically active cosidic linkages to their constituent protein. The N- and human shASEGP glycoprotein wherein the shASEGP pos O-linked glycans are attached to polypeptides through aspar sesses at least one N-linked Sugar moiety. The studies shown agine-N-acetyl-D-glucosamine and serine (threonine)-N- herein demonstrate that human PH20 requires N-linked gly acetyl-D-galactosamine linkages, respectively. Complex 30 cans for catalytic activity, whereas bovine and bee venom N-linked oligosaccharides do not contain terminal mannose hyaluronidases remain active without Such N-linked glycans. residues. They contain only terminal N-acetylglucosamine, A human hyaluronidase PH20 domain devoid of N-linked galactose, and/or sialic acid residues. Hybrid oligosaccha moieties is catalytically inactive. Thus classic recombinant rides contain terminal mannose residues as well as terminal DNA technology does not permit the production of a cata N-acetylglucosamine, galactose, and/or sialic acid residues. 35 lytically active human shASEGP, unlike bee venom hyalu With N-linked glycoproteins, an oligosaccharide precursor ronidase, which can be produced in E. coli. is attached to the amino group of asparagine during peptide The invention includes methods and cells for generation of synthesis in the endoplasmic reticulum. The oligosaccharide an N-linked shASEGP glycoprotein polypeptide, by using of moiety is then sequentially processed by a series of specific a cell capable of introducing said N-linked Sugar moieties or enzymes that delete and add Sugar moieties. The processing 40 by introduction of said N-linked moieties on a shASEGP occurs in the endoplasmic reticulum and continues with pas polypeptide. Methods of identifying properly glycosylated sage through the cis-, medial- and trans-Golgi apparatus. SHASEGPs are further disclosed. Catalytically active PEGylated and/or super-Sialated shA SUMMARY OF THE INVENTION SEGP glycoproteins are also provided. PEGylated and/or 45 super-sialated shASEGPs possess greater serum half-lives Provided herein are soluble glycosaminoglycanase compared to naturally occurring non-Sialated bovine and enzymes particularly members of the soluble neutral-active ovine testes shASEGPs, and are thus preferable for both Hyaluronidase Glycoprotein family (also referred to hereinas enzyme stability and use as drugs or adjuvants in circum sHASEGPs), preferred members being human soluble neu stances in which prolonged half-lives are desirable (such as is tral-active Hyaluronidase Glycoproteins, particularly the 50 commonly the case with intravenous delivery). The invention human soluble PH-20 Hyaluronidase Glycoproteins (also provides methods for the preparation of PEGylated and/or referred to herein as rhuPH20s). The soluble neutral-active super-Sialated shASEGPs, compositions and uses thereof. Hyaluronidases provided herein are each a shASEGP family Without being restricted to a particular application or mecha member, designated herein as a shASEGP. The soluble nism of action, it is generally considered that the attachment Hyaluronidase domain, and uses thereof are also provided. 55 of PEG moieties to shASEGPs and other glycosaminoglyca While a number of uses and applications of soluble gly nases can be used to effectively shield the molecules, decreas cosaminoglycanases (sometimes referred to herein as GAG ing its relative sensitivity to proteases and potentially also Enzymes) are illustrated herein using rHuPH20 as an exem decreasing the extent and rate of its clearance and elimination plary glycosaminoglycanase (e.g. in promoting the delivery from the body. Applying these principles and techniques to of pharmacologics and other agents within tissues of the 60 other glycosaminoglycanases, PEGylated, Super-Sialated mammalian body), it will be appreciated by those of skill in and/or otherwise modified versions of such other glycosami the art that other glycosaminoglycanases such as those noglycanases can likewise be generated and used in the con described herein or others known in the art can be applied to text of methods and techniques as described and illustrated a number of such applications. Presently preferred soluble herein (e.g. techniques for promoting the dispersion of phar glycosaminoglycanases, such as SHASEGPs, exhibit some 65 macologics and other agents within tissues of the body). hyaluronidase activity and can exhibit other glycosaminogly Proteins encoded by naturally GPI anchor deficient shA canase activities as well. Human soluble glycosaminoglyca SEGP splice variants are also provided. US 7,871,607 B2 5 6 Further provided are compositions of the shASEGP com otides, and generally less than 1000, typically less than about prising, a soluble shASEGP glycoprotein with a metal ion, 100 nucleotides, set forth in SEQID NO. 6 (or the comple wherein the metal ion is Calcium, Magnesium or Sodium. ment thereof); or contain at least about 30 nucleotides (or the sHASEGPs are generally optimally active in the presence of complement thereof) or contain oligonucleotides that hybrid said metals. Formulations consisting of shASEGP in the ize along their full-length (or at least about 70, 80 or 90% presence of said metal ions are also provided. thereof) to any such fragments or oligonucleotides. The Modifications of shASEGPs and other glycosaminoglyca length of the fragments are a function of the purpose for nases to further prolong their half life are provided. Chemical which they are used and/or the complexity of the genome of modifications of a shASEGPs and other glycosaminoglyca interest. Generally probes and primers contain less than about nases with polymers such as polyethylene glycol and dextran 10 50, 150 or 500 nucleotides. are provided. Such modifications shield shASEGPs and Also provided are plasmids containing any of the nucleic other glycosaminoglycanases from removal from circulation acid molecules provided herein. Cells containing the plas and the immune system as well as glycosylation receptors for mids are also provided. Such cells include, but are not limited mannose and asialoglycoprotein. Further provide are meth to, bacterial cells, yeast cells, fungal cells, plant cells, insect ods to link to specific functional groups such as glycosylation 15 cells and animal cells. sites, positively charged amino acids and cysteines. Also provided are enhanced mammalian expression sys Assays for identifying effectors, such as compounds, tems using signal leaders capable of efficient secretion of including Small molecules, and conditions, such as pH, tem sHASEGPs. An example of such efficient secretory leader perature and ionic strength, that modulate the activation, peptide amino acid sequence and fusion protein with a SHA expression or activity of shASEGPs, are also provided SEGP is found in SEQID Nos. 43 and 46. herein. In exemplary assays, the effects of test compounds on Also provided is a method of producing shASEGPs by the ability of a Hyaluronidase domain of shASEGP to cleave growing the above-described cells under conditions whereby a known Substrate, typically a glycosaminoglycan or pro the shASEGP is expressed by the cells, and recovering the teoglycan, are assessed. Agents, generally compounds, par expressed shASEGP polypeptide or glycoprotein. Methods ticularly small molecules, that modulate the activity of the 25 for isolating nucleic acid encoding other shASEGPs are also Hyaluronidase domain are candidate compounds for modu provided. lating the activity of the shASEGP. The Hyaluronidase Also provided are cells, generally eukaryotic cells, such as domains can also be used to produce Hyaluronidase-specific mammalian cells and yeast cells, in which a shASEGP antibodies with function-perturbing activity. The Hyalu polypeptide is expressed on the surface of the cells. Such cells ronidase domains provided herein include, but are not limited 30 can be used in drug screening assays to identify compounds to, the N-terminal glycsoyl-hydrolase domain with C-termi that modulate the activity of the shASEGP polypeptide. nal truncated portions thereof that exhibit catalytic activity in These assays, including invitrobinding assays, and transcrip vitro. tion based assays in which signal transduction mediated Nucleic acid molecules encoding the proteins and Hyalu directly or indirectly, such as via activation of pro-growth ronidase domains are also provided. Nucleic acid molecules 35 factors, by the shASEGP is assessed. that encode a soluble Hyaluronidase domain or catalytically Also provided are peptides encoded by Such nucleic acid active portions thereof and also those that encode the full molecules. Included among those polypeptides is the SHA length shASEGP are provided. Nucleic acid encoding an SEGP Hyaluronidase domain or a polypeptide with amino exemplary Hyaluronidase domain and downstream nucleic acid changes Such that the specificity and/or Hyaluronidase acid is set forth in SEQ ID No. 6; and the Hyaluronidase 40 activity remains substantially unchanged. In particular, a Sub domain of an exemplary shASEGP is set forth in SEQID No. stantially purified mammalian shASEGP glycoprotein is 1 (amino acids 35-464). The protein sequence and encoding provided that comprises a secreted neutral-active enzyme. nucleic acid sequence of an exemplary full-length shASEGP The invention also includes a Hyaluronidase catalytic are set forth in SEQID Nos. 1 and 6. domain and may additionally include other domains. The Also provided are nucleic acid molecules that hybridize to 45 sHASEGP may form homodimers and can also form het such shASEGP-encoding nucleic acid along their full-length erodimers with some other protein, Such as a membrane or along at least about 70%. 80% or 90% of the full-length and bound protein. Also provided is a Substantially purified gly encode the Hyaluronidase domain or portion thereofare pro coprotein including a sequence of amino acids that has at least vided. Hybridization is generally effected underconditions of 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, at least low, generally at least moderate, and often high Strin 50 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, gency. 98%, or 99% identity to an exemplary shASEGP where the The isolated nucleic acid fragment is DNA, including percentage identity is determined using standard algorithms genomic or cDNA, or is RNA, or can include other compo and gap penalties that maximize the percentage identity. nents, such as protein nucleic acid or other nucleotide ana Splice variants of shASEGPs, particularly those with a logs. The isolated nucleic acid may include additional com 55 catalytically active Hyaluronidase domains, are contem ponents, such as heterologous or native promoters, enhancers plated herein. and other transcriptional and translational regulatory In other embodiments, substantially purified polypeptides sequences, these genes may be linked to other genes, such as that include a Hyaluronidase domain of a shASEGP reporter genes or other indicator genes or genes that encode polypeptide or a catalytically active portion thereof, but that indicators. 60 do not include the entire sequence of amino acids set forth in Also provided is an isolated nucleic acid molecule that SEQ ID No. 1 are provided. Among these are polypeptides includes the sequence of molecules that is complementary to that include a sequence of amino acids that has at least 70%, the nucleotide sequence encoding shASEGP or the portion 80%, 85%, 90%. 95% or 100% sequence identity to SEQID thereof. No. 1 or 3. Also provided are fragments thereof or oligonucleotides 65 In a specific embodiment, a nucleic acid that encodes a that can be used as probes or primers and that contain at least eukaryotic Hyaluronidase glycoprotein, designated a eukary about 10 to 16 nucleotides, typically at least about 20 nucle otic shASEGP is provided. In particular, the nucleic acid US 7,871,607 B2 7 8 includes the sequence of nucleotides set forth in SEQID No. ably, sialic acid residues are found in the terminal residues of 6, particularly set forth as nucleotides 106-1446 of SEQ ID N-linked glycosylation on shASEGPs. NO. 6, or a portion thereof that encodes a catalytically active N-linked oligosaccharides fall into several major types polypeptide. (oligomannose, complex, hybrid, Sulfated), all of which have Also provided are nucleic acid molecules that hybridize 5 (Man) 3-GlcNAc-GlcNAc-cores attached via the amide under conditions of at least low stringency, generally moder nitrogen of Asn residues that fall within-Asn-Xaa-Thr/Ser ate stringency, more typically high Stringency to the SEQID sequences (where Xaa is not Pro). Glycosylation at an -Asn NO. 6 or degenerates thereof. Xaa-Cys- site has been reported for coagulation protein C. In one embodiment, the isolated nucleic acid fragment N-linked sites are often indirectly assigned by the appearance hybridizes to a nucleic acid molecule containing the nucle 10 of a “blank cycle during sequencing. Positive identification otide sequence set forth in SEQ ID No. 6 (or degenerates can be made after release of the oligosaccharide by PNGase F. thereof) under high Stringency conditions. A full-length SHA which converts the glycosylated Asn to Asp. After PNGase F SEGP is set forth in SEQID No. 1 and is encoded by SEQID release, N-linked oligosaccharides can be purified, for NO. 6 or degenerates thereof. example by using Bio-Gel P-6 chromatography, with the 15 oligosaccharide pool Subjected to preparative high pH anion Also provided are muteins of the Hyaluronidase domain of exchange chromatography (HPAEC) (Townsend et al., sHASEGPs, particularly muteins in which one or more Cys (1989) Anal. Biochem. 182, 1-8). Certain oligosaccharide residues in the Hyaluronidase domain that are free (i.e., do not isomers can be resolved using HPAEC. Fucose residues will form disulfide linkages with any other Cys residue in the shift elution positions earlier in the HPAEC chromatogram, Hyaluronidase domain) are substituted with another amino while additional sialic acid residues will increase the reten acid, typically, although not necessarily, with a conservative tion time. Concurrent treatment of glycoproteins whose oli amino acid substitution or a Substitution that does not elimi gosaccharide structures are known (e.g., bovine fetuin, a-l nate the activity, and muteins in which a specific glycosyla acid glycoprotein, ovalbumin, RNAse B, transferrin) can tion site (or sites) is eliminated. facilitate assignment of the oligosaccharide peaks. The col sHASEGP polypeptides, including, but not limited to 25 lected oligosaccharides can be characterized by a combina splice variants thereof, and nucleic acids encoding SHA tion of compositional and methylation linkage analyses SEGPs, and domains, derivatives and analogs thereof are (Waeghe et al., (1983) Carbohydr Res. 123, 281-304), with provided herein. Single chain secreted Hyaluronidase glyco anomeric configurations assigned by NMR spectroscopy proteins that have an N-terminus functionally equivalent to (Van Halbeek (1993) in Methods Enzymol 230). that generated by activation of a signal peptidase to form 30 In the case of some shASEGPs, exemplified by the human sHASEGP are also provided. There are seven potential sHASEGP rHuPH20 described herein, the SHASEGP can N-linked glycosylation sites at N82, N166, N235, N254, comprise both N-glycosidic and O-glycosidic linkages. For N368, N393, N490 of the human PH20 shASEGP exempli example, rhuPH20 (as produced in the DG44 CHO line3D3, fied in SEQID NO: 1. Disulfide bonds form between the Cys as described in the Examples below) has been discovered to residues C60-C351 and Cys residues C224 to C238 to form 35 have O-linked oligosaccharides as well as N-linked oligosac the core Hyaluronidase domain. However, additional cys charides. Glycosidic modifications of such shASEGPs (for teines are required in the carboxy terminus for neutral example, modifications comprising one or more additions, enzyme catalytic activity such that the shASEGP domain removals or alterations of such N-linked and/or O-linked from amino acids 36 to Cys 464 in SEQID No. 1 comprises oligosaccharides can be used to generate glycosidic variant the minimally active human PH20 shASEGP hyaluronidase 40 sHASEGPs exhibiting altered pharmacokinetic and/or phar domain. Thus, N-linked glycosylation site N-490 is not macodynamic profiles that make them desirable for particular required for propersFIASEGP activity. As will be appreciated applications. By way of illustration, but without being by those of skill in the art, minor changes can be made to Such restricted to a particular mechanism of action, removal or compositions as described and illustrated herein without sub alteration of (such as by capping or otherwise de-exposing) stantially eliminating or in some cases without Substantially 45 one or more N-linked and/or O-linked oligosaccharides that reducing (or potentially even improving) their useful activity, are involved in binding to cellular or other receptors can be and can thus be similarly employed in various applications as used to alter the extent and/or rate at which a shASEGP binds described herein. to or is taken up by a particular tissue, which can in turn be N-linked glycosylation of some shASEGPs (such as the used to alter for example its pharmacokinetic profile (e.g., by sHASEGP comprising the amino acid sequence of SEQ ID 50 increasing its serum half life or otherwise altering its bioab No. 1) can be very important for their catalytic activity and Sorption). stability. While altering the type of glycan modifying a gly Formulations of shASEGPs are also provided. SHA coprotein can have dramatic affects on a proteins antigenic SEGPs may beformulated in lyophilized forms and stabilized ity, structural folding, Solubility, and stability, most enzymes Solutions for example. Formulations containing specific are not thought to require glycosylation for optimal enzyme 55 metal ions, such as calcium, magnesium, or Sodium, are use activity. Such shASEGPs are thus unique in this regard, in ful for optimal activity at neutral pH. In addition to stabilized that removal of N-linked glycosylation can result in near Solution formulations, slow release formulations are contem complete inactivation of the Hyaluronidase activity. For such plated herein for extended removal of glycosaminoglycans or sHASEGPs, the presence of N-linked glycans is critical for extended promotion of spreading or diffusion of agents such generating an active enzyme. Protein expression systems 60 as pharmacologics. Also provided herein are kits providing suitable for the introduction of critical N-linked glycosylation for pre-packaged syringes of shASEGPs for the administra residues on shASEGPs are included. Additionally, the intro tion of small volumes of shASEGP for intraocular surgical duction of deglycosylated shASEGP polypeptide in the pres procedures and other Small Volume procedures. Balanced salt ence of extracts capable of introducing N-linked glycans are formulations for ex vivo use in artificial reproductive tech included. In one aspect of the invention, complex glycosyla 65 nology procedures are also provided. tion capped with Sialation is described whereas others capped Methods for the use of glycosaminoglycanases including with free mannose residues are contemplated as well. Prefer sHASEGPs in the removal of glycosaminoglycans are also US 7,871,607 B2 10 provided. Glycosaminoglycanases including shASEGPs enhance the delivery of Solutions and drugs into and/or open channels in the interstitial space through degradation of through interstitial spaces. This can be useful for the diffusion glycosaminoglycans that generally permit the diffusion of ofanesthesia and for the administration of therapeutic fluids, molecules less than about 500 nm in size. These channels can molecules and proteins. Subcutaneous, intradermal and intra remain relatively open for a period of 24-48 hours depending muscular administration of molecules in the presence of on dose and formulation. Such channels can be used to facili SHASEGPs (and/or other glycosaminoglycanases) also tate the diffusion of exogenously added molecules Such as facilitate their systemic distribution more rapidly. Such meth fluids, Small molecules, proteins, nucleic acids and gene ods are very useful when intravenous access is not available therapy vectors and other molecules less than about 500 nm in or where more rapid systemic delivery of molecules is size. In addition, without being restricted to a particular 10 needed. By way of illustration, delivery of other large mol theory or mechanism of action, it is believed that the forma ecules such as Factor VIII, that are poorly bioavailable upon tion of such channels can facilitate bulk fluid flow within an Subcutaneous administration, may be injected with SHA interstitial space, which can in turn promote the dispersion or SEGPs to increase their availability. movement of a solute (such as a detectable molecule or other Uses of shASEGPs for enzymatic removal of the cumulus diagnostic agent, an anesthetic or other tissue-modifying 15 matrix Surrounding oocytes are also provided. The removal of agent, a pharmacologic or pharmaceutically effective agent, the cumulus matrix using a purified shASEGP without the or a cosmetic or otheresthetic agent) that is effectively carried toxic contaminants of extract derived hyaluronidase permits by the fluid in a process sometimes referred to herein as more gentle recover of the oocyte with greater viabilities. “convective transport” or simply convection. Such convective Moreover, shASEGPs can be manufactured without the use transport can Substantially exceed the rate and cumulative of cattle extracts or other organisms that carry viruses and effects of molecular diffusion and can thus cause the thera other pathogens such as transmissible spongiform encepha peutic or other administered molecule to more rapidly and lopathies. effectively perfuse a tissue. Furthermore, when a molecule Injections of small volumes of shASEGP for intraocular Such as a therapeutic or other agent (Such as a small molecule use may also be used for Small spaces. For example, SHA drug or a larger molecule or complex) is co-formulated or 25 SEGPs and/or other glycosaminoglycanases may be injected co-administered with a shASEGP (or other glycosaminogly into the anterior chamber of the eye to remove excess vis canase) and both are injected into a relatively confined local coelastic Substrates that are administered during Surgery. site. Such as a site of non-intravenous parenteral administra Intraocular injection of shASEGPs can also be used to reduce tion (e.g., intradermal, Subcutaneous, intramuscular, or into intraocular pressure in glaucoma, to dissolve vitreous aggre or around other internal tissues, organs or other relatively 30 gates, or “floaters', to clear vitreous hemorrhage, for the confined spaces within the body), then the fluid associated treatment of macular degeneration, to promote vitreo retinal with the administered dose can both provide a local driving detachment in diabetic retinopathy and to be mixed with other force (i.e. hydrostatic pressure) as well as lower impedance to enzymes to promote reshaping of the cornea along with cor flow (by opening channels within the interstitial matrix)— rective lenses. As described herein, shASEGPs and/or other both of which would tend to increase fluid flow, and with it 35 glycosaminoglycanases can also be injected into the eye or convective transport of the therapeutic agent or other mol into Surrounding periorbital spaces as a form of “spreading ecule contained within the fluid. As discussed and illustrated agent, i.e. to promote the delivery of diagnostic, anesthetic or in more detail herein, and as will be appreciated by those of pharmacologic agents to sites within and/or around the target skill in the art, these aspects of the use of shASEGPs and tissue. For example, injection of a shASEGP concomitant other glycosaminoglycanases can have Substantial utility for 40 with or in temporal proximity to injection of diagnostic, anes improving the bioavailability as well as manipulating other thetic or pharmacologic agents into the Sub-Tenon's or epis pharmacokinetic and/or pharmacodynamic characteristics of cleral space can be used to promote transcleral delivery of the co-formulated or co-administered agents. pharmacologic to interior portions of the eye (such as the sHASEGPs and other glycosaminoglycanases can also be choroid, retina and vitreous body). The episcleral space is a used to remove excess glycosaminoglycans such as those that 45 lymph-containing space between the Fascia Bulbi (capsule of occur following ischemia reperfusion, inflammation, arterio Tenon) and the sclera which is a relatively tough protective Sclerosis, edema, cancer, spinal cord injury and otherforms of tissue Surrounding the choroid and retina and the interior of scarring. In some instances, SHASEGPs and otherglycosami the eye. The Sub-Tenon's or episcleral space, which is some noglycanases can be delivered systemically by intravenous times referred to as the periscleral lymph space, is generally infusion. This can be helpful when local access is not readily 50 also continuous with the Subdural and Subarachnoid cavities available such as the heart or brain or in the case of dissemi and is traversed by bands of connective tissue extending from nated neoplasm wherein the disease is through the body. For the fascia to the sclera. As will be appreciated by those of skill Such intravenous or other intravascular applications, Super in the art, the ability to promote delivery of compositions Sialated shASEGPs are often preferable to increase serum across the protective Scleral layer covering the eye allows for half-life and distribution over native hyaluronidase enzymes 55 a great variety of pharmacologic and other agents to be con that lack terminal sialic acids. veniently and efficiently delivered to underlying tissues In some circumstances, such as spinal cord injury and other within the eye, Such as the choroid, retina or vitreous humor. nervous system disorders, glaucoma and certain other disor By applying compositions of the present invention to such in ders of the eye, as well as various autoimmune, inflammatory vivo delivery routes even relatively difficult to reach tissues, conditions, cancers and other chronically progressive dis 60 such as those at the back of the eye, can be treated by mini eases and conditions, and cosmetic treatments, Sustained mally invasive means. It will be recognized that in some delivery is preferred. As will be appreciated by those of skill instances, the use of a longer lasting shASEGP Such as a in the art, a number of different compositions and techniques pegylated-shASEGP will be desirable. have been developed to provide slow-release or sustained It is important to note that while animal-derived hyalu release formulations or depots of molecules of interest. 65 ronidases can and have been applied to treat certain condi In otherindications, a single short acting dose is preferable. tions of the eye, and as a spreading factor to promote the Temporary removal of glycosaminoglycans can be used to delivery of anesthetics and other therapeutic or pharmaco US 7,871,607 B2 11 12 logic agents, several important considerations apply to the ration, which are commonly used to reduce the amount of use of animal-derived enzymes and potentially either limit applied Viscoelastic remaining in the eye after Surgery is their use or introduce safety concerns or additional risks when completed. Viscoelastics are commonly used during anterior they are used for treating humans. Among these consider segment Surgeries to maintain a deep anterior chamber allow ations is the concern that proteins and other impurities present ing for more efficient manipulations and potentially protect in the formulations can lead to immunogenic and/or other ing the corneal endothelium and other tissues during Surgery. problems when applied to human tissues in vivo. In addition, A variety of Viscoelastics containing hyaluronate and/or to the extent that non-human animal enzymes are employed, chondroitin Sulfate are used in Surgeries such as anterior eye even purified enzymes can themselves further contribute to segment procedures (see, e.g., ProviscTM, ViscoatTM and Duo immunogenicity since they are of non-human origin. Further 10 viscTM (available from Alcon Laboratories, Inc., www.alcon in that regard, the potential for degradative enzymes such as labs.com); HealonTM, Healon 5TM and Healon GVTM (avail hyaluronidases to effectively promote their own dispersal (as able from Advanced Medical Optics, www.healon.com); described herein), could increase the probability that immu AmviscTM and AmviscTM Plus (available from Bausch & nogenic versions or formulations of Such animal-derived Lomb, www.bausch.com); I-ViscTM, I-ViscTM Plus, I-ViscTM products are contacted by the immune system. 15 18, and I-ViscTM Phaco (available from I-MED Pharma, Concerns with animal-derived and/or impure factors are www.imedpharma.com); LA LONTM (available from Gen also of increased significance in a number of situations; for eral Innovations, www.general trade.net)). However, as is example, situations in which the targettissue has already been known in the art, retained viscoelastic in the anterior chamber Subjected to Surgical or other procedures, situations in which following Surgical procedures such as cataract Surgery can the agent is being introduced into a relatively immune-privi lead to compromised outflow from the anterior chamber (po leged space (such as portions of the eye, the heart, the nervous tentially by reducing outflow through the trabecular mesh system and many other tissues and organs that are not typi work) which can cause elevations in intraocular pressure cally exposed to the external environment), and/or situations (IOP spikes) that can lead to glaucoma. in which the patient is immuno-compromised or otherwise at Thus shASEGPs of the present invention can be intro elevated risk associated with infection. Use of such animal 25 duced, for example, by injection into the anterior chamber of derived formulations may thus be avoided in a number of the eye as a “viscoelastic antidote for previously-applied applications in which the risk of infection or other complica Viscoelastic formulations containing glycosaminglycans tions undercut their potential utility. In those situations and in Such as hyaluronan. For use as Such a viscoelastic antidote, others, the use of recombinantly-prepared shASEGPs of the the shASEGP will generally be introduced by post-surgical present invention can provide applications that are both 30 injection into the anterior chamber (with or without prior highly effective and have favorable safety profiles, and thus irrigation or aspiration to remove Some portion of the previ improve and expand the conditions and situations in which ously applied viscoelastic) following anterior segment pro these enzymes are beneficially used. cedures such as cataract Surgery and intraocular lens implan In the context of non-intravenous parenteral injections tation (e.g. by phacoemulsification or extracapsular Surgery), (such as intradermal, Subcutaneous, intramuscular and other 35 glaucoma filtration Surgery (trabeculectomy), corneal trans injections into spaces other than the vasculature), a SHA plant Surgery (e.g. penetrating keratoplasty), post-traumatic SEGP (and/or another glycosaminoglycanase) and another eye surgery, and the like. As will be appreciated by those of agent (e.g. a co-formulation or a mixture comrising a SHA skill in the art, the amount of a shASEGP applied in such a SEGP and another agent Such as a diagnostic agent, an anes context will depend, interalia, on its specific activity as well thetic agent, a pharmacologic agent, an esthetic agent, or 40 as the amount of viscoelastic used and whether or not some of combinations therefo) in a volume of liquid (e.g. a pharma the viscoelastic is first physically removed (e.g. by irrigation ceutical excipient or other Solution) can be introduced into a and aspiration). Each application will thus generally be opti site or sites within the body by injection or infusion. Without mized for the particular circumstances, as will be appreciated wishing to be bound by theory, it is believed that several by those of skill in the art in view of the descriptions and forces can be brought into motion to enhance delivery of the 45 illustrations provided herein. pharmacologic or other agent (the extent of which depends in Some viscoelastics, such as ViscoatTM and DuoViscTM part on the particular composition, Volume and site of admin (available from Alcon Labs) comprise both hyaluronate and istration for example). These driving forces can include an chondroitin sulfate. Advantageously, many shASEGPs of the increase in hydrostatic pressure as a Volume of fluid is effec present invention are enzymatically active both as hyalu tively forced into a contained space (such as the Sub-Tenon's 50 ronidases and as chondroitinases, making them particularly space referred to above, or a site of intradermal, Subcutane suitable for such dual formulation viscoelastics. ous, intramuscular or other non-IV parenteral injection), a Hyaluronidases including shASEGPs can also be com Subsequent increase in convective transport of Solutes (or bined with one or more additional glycosaminoglycanases convection) as fluid flow is increased down its pressure gra (such as chondroitinases) for applications such as the forego dient (and dissolved molecules or macromolecular com 55 ing or to further open channels within interstitial spaces for plexes are carried with it), as well as an increase in diffusion other applications such as those described and illustrated and/or permeation mediated by degradation of glycosami herein. noglycans and concomitant channel formation within the To the extent that viscoelastics are also used in other pro downstream intercellular matrix or interstitial space. cedures, shASEGPs of the present invention can likewise be In the case of shASEGPs being injected into the anterior 60 used in conjunction with or as replacement of techniques for chamber of the eye to remove excess viscoelastic substrates reducing or eliminating amounts of applied viscoelastic, i.e. Such as glycosaminoglycan-containing formulations (which as a viscoelastic antidote. are commonly used as opthalmosurgical aids in a variety of In applications of SHASEGPs as spreading agents in tis anterior segment procedures), it will be appreciated by those Sues such as those of the eye, a formulation comprising one or of skill, in view of the teachings of the present application, 65 more shASEGPs as described herein is typically applied to a that shASEGP can be used to supplement or to obviate the target tissue prior to or concomitant with application of an need for post-Surgical procedures Such as irrigation and aspi anesthetic, diagnostic, pharmacologic or other agent that is US 7,871,607 B2 13 14 desired to be delivered into or to the vicinity of the target in turn tends to increase interstitial fluid flow and to concomi tissue. In some contexts, shASEGPs can be used to promote tantly facilitate the diffusion and/or convective solute trans delivery of a pharmacologic or other agent across a first tissue port (convection) of dissolved components within the inter to target one or more tissues distal to the first. By way of Stitial fluid (Such as anesthetics, drugs and other illustration, introduction of shASEGPs into the episcleral 5 pharmacologic agents, labels and diagnostic agents, and the space Surrounding the eye prior to or concomitantly with like). As will be appreciated by those of skill in the art, introduction of a pharmacologic or other agent into the epis sHASEGPs of the present invention can be applied to cleral space can be used to promote delivery of the agent enhance the bioavailability (and potentially improve other across the protective Scleral tissue and into the choroid, the pharmacokinetic and/or pharmacodynamic properties) of a retina and the vitreous body. In this manner, a number of 10 number of pharmacologic and other agents that are useful for pharmacologic and/or other agents which are useful for treat treating or diagnosing various disease conditions or other ing conditions of the choroid, retina and/or vitreous can be wise modifying one or more tissues in vivo. Illustrative cat delivered relatively locally to the target tissues but at the same egories of such agents include: anti-cancer agents, anti-infec time using a relatively non-invasive approach Such as a Sub tives, anesthetics, anti-inflammatories, cytokines, antibodies Tenon's injection. Analogous situations exist in other situa 15 and other proteins, nucleic acids, macromolecular com tions in which shASEGPs can be applied to one tissue or plexes, and numerous other molecules and pharmacologic location in the body in order to facilitate the delivery of a agents which modify cellular or other physiologic activities, pharmacologic or other agent within that tissue or location including various categories of agents (and exemplary mem and/or to distal sites, as described and illustrated herein. bers thereof) described herein and in the art. While the extent and rate of dispersal of a co-formulated or While the diffusion and convective transport of even small co-administered agent depends in part on the tissue and agent molecules can be enhanced by opening interstitial channels involved, such disperal can generally be increased by, inter and increasing fluid flow, in the case of larger pharmacologic alia, increasing the amount of SHASEGP applied, employing or other agents, such as many biotherapeutics (including anti a SEHASEGP having higher specific activity, employing a bodies and other proteins, large nucleic acids, macromolecu sHASEGP that is more resistant to degradation or other inac 25 lar complexes (such as liposomes and other macromolecular tivation (such as a PEGylated, super-Sialated or other such carriers), as well as gene therapy vectors and the like)), the modified shASEGP), providing a sustained release or depot size of the molecules and the presence of interstitial compo formulation of the shASEGP, and other such approaches to nents such as glycosaminoglycans Substantially impairs the increasing the effective activity and/or duration of the applied diffusion and/or convection of the agents. Several conse sHASEGP. In addition, where it is desired that the SHASEGP 30 quences potentially limiting the agents usefulness can result. faciliate the dispersion of an agent into a tissue or location For example, the pharmacokinetics of the agent can be effec distalto, e.g., a site of injection, then providing thesHASEGP tively impaired by a slowing of absorption and thus distribu and/or agent in a significant Volume of liquid (such as would tion of the agent. In addition, trapping of a portion of the agent partially fill or even slightly distend the site of injection), can at or near the site of administration both limits its bioavail be used to further promote dispersion through a process of 35 ability and can also cause toxicity as a result of a potentially convective transport, as described and illustrated herein. Sustained and high local dose. In the latter regard, local tox As will be appreciated by those of skill in the art, shA icity that may be associated with painful or other side effects SEGPs can thus be used to effectively promote delivery of a is a problem with many large biomolecules that are adminis number of anesthetics, diagnostics, pharmacologics and/or tered by non-intravenous injection, such as by Subcutaneous, other agents to the posterior segments of the eye for treating 40 intradermal or intramuscualar injection. As a result, a number conditions such as retinal detachments, retinal vein occlu of pharmacologic agents have pharacokinetic (PK) and/or sions, proliferative retinopathies, diabetic retinopathies, pharmacodynamic (PD) profiles that can be enhanced by inflammatory conditions (such as uveitis, choroiditis, retinitis co-formulating the agent with a shASEGP (or other gly and the like), as well as degenerative diseases, vascular dis cosaminoglycanase) and/or co-administering the agent with a eases and various tumors. Again as will be appreciated by 45 sHASEGP (or other glycosaminoglycanase), which may be those of skill in the art, a variety of pharmacologic or phar provided before, coincident with or after the agent, and maceutically effective agents can be usefully applied to treat administered at the same or a different site, which parameters ing Such posterior segment conditions and diseases, includ would be the subject of optimization instandard models (such ing, by way of illustration, anesthetic and pharmacologic as animal models typically used to assess the pharmacokinet agents such as those described and illustrated below. 50 ics and pharmacodynamics of the agent). Any of a variety of Co-formulations or co-administrations of shASEGP with therapeutics and pharmacologics as well as other agents (such other Substances may also be envisioned for injectable pens as cosmetic or esthetic formulations) that are typically admin for Small Volume or rapid subcutaneous administration. istered by parenteral administration can thus be enhanced Examples such as EpipenTM, insulin, and other fluids can be using shASEGPs. formulated. The methods of the invention include adminis 55 While intravascular administration, particularly by intra tration of the shASEGP polypeptide or pharmaceutical com venous (IV) injection, can generally provide for rapid bio positions containing shASEGP prior to, simultaneously with availability, IV injections are often inconvenient, associated or following administration of other therapeutic molecules. with risks or inapplicable (and even where practicable can be The shASEGP may be administered at a site different from associated with less than optimal overall PK/PD profiles). the site of administration of the therapeutic molecule or the 60 Since shASEGPs can be applied to enhance the bioavailabil sHASEGP may be administered at a site the same as the site ity of agents administered by non-IV parenteral routes (such of administration of the therapeutic molecule. as Subcutaneous, intradermal, intramuscular and other routes Without wishing to be bound by theory, it is believed that delivering agents into the interstitium), agents that have been the ability of shASEGPs and other glycosaminoglycanases delivered by IV injection can be reformulated for delivery by to cause the degradation of a portion of the glycosaminogly 65 non-IV parenteral routes. Such reformulations can provide cans in the interstitial spaces between cells results in a tem Substantial benefits such as allowing use of agents in which porary opening up of channels within the interstitium, which IV administration is inconvenient, inapplicable, unsafe, too US 7,871,607 B2 15 16 expensive or otherwise limited. The reformulation of IV advantageous to deliver the agent into the dermal as opposed agents as non-IV parenterals can also allow patients to self to sub-dermal layers since there tends to be a relatively high administer the agents, and can allow dosing to be optimized concentration of antigen-presenting cells (APCs) localized (e.g. better tailored to the agent's PK/PD parameters) by within the dermis. avoiding the need for administration by intravenous injection. As will be appreciated by those of skill in the art, PK/PD In the context of those agents that are typically delivered by parameters that can be improved by using shASEGPs (and/or intradermal injection (whether by standard needles or other other glycosaminoglycanases) include Such measures as newer devices), or those many other agents which are not Cmax (the maximal concentration of agent achieved follow currently but could be delivered by intradermal injection, the ing absorption in, e.g., the bloodstream), Tmax (the time 10 local co-introduction of a shASEGP (and/or other gly required to achieve maximal concentration), T/2 (the time cosaminoglycanase) can be used to enhance delivery of the required for the concentration to fall by half). Cmin (the pharmacologic or other agent delivered and to promote its minimal concentration of agent following metabolism and dispersal or spreading within the dermis. In the case of a excretion), AUC (area under the curve of concentration ver vaccine, where the dermis may be the primary target tissue, sus time, a measure of the overall amount of bioavailability), 15 given the local abundance of APCs, the shASEGP (and/or concentrations in various tissues of interest (including, e.g., other glycosaminoglycanase) can thus facilitate dispersal of the rate of achieving desired concentrations, the overall lev the vaccine within the target tissue, thereby increasing the els, and the duration of maintaining desired levels), and Emax probability and extent of interactions between a vaccine and (the maximal effect achieved). By way of illustration, refor APCs (which can significantly potentiate the generation of an mulating a drug or other pharmacologic agent with a SHA immunomodulatory response). In the case of other agents, the SEGP or co-administering the agent with a shASEGP (which dermis may not be the primary target tissue but is rather a can be introduced locally or systemically, and before, tissue into which a dose of the agent is introduced from which together with or after the agent) can be used for example to it is desired that the agent be absorbed into another tissue, increase an agent's Cmax, decrease its Tmax, decrease its typically the bloodstream. In the latter case, the bloodstream T/2, increase its AUC and/or increase its Emax. As will be 25 may itself be the target tissue of interest (e.g. for blood appreciated, the ability to readily and proportionately modulatory factors as described herein and in the art), or the manipulate such key PK and PD parameters (e.g. by applying bloodstream may itselfbe a delivery tissue by which the agent more or less units of shASEGP and altering the timing and/or is transported to a distal target tissue (e.g. a tissue in the body location of administration) enables the improvement of a supplied by the bloodstream). In either of these latter cases (in number of a pharmacologic and other agents. Evaluating the 30 which delivery is intradermal but it is desired that the agent be levels of these parameters can be performed in models such as delivered to the bloodstream), the shASEGP (and potentially animal models used for PK/PD measurements of the agent, a volume of liquid in which it is injected) can facilitate dis and co-formulations and/or co-administrations that enhance persal of the agent first within the dermis and thence into the the relative efficacy to safety profile thus selected for a par vasculature draining the dermis and eventually into the larger ticular application. 35 blood supply), as described and illustrated herein. Among non-IV parenterals, shASEGPs (and/or other gly The use of shASEGPs to enhance the pharmacokinetics cosaminoglycanases) can also be used to allow the agents to and/or pharmacodynamics of other therapeutic or pharmaco be administered by more convenient routes, and/or with logic agents can also be applied to agents delivered by routes greater efficiency. By way of illustration (and without limita other than non-IV parenteral administration. For example, tion), by reformulating and/or co-administering agents with 40 without wishing to be bound by theory, it is believed that the sHASEGP (either local or systemic and either before, coin presence of shASEGPs in interstitial spaces within the body cident with or after administration of the agent) agents that are (which can be achieved by local and/or systemic administra typically administered by Subcutaneous injection can instead tion of shASEGPs) tends to promote interstitial channels and be administered by intradermal injection, and agents that are an increase in fluid flow within the interstitial space that in typically administered by intramuscular injection can instead 45 turn facilitates both diffusion and convective transport of be administered by Subcutaneous or intradermal injection. agents dissolved with the interstitial fluid (Such as pharma Alternatively, agents can be administered by the same route, cologic and other agents). By way of illustration, agents that but with improved pharmacokinetics and/or pharmacody are administered directly into the bloodstream (e.g. by intra namics using shASEGPs. venous injection), or orally (and thus enter the bloodstream The use of shASEGPs (and/or other glycosaminoglyca 50 after uptake from the gastrointestinal tract for example), can nases) to reformulate IV drugs and other agents as non-IV still be subject to constraints in the post-absorption i.e. dis parenterals also enables delivery of the agents using any of a trubution phase of their pharmacokinetics. Without wishing variety of new injection devices designed to ease and/or speed to be bound by theory, it is believed that shASEGPs can delivery, and to facilitate self-administration. Such devices enhance delivery to target cells by increasing interstitial per include, for example, ultra-sharp and microneedle devices 55 meability and fluid flow and thus increasing the diffusion (such as those being developed by Becton Dickinson and and/or convection of agents within the interstitial space others) as well as needle-free injection devices (such as (which effectively forms the intermedium between almost all BiojectorTM and other devices available from Bioject: Intra pharmacologic delivery routes and the target cells). JectTM and other devices available from Aradigm; Medijec In addition, it is believed that changes in oncotic pressure torTM devices and the like). A number of devices (such as the 60 brought about by the administration of shASEGPs can con Biojector) are particularly useful for facilitating intradermal tribute to enhancing the delivery of pharmacologic agents. injections which tend to require more experience when using Again, without wishing to be bound by theory, the presence of standard needles (due to the potential for penetrating the sHASEGP and concomitant degradation along the interstitial dermis during needle placement, and delivering the agent to side of a vascularized tissue, would tend to decrease the an underlying tissue site such as the Subcutaneous layer). For 65 oncotic pressure within the interstitial space which in turn Some pharmacologic agents, such as Vaccines for example would enhance fluid filtration from the vasculature into the (e.g. a DNA-based or other vaccine), it can be particularly interstitial space. US 7,871,607 B2 17 18 The potential to decrease interstitial pressure is considered lems and limitations associated with systemic administration to be particularly important in the context of many cancers in in which concentrations at undesired sites may equal or even which interstitial pressure within the tumor (tumor interstitial exceed concentrations at the desired target site (potentially pressure of TIF) is elevated. High TIF can result in relative triggering undesired side effects as well as wasting agent). In impedance to fluid flow from the vasculature toward the cen Some cases, agents that are not widely used or are not used for ter of a tumor, thereby limiting the amount of an anti-cancer certain indications or in certain patients, for example, may be agent that effectively reaches a tumor, particulary sites that effectively applied to benefit additional patients by being are deeper within a tumor mass. The introduction of shA co-formulated or co-administered with sEASEGPs as SEGPs to tumor interstitia would thus tend to enhance the described and illustrated herein. delivery of locally delivered as well as systemically available 10 As will be appreciated by those of skill in the art, the ability anti-cancer agents which can more readily penetrate the to employ shASEGPs to enhance, speed and/or target bio tumor when interstitial oncotic pressure is reduced and dif distribution of co-formulated and/or co-administered agents, fusion and/or convective transport increased. Measurements and to manipulate other aspects of their pharmacokinetics or of TIF and hydraulic conductivity (K) within tumors (e.g. pharmacodynamics (e.g. in order to improve their risk: ben using tagged molecules Such as albumin labeled with Evan's 15 efit profile or facilitate their use by patients, family or health Blue dye) can be used to quantitatively assess the effect of care professionals), provides a major opportunity for improv various concentrations of shASEGPs on the fluid dynamics ing drugs and other agents that are used treat, diagnose or of tumors in vivo, as illusrated herein and/or in the art. prevent diseases. Further exacerbating the reduced fluid dynamics within Without being limited to a particular set of applications, many tumors, and highlighting an additional potential benefit glycosaminoglycanases, including SHASEGPs as described of applying shASEGPs to tumors, is the fact that many herein (as well as other hyaluronidases and other glycosami tumors exhibit accumulation of glycosaminoglycans, par noglycanases) can be used to effectively achieve bolus or ticularly hyaluronan (which may be due to the fact that lym bolus-like delivery of any number of pharmacologic and phatics, which are the predomint route for hyaluronan catabo other agents by non-intravenous parenteral routes, as well as lism, are impaired or lacking in many tumors). Such excess 25 by other routes of administration (e.g. by enhancing the deliv hyaluronan can contribute to impeding hydraulic conductiv ery of agents into and/or through a target tissue after they ity. The introduction of shASEGPs can thus be used to coun leave the bloodstream (whether they were introduced into the teract the accumulation of glycosaminoglycans in many bloodstream directly (such as by IV administration) or indi tumors, improving hydraulic conductivity within the tumor rectly (such as by oral or non-IV parenteral administration)). and effectively rendering them more susceptible to anti-tu 30 Without being restricted to a specific mechanism of action mor agents (whether locally or systemically delivered). or aspect thereof, it is believed that the ability of these As will be appreciated by those of skill in the art, the enzymes to temporarily degrade components of the intersti principles described herein can also be applied to numerous tial matrix between cells (which accounts for a substantial other pharmacologics and other agents that are desired to be portion of the fluid space in the body and a correspondingly delivered to sites within the body, such as for the prevention, 35 large space that must be traversed in order for pharmacologics diagnosis and/or treatment of disease or to otherwise modu and other agents to reach most target cells) can significantly late physiological functions. Illustrative classes of Such promote the delivery of agents to target cells by one or more agents (and exemplary members thereof) are provided herein, of several potentially synergistic means. First, while the bulk and many more are known in the art or under development. of the interstitial fluid exhibits some degree of flow, that flow In addition to their use in potential improvements and/or 40 is often restrained or impeded by glycosaminoglycans such as reformulations of a variety of parenterally-administered hyaluronan and other interstitial components. The ability of pharmacologics and/or agents, SHASEGPs can also be use glycosaminoglycanases including SHASEGPS (as well as fully employed in connection with non-parenteral agents other hyaluronidases and other glycosaminoglycanases) to (such as agents formulated as pills, liquids or other forms for open channels within Such interstitial spaces, as described ingestion and typical absorption through the gastrointestinal 45 and illustrated herein, can be used to decrease impedance and tract). For example, since most non-parenteral drugs must increase the extent and rate of “downstream” flow resulting ultimately reach cells within the interstitium in order to exert from any given “upstream' pressure. Second, in the case of their desired effects, the use of shASEGPs to enhance diffu non-IV parenteral injections of shASEGPs (or other gly sion and/or convective transport within the interstitium (ei cosaminoglycanase) and another pharmacologic or other ther systemically or locally (e.g., by local or targeted admin 50 agent, the Volume of the non-IV parenteral injection can be istration of shASEGP)) can be applied to improve the extent used to increase the upstream driving pressure or pressure and/or rate at which non-parenterals (as well as parenterals) head (e.g. by increasing the hydrostatic pressure within a reach desired target cells. confined space), which can further promote flow. Third, since In addition, a number of agents that are typically adminis the volume of fluid comprising the introduced shASEGP (or tered non-parenterally (e.g. orally) may be reformulated, or 55 other glycosaminoglycanase) and other agent is effectively their active ingredients reformulated, for use in parenteral driven down the increased pressure gradient (i.e. the administrations that are rendered more effective and/or safe increased gradient created by elevating the hydrostatic pres in combination with shASEGPs. To illustrate this broadly sure associated within the injected “bolus' and simulta applicable approach, the ability of shASEGPs to facilitate neously decreasing the interstitial pressure within the Sur targeted delivery to particular tissues (such as the ability of 60 rounding tissue), solutes within the bolus can be effectively sHASEGPs to provide for transcleral delivery to tissues carried (e.g. by convective transport) into the adjacent tissue. within the posterior of the eye) can be used to deliver any of This injected fluid or bolus can thus be efficiently and rela a variety of agents (including agents previously administered tively quickly moved into the adjacent tissue and deliver systemically) directly and preferentially to a localized site of whatever pharmacologic or other agent was introduced at or interest within the body. This can not only provide for more 65 near the same site of introduction (e.g. by co-formulating or desirable and/or more rapidly achieved concentrations at the co-administering the agent in combination with the SHA site of interest, but can substantially reduce potential prob SEGP or other glycosaminoglycanase). US 7,871,607 B2 19 20 By way of illustration, using glycosaminoglycanases such GAG Enzyme is introduced. As will be appreciated by those as shASEGPs to open channels within interstitial spaces, as of skill in the art, the volume (V) that can be accommodated described and illustrated herein, can be used to increase inter and the resulting extent of distension (D) depends on the Stitial flow into and through tissues such as tumors and others particular tissue. For purposes of illustration and not limita in which flow is typically impeded and interstitial pressures tion, the extent to which distention occurs varies, for example, elevated. Illustrative examples of these principles as from situations in which (i) the Dosing Tissue is a relatively described herein include the use of shASEGPs to reduce the large and/or rapidly moving fluid-filled space (such as the interstitial pressure within a tumor in vivo. bloodstream in connection with intravenous or intraarterial Increased flow can likewise be applied to normal tissue to, delivery), in which case L can be large (e.g. many ml if e.g., allow an anesthetic, diagnostic, cosmetic or other 10 desired) but D tends to be relatively minimal; (ii) the Dosing esthetic, or a pharmacologic or other agent to be delivered Tissue is a relatively smaller and/or less rapidly moving fluid into a tissue (e.g. to modify the tissue or introduce a depot filled space (such as cerebrospinal fluid in connection with formulation into a tissue). Increased flow can likewise be intrathecal delivery), in which case L can still be relatively applied to a first or proximal tissue to promote delivery of an large but D may be more than minimal depending on the agent through the tissue of introduction to a distal tissue, 15 precise site and Volume of injection); (iii) the Dosing Tissue which distal tissue may itself by the desired target tissue or a is a relatively smaller but somewhat distensible space (such as route to the desired target tissue. Illustrative examples of the episcleral space Surrounding the eye for purposes of illus these principles as described herein include, for example: (i) tration); or (iv) the Dosing Tissue is a relatively more dense the use of shASEGPs introduced into the episcleral space and/or more confined space with higher resisting pressure Surrounding the eye to promote the delivery of agents across (such as the dermis in connection with intradermal delivery the Sclera in order to target distal target tissues within the for purposes of illustration). interior of the eye such as the retina and choroid; and (ii) the As will be appreciated by those of skill in the art, V can use of shASEGPs introduced intradermally to promote the range from Volumes of less than 0.1 ml to Volumes of greater delivery of agents through the Subdermal layers and into the than 100 ml, formany applications being in the range of about bloodstream, which can itself be the target tissue (e.g. for 25 0.5 ml to 20 ml, and for many in the range of 1 ml to 10 ml, blood-modifying agents) or a route by which the agent(s) can frequently from 2 to 5 ml; but can be varied for particular be delivered to other target tissues within the body situations as illustrated herein. V can also be specifically By way of further illustration of these techniques and com optimized within Such ranges for a particular application as positions for promoting delivery of pharmacologics and other desired; e.g. by comparing standard pharmacokinetic and/or agents to desired tissues within the body, the following addi 30 pharmacodynamic profiles over a range of test Volumes. tional examples are provided. By means such as described As will be appreciated by those of skill in the art in view of and illustrated herein, the pharmacokinetic and/or pharmaco these teachings, by combining the rapidly acting and reiniti dynamic profiles of pharmacologic agents, and the absorp ating channel-opening activity of the GAG Enzyme (which tion, distribution and/or effectiveness profiles of other agents, both promotes and is promoted by its own delivery), together can be enhanced or otherwise modified. In addition, agents 35 with a delivery system that both promotes and is promoted by that were essentially or practically limited to delivery by one the catalytic activity, a multiply synergistic drug delivery route (such as by IV injection) can be delivered by other safer, system can thus be generated; as further described and illus less intrusive, less costly, and/or more convenient routes. trated herein. By way of further illustration and without being limited to In a first illustrative aspect, a volume of liquid (V) com a particular type of application, a Volume (V) of liquid (L) 40 prising a shASEGP or other glycosaminoglycanase (GAG comprising a shASEGP or other glycosaminoglycanase Enzyme) and one or more pharmacologic or other agents (GAG Enzyme) can be introduced into a tissue site in the body (Agent(s) (Such as detectable molecules or other diagnostic (a Dosing Tissue). By means as described and illustrated agents, anesthetics or other tissue-modifying agents, pharma herein, the GAG Enzyme can be delivered into and to some cologics or pharmaceutically effective agents, cosmetics or extent through the Dosing Tissue. Without being restricted to 45 other esthetic agents, and other agents desired to be intro a particular mechanism of action, the GAG Enzyme can be duced into one or more tissues within the body) is introduced effectively carried into the Dosing Tissue by convective trans into a first tissue site in the body (a Dosing Tissue) which is port by the volume of liquid (L). The convective transport can itself a desired target tissue (a Target Tissue) for the agent. By in turn be promoted in part by the hydrostatic pressure asso means as described and illustrated herein, the Agent(s) are ciated with L (which can provide a driving pressure) and in 50 delivered into and throughout the Target Tissue to a greater part by the ability of the GAG Enzyme to open channels extent and/or rate than would occur in the absence of the GAG within the interstitial spaces of the Dosing Tissue (which can Enzyme. reduce downstream impedance to flow and thereby lower the As will be appreciated by those of skill in the art in con resisting pressure). A fluid-driving pressure differential can nection with this and the other illustrative aspects herein, the thus be created and to the extent desired it can be increased, 55 Dosing Tissue can be any of a variety of tissues that can be for example, by one or both of the following: (i) introducing readily reached from outside of the body (e.g. using various and if desired increasing a driving hydrostatic pressure (e.g. routes of administration as described and illustrated herein by introducing Linto a confined space and if desired increas and/or as known in the art) or from inside the body (e.g. in ing V); and (ii) reducing the downstream impedance to flow connection with Surgery or using any of a variety of non (e.g. by introducing GAG Enzyme, and if desired increasing 60 Surgical or minimally-invasive approaches and devices to its effectiveness by for example increasing its concentration, reach tissues within the body). Such Dosing Tissues include increasing its resistance to degradation (e.g. by PEGylation, those described and illustrated herein as well as others known Super-Sialation or other modification), and/or increasing its in the art. delivery by convective transport (e.g. by increasing V)). As will likewise be appreciated by those of skill in the artin In certain embodiments (e.g. in which increased extent or 65 connection with this and the other illustrative aspects herein, rate of delivery is desired), V is a volume sufficient to tem the Target Tissue can be any of a variety of tissues to which it porarily distend the site within the Dosing Tissue at which the is desired that a GAG Enzyme and potentially other Agent(s) US 7,871,607 B2 21 22 are delivered (e.g. (i) tissues that are the subject of efforts to In a fifth illustrative aspect, a volume of liquid (V) com diagnose, prevent or treat a disease condition (whether intrin prising a SHASEGP (or other glycosaminoglycanase) (GAG sically caused such as an inherited or developed disease con Enzyme) and one or more pharmacologic or other agents dition or extrinsically caused such as an infection); (ii) tissues (Agent(s)) is introduced into a tissue site in the body (Dosing that are desired to be anesthetized or otherwise modulated Tissue) which is proximal to one or more delivery tissues, (e.g. in the context of Surgery and/or other procedures); (iii) such as the bloodstream, lymph or cerebrospinal fluid (Deliv tissues in which it is desired to trigger an immune response ery Tissues), which deliver fluid to one or more desired target (such as associated with vaccination); (iv) tissues which are to tissues (Target Tissues) within the body. By means as serve as a depot site for releasing agent(s) to other tissues; (v) described and illustrated herein, the Agent(s) are delivered tissues to be modified for esthetic or cosmetic purposes; and 10 from Tissue A to one or more Delivery Tissues and from there (vi) tissue or sites in the body to be modified for other pur to one or more downstream Target Tissues to a greater extent poses). SuchTarget Tissues include those described and illus and/or rate than would occur in the absence of the GAG trated herein as well as others known in the art. Enzyme(s). As will likewise be appreciated by those of skill in the artin In a sixth illustrative aspect, a Volume of liquid (V) com connection with the forgoing and with other illustrative 15 prising a SHASEGP (or other glycosaminoglycanase) (GAG aspects herein, the Agent(s) can be any of a variety of mol Enzyme) together with one or more pharmacologic or other ecules, macromolecules and macromolecular complexes, that agents (Agent(s)) is introduced into one or more delivery are or can be used for altering or modulating conditions tissues, such as the bloodstream, lymph or cerebrospinal fluid within the body (e.g. (i) for purposes of diagnosing, prevent (Delivery Tissues) which supplies fluid to one or more desired ing or treating a disease condition (whether intrinsically Target Tissues (e.g. via the bloodstream Supplying a Target caused such as an inherited or developed disease condition or Tissue). By means as described and illustrated herein, the extrinsically caused such as an infection) affecting the Target Agent(s) permeate the Target Tissue to a greater extent and/or Tissue and/or adjacent tissues; (ii) for purposes of anesthe rate than would occur in the absence of the GAG Enzyme(s). tizing or otherwise modulating the physiology of the Target In a seventh illustrative aspect, a volume of liquid (V) Tissue; (iii) for purposes of triggering an immune response as 25 comprising a SEIASEGP (or other glycosaminoglycanase) associated with vaccination; (iv) for purposes of using the (GAG Enzyme) is introduced into a tissue site in the body Target Tissue as a depot site for releasing agent(s) to other (Tissue A) which is a desired target tissue or near a desired tissues; (V) for esthetic or cosmetic purposes; and/or (vi) for target tissue (Target Tissue), and one or more pharmacologic other purposes served by delivering a GAG Enzyme and or other agents (Agent(s)) are introduced into the body by potentially other Agent(s) to a tissue or other site in the body). 30 another route of administration (such as by oral delivery or Such Agents include the various classes of agents and mem intravenous injection) which allows the Agent(s) to reach the bers thereof described and illustrated herein, as well as other Target Tissue (e.g. via the bloodstream supplying the Target members of such classes of agents, and other classes of agents Tissue, following absorption or injection into the blood). By (and members thereof) known in the art. means as described and illustrated herein, the Agent(s) per In a second illustrative aspect, a Volume of liquid (V) 35 meate the Target Tissue to a greater extent and/or rate than comprising a shASEGP or other glycosaminoglycanase would occur in the absence of the GAG Enzyme(s). (GAG Enzyme) and one or more pharmacologic or other In an eighth illustrative aspect, a Volume of liquid (V) agents (Agent(s)) is introduced into a tissue site in the body comprising a SEIASEGP (or other glycosaminoglycanase) (Dosing Tissue) which is adjacent or proximal to a tissue (GAG Enzyme) is introduced into a tissue site or sites within (Tissue B) which is itself a desired target tissue. By means as 40 the body (Tissue Site(s)) which has an elevated interstitial described and illustrated herein, the Agent(s) are delivered pressure. By means as described and illustrated herein, the from Tissue A to the target Tissue B to a greater extent and/or interstitial pressure within the Tissue Site(s) is reduced by the rate than would occur in the absence of the GAG Enzyme. degradative activity of the GAG Enzyme(s). In a third illustrative aspect, a volume of liquid (V) com In a ninth illustrative aspect, a Volume of liquid (V) com prising a SEIASEGP (or other glycosaminoglycanase) (GAG 45 prising a SHASEGP (or other glycosaminoglycanase) (GAG Enzyme) and one or more pharmacologic or other agents Enzyme) is introduced into a tissue site or sites within the (Agent(s)) is introduced into a tissue site in the body (Dosing body (Tissue Site(s)) which has an excess of hyaluronan or Tissue) which is proximal to one or more tissues (Intervening other glycosaminoglycan. By means as described and illus Tissues) located between Tissues A and another tissue or trated herein, the excess of hyaluronan or other glycosami tissues (Distal Tissues) which are themselves desired target 50 noglycan within the Tissue Site(s) is reduced by the degrada tissues or include a desired target tissue (Target Tissues). By tive activity of the GAG Enzyme(s). means as described and illustrated herein, the Agent(s) are In antenth illustrative aspect, a Volume of liquid (V) com delivered from Tissue A and through one or more Intervening prising a SHASEGP (or other glycosaminoglycanase) (GAG Tissues to reach one or more Target Tissues to a greater extent Enzyme) is introduced into a tissue site or sites within the and/or rate than would occur in the absence of the GAG 55 body (Tissue Site(s)) which are at or near a desired access Enzyme(s). point for hypodermoclysis (a Clysis Site). By means as In a fourth illustrative aspect, a volume of liquid (V) com described and illustrated herein, fluids can then be delivered prising a SEIASEGP (or other glycosaminoglycanase) (GAG into and through the Clysis Site(s) to a greater extent and/or Enzyme) and one or more pharmacologic or other agents rate than would occur in the absence of the GAG Enzyme(s). (Agent(s)) is introduced into a tissue site in the body (Dosing 60 As will be appreciated by those of skill in view of the Tissue) which is proximal to one or more delivery tissues, detailed descriptions and illustrative embodiments provided such as the bloodstream, lymph or cerebrospinal fluid (Deliv herein, any of a large variety of agents can be co-introduced ery Tissues), which are desired target tissues or include a (e.g. by co-formulation or co-administration with the GAG desired target tissue. By means as described and illustrated Enzyme) and any of a variety of GAG Enzymes can be herein, the Agent(s) are delivered from Tissue A to one or 65 employed at concentrations in ranges such as those provided more Delivery Tissues to a greater extent and/or rate than herein but typically optimized for the particular tissue and would occur in the absence of the GAG Enzyme(s). objective involved. The volume of liquid (V) applied in the US 7,871,607 B2 23 24 above illustrative aspects will generally depend on the nature commonstructural features as described herein. As described of the site of introduction and will likewise typically be opti and illustrated herein, hyaluronidases (i.e. glycosaminogly mized for the particular tissue and objective involved. As canases capable of breaking down hyaluronan, preferably described and illustrated herein, in the case of non-IV those exhibiting at least Some activity in the ranges of neutral parenteral administrations, V can be increased (within the 5 pH) that are normally membrane anchored can be converted particular constraints of the tissue site) in order to provide an to soluble HASEGPs or shASEGPs by removing or other enhanced hydrostatic driving pressure which, coupled with wise modifying one or more of the regions that are associated decreased impedance associated with degradation of hyalu with anchoring the hyaluronidase in the membrane. roman or other glycosaminoglycan, can effectively cause the As used herein, Soluble hyaluronidase refers to a polypep Volume of liquid to function as a bolus—carrying any solute 10 tide characterized by its solubility under physiologic condi it contains into the downstream interstitial space and poten tions. Soluble HASEGP can be distinguished for example by tially beyond. its partitioning into the aqueous phase of a Triton X-114 Hence, provided herein are a variety of techniques employ solution warmed to 37C (Bordier et al J. Biol. Chem. 1981 ing hyaluronidases (and/or other glycosaminoglycanases) to Feb. 25: 256(4): 1604-7). Lipid anchored HASEGP on the promote the delivery of pharmacologic and other agents to 15 other hand will partition into the detergent rich phase, but will sites within the body, as well as a family of eukaryotic partition into the detergent poor or aqueous phase following secreted neutral active hyaluronidase glycoproteins desig treatment with Phospholipase-C. nated shASEGPs, and functional domains thereof, especially Thus, reference, for example, to "sHASEGP encom Hyaluronidase (or catalytic) domains thereof, muteins and passes all glycoproteins encoded by the shASEGP gene fam other derivatives and analogs thereof. Also provided herein ily, including but not limited to: Human shASEGP, mouse are nucleic acids encoding the shASEGPs. Additionally pro sHASEGP, or an equivalent molecule obtained from any vided are formulations, co-formulations and therapeutic uses other source or that has been prepared synthetically or that of said shASEGPs (including by co-administration with exhibits the same activity. Sequences of encoding nucleic other agents) to treat, prevent or diagnose disease, and for use acid molecules and the encoded amino acid sequences of as tissue modifying enzymes. 25 exemplary shASEGPs and/or domains thereof are set forth, for example in SEQ ID NO: 4. The term also encompasses BRIEF DESCRIPTION OF THE DRAWINGS sHASEGP with amino acid substitutions that do not substan tially alter activity of each member and also encompasses FIG. 1 is a vector map of shASEGP Vector HZ24. splice variants thereof. Suitable Substitutions, including, 30 although not necessarily, conservative Substitutions of amino DETAILED DESCRIPTION OF THE INVENTION acids, are known to those of skill in this art and can be made without eliminating the biological activity, such as the cata A. DEFINITIONS: Unless defined otherwise, all technical lytic activity, of the resulting molecule. and Scientific terms used herein have the same meaning as is As used herein, a shASEGP, whenever referenced herein, commonly understood by one of skill in the art to which the 35 includes at least one or all of or any combination of a invention(s) belong. All patents, patent applications, pub polypeptide encoded by the sequence of nucleotides set forth lished applications and publications, Genbank sequences, in SEQID NO. 6 or by a sequence of nucleotides that includes websites and other published materials referred to throughout nucleotides that encode amino acids 1-509 of SEQID No. 1; the entire disclosure herein, unless noted otherwise, are incor a polypeptide encoded by a sequence of nucleotides that porated by reference in their entirety. In the event that there is 40 hybridizes under conditions of low, moderate or high strin a plurality of definitions for terms herein, those in this section gency to the sequence of nucleotides set forth in SEQID NO. prevail. 6; a polypeptide that includes the sequence of amino acids set Where reference is made to a URL or other such identifier forth as amino acids 1-509 of SEQID No. 1; a polypeptide or address, it understood that such identifiers can change and that includes a sequence of amino acids having at least about particular information on the internet can come and go, but 45 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, equivalent information can be found by searching the inter 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, net. Reference thereto evidences the availability and public 97%, 98% or 99% sequence identity with the sequence of dissemination of Such information. amino acids set forth in SEQID No. 1 or as amino acids 1-448 As used herein, the abbreviations for any protective groups, of SEQID No. 4. amino acids and other compounds, are, unless indicated oth 50 In particular, the shASEGP polypeptide, with the Hyalu erwise, in accord with their common usage, recognized ronidase domains as indicated in SEQID No. 4 is provided. abbreviations, or the IUPAC-IUB Commission on Biochemi The polypeptide is a single or two chain polypeptide. Smaller cal Nomenclature (see, (1972) Biochem. 11:942-944). portions thereof that retain Hyaluronidase activity are also As used herein, eukaryotic Hyaluronidase refers to a provided. The Hyaluronidase domains from sFHASEGPs vary diverse family of glycosaminoglycan endoglucosaminidases, 55 in size and constitution, including insertions and deletions in wherein a glutamate residue in the Hyaluronidase hydrolyzes Surface loops. Thus, for purposes herein, the catalytic domain the beta 1.4 linkages of hyaluronan and chondroitin Sulfates is a portion of a shASEGP, as defined herein, and is homolo through an acid-base catalytic mechanism. gous to a domain of other hyaluronidase like sequences. Such Of particular interest are soluble neutral-active hyalu as HYAL1, HYAL2, HYAL3, which have been previously ronidases or SEIASEGPs of mammalian, including human, 60 identified; it was not recognized, however, that an isolated origin. Those of skill in this art recognize that, in general, single chain form of the human Hyaluronidase domain could single amino acid substitutions in non-essential regions of a function in in vitro assays. The Aspartate and Glutamate polypeptide do not substantially alter biological activity (see, residues necessary for activity are present in conserved e.g., Watson et al., (1987) Molecular Biology of the Gene, 4th motifs. Edition, The Benjamin/Cummings Pub. co., p. 224). 65 As used herein, a “neutral hyaluronidase domain of a As used herein, membrane anchored HASEGP, refers to a solublesHASEGP” refers to a beta 1.4 endoglucosaminidase family of membrane anchored Hyaluronidases that share domain of a shASEGP that exhibits Hyaluronidase activity at US 7,871,607 B2 25 26 neutral pH, is soluble under conditions as described and be construed as referring to a nucleic acid encoding only the shares homology and structural features with the hyalu recited single chain Hyaluronidase domain or active portion ronidase glycosyl-hydrolase family domains but contains thereof, and not the other contiguous portions of the shA additional sequences in the carboxy terminus that are SEGP as a continuous sequence. required for neutral activity. Hence it is at least the minimal As used herein, “disease' or “disorder” refers to a patho portion of the domain that exhibits Hyaluronidase activity as logical condition in an organism resulting from, e.g., infec assessed by standard in vitro assays and remains soluble. tion or genetic defect, and characterized by identifiable symp Contemplated herein are such Hyaluronidase domains and tOmS. catalytically active portions thereof. Also provided are trun As used herein, a splice variant refers to a variant produced cated forms of the Hyaluronidase domain that include the 10 by differential processing of a primary transcript of genomic Smallest fragment thereof that acts catalytically as a single nucleic acid, Such as DNA, that results in more than one type chain form. As used herein and in the art, neutral or neutral of mRNA. Splice variants of shASEGPs are provided herein. active refers to a protein exhibiting activity at neutral pH (e.g. As used herein, the Hyaluronidase domain of a shASEGP exhibiting activity at about pH 7) and which is therefore protein refers to the Hyaluronidase domain of a shASEGP active at a pH range characteristic of many physiological 15 that exhibits neutral endoglucosaminidase activity. Hence it tissues. As will also be appreciated by those of skill in the art, is at least the minimal portion of the protein that exhibits a protein generally exhibits a range of activity around its pH endoglucosaminidase activity as assessed by standard assays optimum. The pH optimum of a neutral active protein will in vitro. Exemplary human Hyaluronidase domains include at typically be within one to several pH units above or below pH least a Sufficient portion of sequences of amino acids set forth 7, but its range of activity may extend over many pH units. in SEQID No. 4 to exhibit endoglucosaminidase activity. A Hyaluronidase domain of an shASEGP, whenever ref Also contemplated are nucleic acid molecules that encode erenced herein, includes at least one or all of or any combi a polypeptide that has endoglucosaminidase activity in an in nation of or a catalytically active portion of an N-linked vitro Hyaluronidase assay and that have at least 70%, 75%, glycoprotein polypeptide that includes the sequence of amino 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, acids set forth in SEQID No. 1; a polypeptide encoded by a 25 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence of nucleotides that hybridizes under conditions of sequence identity with the full-length of a Hyaluronidase low, moderate or high Stringency to the sequence of nucle domain of an shASEGP polypeptide, or that hybridize along otides set forth in SEQID NO. 6; a polypeptide that includes their full-length or along at least about 70%, 80% or 90% of the sequence of amino acids set forth in SEQ ID No. 1; a the full-length to a nucleic acids that encode a Hyaluronidase polypeptide that includes a sequence of amino acids having at 30 domain, particularly under conditions of moderate, generally least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, high, Stringency. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, For the Hyaluronidase domains of PH20 for example, resi 96%, 97%, 98% or 99% sequence identity with the sequence dues within the N-terminal region can be critical yet not of amino acids set forth in SEQ ID No. 1; and/or a Hyalu Sufficient for activity. By application of techniques such as ronidase domain of a polypeptide encoded by a splice variant 35 shown herein one can generate Hyaluronidase domains of the of the SHASEGP. sHASEGP that are catalytically active. By way of illustration, Thus, for purposes herein, the Hyaluronidase domain is a while the Hyaluronidase domain of human PH20 generally portion of a shASEGP, as defined herein, and is homologous requires the N-terminal amino acids thereof for activity; the to a domain of other shASEGPs. As with the larger class of C-terminus portion can be truncated until the last Cysteine enzymes of the hyaluronidase family, the shASEGP catalytic 40 residue, yet requires additional amino acids to be optimally domains share a high degree of amino acid sequence identity. active. The amount that can be removed can be determined The Asp and Glu residues necessary for activity are present in empirically by testing the polypeptide for Hyaluronidase conserved motifs. activity in an in vitro assay that assesses catalytic cleavage. By active form is meant a form active in vivo and/or in Hence Smaller portions of the Hyaluronidase domains, par vitro. As described herein, the Hyaluronidase domain also 45 ticularly the single chain domains, thereof that retain Hyalu can exist as a soluble secreted glycoprotein. It is shown herein ronidase activity are contemplated. Such smaller versions that, at least in vitro, the single chain forms of the shASEGPs generally are C-terminal truncated versions of the Hyalu and the catalytic domains or enzymatically active portions ronidase domains. The Hyaluronidase domains vary in size thereof (typically C-terminal truncations) exhibit Hyalu and constitution, including insertions and deletions in Surface ronidase activity. Hence provided herein are isolated forms of 50 loops. Such domains exhibit conserved structure, including at the Hyaluronidase domains of shASEGPs and their use in in least one structural feature, Such as the proton donor, and/or vitro drug screening assays for identification of agents that other features of Hyaluronidase domains of endoglu modulate the activity thereof. cosamimidases. Thus, for purposes herein, the Hyaluronidase As used herein, the catalytically active domain of a SEHA domain is a single chain portion of a shASEGP, as defined SEGP refers to the neutral active endoglucosaminidase 55 herein, but is homologous in its structural features and reten domain as defined by activity in vitro towards a glycosami tion of sequence of similarity or homology the Hyaluronidase noglycan Substrate. domain of otherhyaluronidase-like sequences. The glycopro sHASEGPs of interest include those that are active against tein exhibits Hyaluronidase activity as a single chain. chondroitin Sulfates and chondroitin Sulfate proteoglycans As used herein, by homologous means about greater than (CSPG's) in vivo and in vitro; and those that are active against 60 25% nucleic acid sequence identity, such as 25% 40%, 60%, hyaluronan. As used herein, a human shASEGP is one 70%, 80%, 90% or 95%. If necessary the percentage homol encoded by nucleic acid, such as DNA, present in the genome ogy will be specified. The terms “homology' and “identity” of a human, including all allelic variants and conservative are often used interchangeably. In general, sequences are variations as long as they are not variants found in other aligned so that the highest order match is obtained (see, e.g.: mammals. 65 Computational Molecular Biology, Lesk, A. M., ed., Oxford As used herein, nucleic acid encoding a Hyaluronidase University Press, New York, 1988: Biocomputing: Informat domain or catalytically active portion of a shASEGP” shall ics and Genome Projects, Smith, D.W., ed., Academic Press, US 7,871,607 B2 27 28 New York, 1993: Computer Analysis of Sequence Data, Part/. proximately 90% identity). Differences are defined as nucleic Griffin, A. M., and Griffin, H. G., eds., Humana Press, New acid or amino acid substitutions, or deletions. At the level of Jersey, 1994; Sequence Analysis in Molecular Biology, von homologies or identities above about 85-90%, the result Heinje, G., Academic Press, 1987; and Sequence Analysis should be independent of the program and gapparameters set; Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Such high levels of identity can be assessed readily, often Press, New York, 1991; Carillo et al. (1998) Slam J Applied without relying on Software. Math 48: 1073). As used herein, primer refers to an oligonucleotide con By sequence identity, the numbers of conserved amino taining two or more deoxyribonucleotides or ribonucleotides, acids is determined by standard alignment algorithms pro typically more than three, from which synthesis of a primer grams, and are used with default gap penalties established by 10 extension product can be initiated. Experimental conditions each Supplier. Substantially homologous nucleic acid mol conducive to synthesis include the presence of nucleoside ecules would hybridize typically at moderate Stringency or at triphosphates and an agent for polymerization and extension, high Stringency all along the length of the nucleic acid or such as DNA polymerase, and a suitable buffer, temperature along at least about 70%, 80% or 90% of the full-length and pH. nucleic acid molecule of interest. Also contemplated are 15 As used herein, animals include any animal. Such as, but nucleic acid molecules that contain degenerate codons in are not limited to, goats, cows, deer, sheep, rodents, pigs and place of codons in the hybridizing nucleic acid molecule. humans. Non-human animals, exclude humans as the con Whether any two nucleic acid molecules have nucleotide templated animal. The shASEGPs provided herein are from sequences that are at least, for example, 80%, 85%, 90%, any source, animal, plant, prokaryotic and fungal. Preferred 95%, 96%, 97%, 98% or 99% “identical can be determined SHASEGPs are of animal origin, including mammalian ori using known computer algorithms such as the “FASTA' pro gin, and more preferably are of human origin in the case of gram, using for example, the default parameters as in Pearson their use in humans. et al (1988) Proc. Natl. Acad. Sci. USA 85: 2444 (other As used herein, genetic therapy involves the transfer of programs include the GCG program package (Devereux, J., et heterologous nucleic acid, Such as DNA, into certain cells, al, Nucleic Acids Research 12: 387 (1984)), BLASTP 25 target cells, of a mammal, particularly a human, with a dis BLASTN, FASTA (Atschul, S. F. et al., JMOLEC BIOL215: order or conditions for which such therapy is sought. The 403 (1990); Guide to Huge Computers, Martin J. Bishop, nucleic acid, such as DNA, is introduced into the selected ED. Academic Press, San Diego, 1994, and CARILLO et target cells in a manner Such that the heterologous nucleic al. (1988) SIAMJApplied Math 48: 1073). For example, the acid, such as DNA, is expressed and a therapeutic product BLAST function of the National Center for Biotechnology 30 encoded thereby is produced. Information database can be used to determine identity. Other Alternatively, the heterologous nucleic acid, such as DNA, commercially or publicly available programs include, can in some manner mediate expression of DNA that encodes DNASTAR “MEGALIGN PROGRAM (Madison, Wis.) the therapeutic product, or it can encode a product, such as a and the University of Wisconsin Genetics Computer Group peptide or RNA that in some manner mediates, directly or (UWG) “Gap' program (Madison Wis.)). Percent homology 35 indirectly, expression of a therapeutic product. Genetic or identity of proteins and/or nucleic acid molecules can be therapy can also be used to deliver nucleic acid encoding a determined, for example, by comparing sequence informa gene product that replaces a defective gene or Supplements a tion using a GAP computer program e.g. Needleman et al. gene product produced by the mammal or the cell in which it (1970), J. Mol. Biol. 48: 443, as revised by Smith and Water is introduced. The introduced nucleic acid can encodeathera man Adv. Appl. Math (1981) 2: 482). Briefly, the GAP pro 40 peutic compound. Such as a growth factor inhibitor thereof, or gram defines similarity as the number of aligned symbols a tumor necrosis factor or inhibitor thereof. Such as a receptor (i.e., nucleotides or amino acids) that are similar, divided by therefore, that is not normally produced in the mammalian the total number of symbols in the shorter of the two host or that is not produced in therapeutically effective sequences. Default parameters for the GAP program can amounts or at a therapeutically useful time. The heterologous include: (1) a unary comparison matrix (containing a value of 45 nucleic acid, such as DNA, encoding the therapeutic product 1 for identities and 0 for non-identities) and the weighted can be modified prior to introduction into the cells of the comparison matrix of Gribskov et al (1986) Nucl. Acids Res. afflicted host in order to enhance or otherwise alter the prod 14: 6745, as described by Schwartz and Dayhoff, eds., Atlas uct or expression thereof. Genetic therapy can also involve Of Protein Sequence And Structure, National Biomedical delivery of an inhibitor or repressor or other modulator of Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 50 gene expression. for each gap and an additional 0.10 penalty for each symbol in As used herein, heterologous nucleic acid is nucleic acid each gap; and (3) no penalty for endgaps. Therefore, as used that encodes proteins (or, if DNA, encodes RNA that encodes herein, the term “identity” represents a comparison between a proteins) that are not normally produced in vivo by the cell in test and a reference polypeptide or polynucleotide. which it is expressed or that mediates or encodes mediators As used herein, the term at least "90% identical to refers 55 that alter expression of endogenous nucleic acid, Such as to percent identities from 90 to 99.99 relative to the reference DNA, by affecting transcription, translation, or other regulat polypeptides. Identity at a level of 90% or more is indicative able biochemical processes. Heterologous nucleic acid. Such of the fact that, assuming for exemplification purposes a test as DNA, can also be referred to as foreign nucleic acid. Such and reference polynucleotide length of 100 amino acids are as DNA. Any nucleic acid, such as DNA, that one of skill in compared. No more than 10% (i.e., 10 out of 100) amino acids 60 the art would recognize or consideras heterologous or foreign in the test polypeptide differ from that of the reference to the cell in which is expressed is herein encompassed by polypeptides. Similar comparisons can be made between a heterologous nucleic acid; heterologous nucleic acid includes test and reference polynucleotides. Such differences can be exogenously added nucleic acid that is also expressed endog represented as point mutations randomly distributed over the enously. Examples of heterologous nucleic acid include, but entire length of an amino acid sequence or they can be clus 65 are not limited to, nucleic acid that encodes traceable marker tered in one or more locations of varying length up to the proteins, such as a protein that confers drug resistance, maximum allowable, e.g. 10/100 amino acid difference (ap nucleic acid that encodes therapeutically effective sub US 7,871,607 B2 29 30 stances, such as anti-cancer agents, enzymes and hormones, Such as promoters, enhancers, transcriptional and transla and nucleic acids (such as DNA and RNA), that indirectly or tional stop sites, and other signal sequences. In order to opti directly encode other types of proteins, such as antibodies. mize expression and/or in vitro transcription, it can be nec Antibodies that are encoded by heterologous nucleic acid can essary to remove, add or alter 5' Untranslated portions of the be secreted or expressed on the surface of the cell in which the 5 clones to eliminate extra, potential inappropriate alternative heterologous nucleic acid has been introduced. translation initiation i.e. start) codons or other sequences that Heterologous nucleic acid is generally not endogenous to can interfere with or reduce expression, either at the level of the cell into which it is introduced, but has been obtained from transcription or translation. Alternatively, consensus ribo another cell or prepared synthetically. Some binding sites (see, e.g., Kozak J. Biol. Chem. 266: Generally, although not necessarily, Such nucleic acid 10 19867-19870 (1991) can be inserted immediately 5' of the encodes RNA and/or proteins that are not normally produced start codon and can enhance expression. The desirability of by the cell in which it is expressed. (or need for) such modification can be empirically deter As used herein, a therapeutically effective product is a mined. product that is encoded by heterologous nucleic acid, typi As used herein, a sequence complementary to at least a cally DNA, that, upon introduction of the nucleic acid into a 15 portion of an RNA, with reference to antisense oligonucle host, a product is expressed that ameliorates or eliminates the otides, means a sequence having Sufficient complementarity symptoms, manifestations of an inherited or acquired disease to be able to hybridize with the RNA generally under mod or that cures the disease. erate or high Stringency conditions, forming a stable duplex; As used herein, recitation that a glycoprotein consists in the case of double-stranded sPASEGP antisense nucleic essentially of the Hyaluronidase domain means that the only acids, a single strand of the duplex DNA (or dsRNA) can thus sHASEGP portion of the polypeptide is a Hyaluronidase be tested, or triplex formation can be assayed. The ability to domain or a catalytically active portion thereof. The polypep hybridize depends on the degree of complementarity and the tide can optionally, and generally will, include additional length of the antisense nucleic acid. Generally, the longer the non-shASEGP-derived sequences of amino acids. hybridizing nucleic acid, the more base mismatches with a As used herein, domain refers to a portion of a molecule, 25 sHASEGP encoding RNA it can contain and still form a e.g., glycoproteins or the encoding nucleic acids that is struc stable duplex (or triplex, as the case can be). One skilled in the turally and/or functionally distinct from other portions of the art can ascertain a tolerable degree of mismatch by use of molecule. standard procedures to determine the melting point of the As used herein, Hyaluronidase refers to an enzyme cata hybridized complex. lyzing hydrolysis of glycosaminoglycans including hyaluro 30 For purposes herein, amino acid substitutions can be made aS. in any of shASEGPs and Hyaluronidase domains thereof For clarity reference to Hyaluronidase refers to all forms, provided that the resulting protein exhibits Hyaluronidase and particular forms will be specifically designated. For pur activity. Amino acid Substitutions contemplated include con poses herein, the Hyaluronidase domain includes the mem servative substitutions, such as those set forth in Table 1, and brane bound and soluble forms of a shASEGP protein. 35 other modifications that do not eliminate proteolytic activity. As used herein, nucleic acids include DNA, RNA and As described herein, substitutions that alter properties of the analogs thereof, including protein nucleic acids (PNA) and proteins, such as removal of cleavage sites and other Such mixture thereof. Nucleic acids can be single or double sites are also contemplated; Such Substitutions are generally stranded. When referring to probes or primers, optionally non-conservative, but can be readily effected by those of skill labeled with a detectable label, such as a fluorescent dye or 40 in the art. radiolabel, single-stranded molecules are generally contem Suitable conservative substitutions of amino acids are plated. Such molecules are typically of a length such that their known to those of skill in this art and can be made generally target is statistically unique or of low copy number (typically without eliminating (and preferably without substantially less than 5, generally less than 3) for probing or priming a altering) the biological activity, for example enzymatic activ library. As is known in the art, generally a probe or primer 45 ity, of the resulting molecule. Those of skill in this art recog contains at least 14, 16, 20 or 30 residues of contiguous or nize that, in general, single amino acid substitutions in non nearly-contiguous sequence complementary to or identical a essential regions of a polypeptide do not substantially alter gene of interest. Probes and primers can be 10, 20, 30, 50, 100 biological activity (see, e.g., Watson et al. Molecular Biology or more nucleic acids long. of the Gene, 4th Edition, 1987. The Benjamin/Cummings As used herein, nucleic acid encoding a fragment or por 50 Pub. co., p. 224). Also included within the definition, is the tion of a shASEGPrefers to a nucleic acid encoding only the catalytically active fragment of a shASEGP, particularly a recited fragment or portion of shASEGP, and not the other single chain Hyaluronidase portion. Conservative amino acid contiguous portions of the shASEGP. Substitutions are made, for example, in accordance with those As used herein, operative linkage of heterologous nucleic set forth in TABLE 1 as follows: to regulatory and effector sequences of nucleotides, such as 55 TABLE 1 Original residue Conservative substitution Ala promoters, enhancers, transcriptional and translational stop (A) Gly; Ser, Abu Arg (R) Lys, Orn Asn (N) Gln: His Cys (C) sites, and other signal sequences refers to the relationship Ser Gln (O) Asn Glu (E) ASP Gly (G) Ala: Pro His (H) ASn: between Such nucleic acid, Such as DNA, and Such sequences Gln Ile (I) Leu; Val; Met; Nile; Nva Leu (L); Val; Met; Nile; of nucleotides. For example, operative linkage of heterolo Nva Lys (K) Arg: Gln: Glu Met (M) Leu: Tyr; Ile: NLe Val gous DNA to a promoter refers to the functional (and typi 60 Orn Lys: Arg Phe (F) Met; Leu: Tyr Ser (S) Thr Thr (T) Ser cally physical) relationship between the DNA and the pro Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) ILE: Leu: Met; Nile; moter such that the transcription of such DNA is initiated Nva. Other substitutions are also permissible and can be from the promoter by an RNA polymerase that specifically determined empirically or in accord with known conservative recognizes, binds to and transcribes the DNA in reading Substitutions. frame. Thus, operatively linked or operationally associated 65 As used herein, Abu is 2-aminobutyric acid; Nle is norleu refers to the functional relationship of nucleic acid, Such as cine; Niva is norvaline; Orn is ornithine. As used herein, the DNA, with regulatory and effector sequences of nucleotides, amino acids, which occur in the various amino acid sequences US 7,871,607 B2 31 32 appearing herein, are identified according to their well pepsin at pH 4.0-4.5; it can be recombinantly expressed to known, three-letter or one-letter abbreviations. The nucle produce the equivalent fragment. otides, which occur in the various DNA fragments, are des As used herein, Fab fragments are antibody fragments that ignated with the standard single-letter designations used result from digestion of an immunoglobulin with papain; they routinely in the art. can be recombinantly expressed to produce the equivalent As used herein, a probe or primer based on a nucleotide fragment. sequence disclosed herein, includes at least 10, 14, typically As used herein, schVs refer to antibody fragments that at least 16 contiguous sequence of nucleotides of SEQID NO. contain a variable light chain V, and variable heavy chain 6, and even more preferably at least 20, 30, 50 or 100 con (VH) covalently connected by a polypeptide linker in any tiguous sequence of nucleotides of SEQID NO. 6. The length 10 order. The linker is of a length such that the two variable of the probe or primer for unique hybridization is a function of domains are bridged without substantial interference. the complexity of the genome of interest. Included linkers are (Gly-Ser) in residues with some Glu or As used herein, amelioration of the symptoms of a particu Lys residues dispersed throughout to increase solubility. lar disorder by administration of a particular pharmaceutical As used herein, humanized antibodies refer to antibodies composition refers to any lessening, whether permanent or 15 that are modified to include human sequences of amino acids temporary, lasting or transient that can be attributed to or so that administration to a human does not provoke an associated with administration of the composition. immune response. Methods for preparation of Such antibod As used herein, antisense polynucleotides refer to syn ies are known. For example, to produce Such antibodies, the thetic sequences of nucleotide bases complementary to hybridoma or other prokaryotic or eukaryotic cell. Such as an mRNA or the sense strand of double-stranded DNA. Admix E. coli or a CHO cell, that expresses the monoclonal antibody ture of sense and antisense polynucleotides under appropriate are altered by recombinant DNA techniques to express an conditions leads to the binding of the two molecules, or antibody in which the amino acid composition of the non hybridization. When these polynucleotides bind to (hybridize variable region is based on human antibodies. Computer pro with) mRNA, inhibition of protein synthesis (translation) grams have been designed to identify such regions. occurs. When these polynucleotides bind to double-stranded 25 As used herein, diabodies are dimeric scEV; diabodies DNA, inhibition of RNA synthesis (transcription) occurs. typically have shorter peptide linkers than ScFVs, and they The resulting inhibition of translation and/or transcription generally dimerize. leads to an inhibition of the synthesis of the protein encoded As used herein, production by recombinant means by using by the sense Strand. Antisense nucleic acid molecule typically recombinant DNA methods means the use of the well known contain a sufficient number of nucleotides to Specifically bind 30 methods of molecular biology for expressing proteins to a target nucleic acid, generally at least 5 contiguous nucle encoded by cloned DNA. otides, often at least 14 or 16 or 30 contiguous nucleotides or As used herein the term assessing is intended to include modified nucleotides complementary to the coding portion of quantitative and qualitative determination in the sense of a nucleic acid molecule that encodes a gene of interest, for obtaining an absolute value for the activity of an shASEGP, example, nucleic acid encoding a single chain Hyaluronidase 35 or a domain thereof, present in the sample, and also of obtain domain of an SHASEGP. ing an index, ratio, percentage, visual or other value indica tive of the level of the activity. Assessment can be direct or As used herein, an array refers to a collection of elements, indirect and the chemical species actually detected need not Such as antibodies, containing three or more members. An of course be the proteolysis product itself but can for example addressable array is one in which the members of the array are 40 be a derivative thereof or some further substance. identifiable, typically by position on a solid phase Support. As used herein, biological activity refers to the in vivo Hence, in general the members of the array are immobilized activities of a compound or physiological responses that on discrete identifiable loci on the surface of a solid phase. result upon in vivo administration of a compound, composi As used herein, antibody refers to an immunoglobulin, tion or other mixture. Biological activity, thus, encompasses whether natural or partially or wholly synthetically produced, 45 therapeutic effects and pharmaceutical activity of Such com including any derivative thereofthat retains the specific bind pounds, compositions and mixtures. Biological activities can ing ability the antibody. Hence antibody includes any protein be observed in in vitro systems designed to test or use Such having a binding domain that is homologous or Substantially activities. Thus, for purposes herein the biological activity of homologous to an immunoglobulin-binding domain. Anti a luciferase is its oxygenase activity whereby, upon oxidation bodies include members of any immunoglobulin claims, 50 of a substrate, light is produced. including IgG, IgM, IgA, Ig|D and IgE. As used herein, functional activity refers to a polypeptide As used herein, antibody fragment refers to any derivative orportion thereof that displays one or more activities associ of an antibody that is less then full-length, retaining at least a ated with a full-length protein. portion of the full-length antibody's specific binding ability. Functional activities include, but are not limited to, bio Examples of antibody fragments include, but are not limited 55 logical activity, catalytic or enzymatic activity, antigenicity to Fab, Fab', F(AB)2, single chain Fvs (scFV), FV, dsEV (ability to bind to or compete with a polypeptide for binding diabody and Fd fragments. The fragment can include multiple to an anti-polypeptide antibody), immunogenicity, ability to chains linked together, such as by disulfide bridges. An anti form multimers, the ability to Specifically bind to a receptor body fragment generally contains at least about 50 amino or ligand for the polypeptide. acids and typically at least 200 amino acids. 60 As used herein, a conjugate refers to the compounds pro As used herein, an FV antibody fragment is composed of vided herein that includes one or more shASEGPs, including one variable heavy domain (VH) and one variable light a shASEGP, particularly single chain Hyaluronidase domain linked by noncovalent interactions. domains thereof, and one or more targeting agents. These As used herein, a dsEV refers to an Fv with an engineered conjugates include those produced by recombinant means as intermolecular disulfide bond. 65 fusion proteins, those produced by chemical means, such as As used herein, an F(AB)2 fragment is an antibody frag by chemical coupling, through, for example, coupling to Sulf ment that results from digestion of an immunoglobulin with hydryl groups, and those produced by any other method US 7,871,607 B2 33 34 whereby at least one shASEGP, or a domain thereof, is lial cells into new functioning capillaries, and the persistence linked, directly or indirectly via linker(s) to a targeting agent. of such functioning capillaries. As used herein, a targeting agent is any moiety, such as a As used herein, pharmaceutically acceptable salts, esters or protein or effective portion thereof, that provides specific other derivatives of the conjugates include any salts, esters or binding of the conjugate to a cell Surface receptor, which, can derivatives that can be readily prepared by those of skill in this internalize the conjugate or shASEGP portion thereof. A art using known methods for Such derivatization and that targeting agent can also be one that promotes or facilitates, for produce compounds that can be administered to animals or example, affinity isolation or purification of the conjugate; humans without substantial toxic effects and that either are attachment of the conjugate to a Surface; or detection of the pharmaceutical active or are prodrugs. conjugate or complexes containing the conjugate. 10 As used herein, a prodrug is a compound that, upon in vivo As used herein, an antibody conjugate refers to a conjugate administration, is metabolized or otherwise converted to the in which the targeting agent is an antibody. biologically, pharmaceutically or therapeutically active form As used herein, derivative or analog of a molecule refers to of the compound. To produce a prodrug, the pharmaceutical a portion derived from or a modified version of the molecule. active compound is modified such that the active compound is As used herein, an effective amount of a compound for 15 regenerated by metabolic processes. The prodrug can be treating a particular disease is an amount that is Sufficient to designed to alter the metabolic stability or the transport char ameliorate, or in some manner reduce the symptoms associ acteristics of a drug, to mask side effects or toxicity, to ated with the disease. Such amount can be administered as a improve the flavor of a drug or to alter other characteristics or single dosage or can be administered according to a regimen, properties of a drug. By virtue of knowledge of pharmacody whereby it is effective. The amount can cure the disease but, namic processes and drug metabolism in Vivo, those of skill in typically, is administered in order to ameliorate the symptoms this art, once a pharmaceutically active compound is known, of the disease. Repeated administration can be required to can design prodrugs of the compound (see, e.g., Nogrady achieve the desired amelioration of symptoms. (1985) Medicinal Chemistry A Biochemical Approach, As used herein equivalent, when referring to two sequences Oxford University Press, New York, pages 388-392). of nucleic acids means that the two sequences in question 25 As used herein, a drug identified by the screening methods encode the same sequence of amino acids or equivalent pro provided herein refers to any compound that is a candidate for teins. When equivalent is used in referring to two proteins or use as a therapeutic or as a lead compound for the design of a peptides, it means that the two proteins or peptides have therapeutic. Such compounds can be Small molecules, includ Substantially the same amino acid sequence with only amino ing Small organic molecules, peptides, peptide mimetics, acid Substitutions (such, as but not limited to, conservative 30 antisense molecules or dsRNA, such as RNAi, antibodies, changes such as those set forth in Table 1, above) that do not fragments of antibodies, recombinant antibodies and other substantially alter the activity or function of the protein or Such compounds that can serve as drug candidates or lead peptide. When equivalent refers to a property, the property compounds. does not need to be present to the same extent (e.g., two 35 As used herein, a peptidomimetic is a compound that mim peptides can exhibit different rates of the same type of enzy ics the conformation and certain stereochemical features of matic activity), but the activities are usually substantially the the biologically active form of aparticular peptide. In general, same. Complementary, when referring to two nucleotide peptidomimetics are designed to mimic certain desirable sequences, means that the two sequences of nucleotides are properties of a compound, but not the undesirable properties, capable of hybridizing, typically with less than 25%, 15%, such as flexibility, that lead to a loss of a biologically active 5% or 0% mismatches between opposed nucleotides. If nec 40 conformation and bond breakdown. Peptidomimetics may be essary the percentage of complimentarity will be specified. prepared from biologically active compounds by replacing Typically the two molecules are selected such that they will certain groups or bonds that contribute to the undesirable hybridize under conditions of high Stringency. properties with bioisosteres. Bioisosteres are known to those As used herein, an agent that modulates the activity of a 45 of skill in the art. For example the methylene bioisostere protein or expression of a gene or nucleic acid either CH2S has been used as an amide replacement in enkephalin decreases or increases or otherwise alters the activity of the analogs (see, e.g. Spatola (1983) pp. 267-357 in Chemistry protein or, in Some manner up- or down-regulates or other and Biochemistry of Amino Acids, Peptides, and Proteins, wise alters expression of the nucleic acid in a cell. Weistein, Ed. volume 7, Marcel Dekker, New York). Mor As used herein, inhibitor of the activity of an shASEGP 50 phine, which can be administered orally, is a compound that encompasses any Substance that prohibits or decrease pro is a peptidomimetic of the peptide endorphin. For purposes duction, post-translational modification (s), maturation, or herein, cyclic peptides are included among pepidomimetics. membrane localization of the shASEGP or any substance As used herein, a promoter region or promoter element that interferes with or decreases the proteolytic efficacy of refers to a segment of DNA or RNA that controls transcription thereof, particularly of a single chain form in an in vitro 55 of the DNA or RNA to which it is operatively linked. The Screening assay. promoter region includes specific sequences that are suffi As used herein, a method for treating or preventing neo cient for RNA polymerase recognition, binding and transcrip plastic disease means that any of the symptoms, such as the tion initiation. tumor, metastasis thereof, the vascularization of the tumors or This portion of the promoter region is referred to as the other parameters by which the disease is characterized are 60 promoter. In addition, the promoter region includes reduced, ameliorated, prevented, placed in a state of remis sequences that modulate this recognition, binding and tran Sion, or maintained in a state of remission. It also means that scription initiation activity of RNA polymerase. These the hallmarks of neoplastic disease and metastasis can be sequences can be cis acting or can be responsive to trans eliminated, reduced or prevented by the treatment. Non-lim acting factors. Promoters, depending upon the nature of the iting examples of the hallmarks include uncontrolled degra 65 regulation, can be constitutive or regulated. Exemplary pro dation of the basement membrane and proximal extracellular moters contemplated for use in prokaryotes include the bac matrix, migration, division, and organization of the endothe teriophage T7 and T3 promoters. US 7,871,607 B2 35 36 As used herein, a receptor refers to a molecule that has an catalogs that describe commonly used laboratory solutions). affinity for a given ligand. Receptors can be naturally occur SspE is pH 7.4 phosphate-buffered 0.18 NaCl. Further, those ring or synthetic molecules. Receptors can also be referred to of skill in the art recognize that the stability of hybrids is in the art as anti-ligands. As used herein, the receptor and determined by TmT which is a function of the sodium ion anti-ligand are interchangeable. Receptors can be used in concentration and temperature (Tm=81.5°C.-16.6+0.41 (% their unaltered State or as aggregates with other species. G+C)-600/L)) so that the only parameters in the wash con Receptors can be attached, covalently or noncovalently, or in ditions critical to hybrid stability are sodium ion concentra physical contact with, to a binding member, either directly or tion in the SspE (or SSC) and temperature. indirectly via a specific binding Substance or linker. Examples It is understood that equivalent Stringencies can be of receptors, include, but are not limited to: antibodies, cell 10 achieved using alternative buffers, salts and temperatures. By membrane receptors surface receptors and internalizing way of example and not limitation, procedures using condi receptors, monoclonal antibodies and antisera reactive with tions of low stringency are as follows (see also Shilo and specific antigenic determinants such as on viruses, cells, or Weinberg, Proc. Natl. AcadSci USA78: 6789-6792 (1981)): other materials, drugs, polynucleotides, nucleic acids, pep Filters containing DNA are pretreated for 6 hours at 40C in a tides, factors, lectins, Sugars, polysaccharides, cells, cellular 15 solution containing 35% formamide, 5xSSC, 50 mM Tris membranes, and organelles. HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll 1% BSA, Examples of receptors and applications using Such recep and 500 ug/ml Denatured Salmon sperm DNA (10x) SSC is tors, include but are not restricted to: a) enzymes: specific 1.5M sodium chloride, and 0.15M sodium citrate, adjusted to transport proteins or enzymes essential to Survival of micro a pH of 7). organisms, which could serve as targets for antibiotic ligand Hybridizations are carried out in the same solution with the selection; b) antibodies: identification of aligand-binding site following modifications: 0.02% PVP 0.02% Ficoll 0.2% on the antibody molecule that combines with the epitope of an BSA, 100VG/M sperm DNA, 10% (wt/vol) dextran sulfate, antigen of interest can be investigated; determination of a and 5-20x10 cpm32P-labled probe is used. Filters are incu sequence that mimics an antigenic epitope can lead to the bated in hybridization mixture for 18-20 hours at 40 C and development of vaccines of which the immunogen is based on 25 then washed for 1.5 hours at 55 C in a solution containing one or more of such sequences or lead to the development of 2xSSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% related diagnostic agents or compounds useful in therapeutic SDS. The wash solution is replaced with fresh solution and treatments such as for auto-immune diseases c) nucleic acids: incubated an additional 1.5 hours at 60 C. Filters are blotted identification of ligand. Such as protein or RNA, binding sites: dry and exposed for autoradiography. If necessary, filters are d) catalytic polypeptides: polymers, including polypeptides, 30 washed for a third time at 65-68 C and reexposed to film. that are capable of promoting a chemical reaction involving Other conditions of low stringency which can be used are well the conversion of one or more reactants to one or more prod known in the art e.g. as employed for cross-species hybrid ucts; Such polypeptides generally include a binding site spe izations). cific for at least one reactant or reaction intermediate and an By way of example and not way of limitation, procedures active functionality proximate to the binding site, in which the 35 using conditions of moderate stringency include, for functionality is capable of chemically modifying the bound example, but are not limited to, procedures using such con reactant (see, e.g., U.S. Pat. No. 5.215,899); e) hormone ditions of moderate Stringency areas follows: Filters contain receptors: determination of the ligands that bind with high ing DNA are pretreated for 6 hours at 55 C in a solution affinity to a receptor is useful in the development of hormone containing 6xSSC, 5x Denhart's solution, 0.5% SDS and 100 replacement therapies; for example, identification of ligands 40 ug/ml denatured salmon sperm DNA. Hybridizations are car that bind to such receptors can lead to the development of ried out in the same solution and 5-20x10632P labeled probe drugs to control blood pressure; and f) opiate receptors: deter is used. Filters are incubated in hybridization mixture for mination of ligands that bind to the opiate receptors in the 18-20 hours at 55C and then washed twice for 30 minutes at brain is useful in the development of less-addictive replace 60 C in a solution containing 1xSSC and 0.1% SDS. Filters ments for morphine and related drugs. 45 are blotted dry and exposed for autoradiography. Other con As used herein, Sample refers to anything that can contain ditions of moderate stringency that can be used are well an analyte for which an analyte assay is desired. The sample known in the art. Washing of filters is done at 37 C for 1 hour can be a biological sample, Such as a biological fluid or a in a solution containing 2xSSC, 0.1% SDS. biological tissue. Examples of biological fluids include urine, By way of example and not way of limitation, procedures blood, plasma, serum, saliva, semen, stool, sputum, cerebral 50 using conditions of high Stringency areas follows: Prehybrid spinal fluid, tears, mucus, sperm, amniotic fluid or the like. ization of filters containing DNA is carried out for 8 hours to Biological tissues are aggregate of cells, usually of a particu overnight at 65 C in buffer composed of 6xSSC, 50 mM lar kind together with their intercellular substance that form Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, one of the structural materials of a human, animal, plant, 0.02% BSA, and 500 ug/ml denatured salmon sperm DNA. bacterial, fungal or viral structure, including connective, epi 55 Filters are hybridized for 48 hours at 65°C. in prehybridiza thelium, muscle and nerve tissues. Examples of biological tion mixture containing 100 ug/ml denatured salmon sperm tissues also include organs, tumors, lymph nodes, arteries and DNA and 5-20x106 CPM P labeled probe. Washing of individual cells. filters is done at 37 C for 1 hour in a solution containing As used herein: Stringency of hybridization in determining 2XSSC, 0.01% PVP 0.01% Ficoll, and 0.01% BSA. This is percentage mismatch is as follows: 1) high Stringency: 0.1X 60 followed by a washin 0.1xSSC at 50 C for 45 minutes before SSPE, 0.1% SDS, 65° C. 2) medium stringency: 0.2xSspE, autoradiography. Other conditions of high Stringency that can 0.1% SDS, 50° C. 3 low stringency: 1.0xSspE, 0.1% SDS, be used are well known in the art. 50° C. Those of skill in this art know that the washing step The term substantially identical or substantially homolo selects for stable hybrids and also know the ingredients of gous or similar varies with the context as understood by those SspE (see, e.g., Sambrook, E. F. Fritsch, T. Maniatis, in: 65 skilled in the relevant art and generally means at least 60% or Molecular Cloning, A Laboratory Manual, Cold spring Har 70%, preferably means at least 80%, 85% or more preferably bor Laboratory Press 1989 Vol 3, p.B. 13, see, also, numerous at least 90%, and most preferably at least 95% identity. US 7,871,607 B2 37 38 As used herein, Substantially identical to a product means As used herein, protein binding sequence refers to a protein sufficiently similar so that the property of interest is suffi or peptide sequence that is capable of specific binding to other ciently unchanged so that the Substantially identical product protein or peptide sequences generally, to a set of protein or can be used in place of the product. peptide sequences or to a particular protein or peptide As used herein, Substantially pure means sufficiently Sequence. homogeneous to appear free of readily detectable impurities As used herein, epitope tag refers to a short stretch of amino as determined by standard methods of analysis, Such as thin acid residues corresponding to an epitope to facilitate Subse layer chromatography (TLC), gel electrophoresis and high quent biochemical and immunological analysis of the epitope performance liquid chromatography (HPLC), used by those tagged protein or peptide. Epitope tagging is achieved by of skill in the art to assess such purity, or Sufficiently pure Such 10 including the sequence of the epitope tag to the protein that further purification would not detectably alter the physi encoding sequence in an appropriate expression vector. cal and chemical properties, such as enzymatic and biological Epitope tagged proteins can be affinity purified using highly activities, of the substance. Methods for purification of the specific antibodies raised against the tags. compounds to produce Substantially chemically pure com As used herein, metal binding sequence refers to a protein pounds are known to those of skill in the art. A substantially 15 or peptide sequence that is capable of specific binding to chemically pure compound can, however, be a mixture of metal ions generally, to a set of metal ions or to a particular Stereoisomers or isomers. In Such instances, further purifica metalion. tion might increase the specific activity of the compound. As used herein, a combination refers to any association As used herein, target cell refers to a cell that expresses a between two or among more items. sHASEGP (in the context of shASEGP production as As used herein, a composition refers to any mixture. It can described herein), or to a cell that is targeted by a shASEGP be a solution, a Suspension, liquid, powder, a paste, aqueous, or co-formulated or co-administered agent in vivo (in the non-aqueous or any combination thereof. context of using shASEGPs to promote the delivery of phar As used herein, fluid refers to any composition that can macologics and other agents to target cells as described flow. Fluids thus encompass compositions that are in the form herein). 25 of semi-solids, pastes, solutions, aqueous mixtures, gels, As used herein, test Substance (or test compound) refers to lotions, creams and other Such compositions. a chemically defined compound (e.g., organic molecules, As used herein, a cellular extract refers to a preparation or inorganic molecules, organic/inorganic molecules, proteins, fraction which is made from a lysed or disrupted cell. peptides, nucleic acids, oligonucleotides, lipids, polysaccha As used herein, an agent is said to be randomly selected rides, Saccharides, or hybrids among these molecules such as 30 when the agent is chosen randomly without considering the glycoproteins, etc.) or mixtures of compounds (e.g., a library specific sequences involved in the association of a protein of test compounds, natural extracts or culture Supernatants, alone or with its associated substrates, binding partners, etc. etc.) whose effect on an shASEGP, particularly a single chain An example of randomly selected agents is the use a chemical form that includes the Hyaluronidase domain or a sufficient library or a peptide combinatorial library, or a growth broth of portion thereof for activity, as determined by an in vitro 35 an organism or conditioned medium. method, such as the assays provided herein. As used herein, an agent is said to be rationally selected or As used herein, the terms a therapeutic agent, therapeutic designed when the agent is chosen on a non-random basis that regimen, radioprotectant or chemotherapeutic mean conven takes into account the sequence of the target site and/or its tional drugs and drug therapies, including vaccines, which are conformation in connection with the agent's action. As known to those skilled in the art. Radiotherapeutic agents are 40 described in the Examples, there are proposed binding sites well known in the art. for Hyaluronidase and (catalytic) sites in the glycoprotein As used herein, treatment means any manner in which the having SEQ ID NO: 1 or SEQ ID NO: 4. Agents can be symptoms of a condition, disorder or disease are ameliorated rationally selected or rationally designed by utilizing the pep or otherwise beneficially altered. tide sequences that make up these sites. For example, a ratio Treatment also encompasses any pharmaceutical use of the 45 nally selected peptide agent can be a peptide whose amino compositions herein. acid sequence is identical to the ATP or calmodulin binding As used herein, vector (or plasmid) refers to discrete ele sites or domains. ments that are used to introduce heterologous nucleic acid Oligosaccharides are considered to have a reducing end into cells for either expression or replication thereof. The and a non-reducing end, whether or not the Saccharide at the vectors typically remain episomal, but can be designed to 50 reducing end is in fact a reducing Sugar. In accordance with effect integration of a gene or portion thereof into a chromo accepted nomenclature, oligosaccharides are depicted herein Some of the genome. Also contemplated are vectors that are with the non-reducing end on the left and the reducing end on artificial chromosomes. Such as yeast artificial chromosomes the right. All oligosaccharides described herein are described and mammalian artificial chromosomes. Selection and use of with the name or abbreviation for the non-reducing saccha such vehicles are well known to those of skill in the art. An 55 ride (e.g., Gal), followed by the configuration of the glyco expression vector includes vectors capable of expressing sidic bond (alpha. or beta.), the ring bond, the ring position DNA that is operatively linked with regulatory sequences, of the reducing saccharide involved in the bond, and then the Such as promoter regions, that are capable of effecting expres name or abbreviation of the reducing Saccharide (e.g., sion of Such DNA fragments. Thus, an expression vector GlcNAc). The linkage between two Sugars may be expressed, refers to a recombinant DNA or RNA construct, such as a 60 for example, as 2.3. 2.fw darw.3, or (2.3). Each saccharide is plasmid, a phage, recombinant virus or other vector that, upon a pyranose. introduction into an appropriate host cell, results in expres As used herein, N-linked Sugar moiety refers to an oli sion of the cloned DNA. Appropriate expression vectors are gosaccharide attached to a shASEGP via the amide nitrogen well known to those of skill in the art and include those that of Asn residues. N-linked oligosaccharides fall into several are replicable in eukaryotic cells and/or prokaryotic cells and 65 major types (oligomannose, complex, hybrid, Sulfated), all of those that remain episomal or those which integrate into the which have (Man) 3-GlcNAc-GlcNAc-cores attached via the host cell genome. amide nitrogen of Asn residues that fall within-Asn-Xaa-Thr/ US 7,871,607 B2 39 40 Ser- sequences (where Xaa is not Pro). N-linked sites are idly cleared from the blood by a mechanism involving the often indirectly assigned by the appearance of a “blank” cycle recognition of the underlying N-acetylgalactosamine (Gal during sequencing. Positive identification can be made after NAc) or galactose (Gal) residues (Goochee et al. (1991) Biol/ release of the oligosaccharide by PNGase F, which converts Technology 9: 1347-1355). For this reason, ensuring the pres the glycosylated Asn to Asp. After PNGase F release, ence of terminal sialic acid on N-linked carbohydrate groups N-linked oligosaccharides can be purified using Bio-Gel P-6 of therapeutic glycoproteins is an important consideration for chromatography, with the oligosaccharide pool Subjected to their commercial development. preparative high pH anion exchange chromatography Circulating glycoproteins are exposed to sialidase(s) (or (HPAEC) (Townsendet al., (1989) Anal. Biochem. 182, 1-8). neuraminidase) which can remove terminal Sialic acid resi Certain oligosaccharide isomers can be resolved using 10 dues. Typically the removal of the Sialic acid exposes galac HPAEC. Fucose residues will shift elution positions earlier in tose residues, and these residues are recognized and bound by the HPAEC chromatogram, while additional sialic acid resi galactose-specific receptors in hepatocytes (reviewed in Ash dues will increase the retention time. Concurrent treatment of well and Harford (1982) Ann. Rev. Biochem. 51:531). Liver glycoproteins whose oligosaccharide structures are known also contains other Sugar-specific receptors which mediate (e.g., bovine fetuin, a-lacid glycoprotein, ovalbumin, RNAse 15 removal of glycoproteins from circulation. Specificities of B, transferrin) can facilitate assignment of the oligosaccha Such receptors also include N-acetylglucosamine, mannose, ride peaks. The collected oligosaccharides can be character fucose and phosphomannose. Glycoproteins cleared by the ized by a combination of compositional and methylation link galactose receptors of hepatocytes undergo Substantial deg age analyses (Waeghe et al., (1983) Carbohydr Res. 123, radation and then enter the bile; glycoproteins cleared by the 281-304.), with anomeric configurations assigned by NMR mannose receptor of Kupffer cells enter the reticuloendothe spectroscopy (Van Halbeek (1993) in Methods Enzymol lial system (reviewed in Ashwell and Harford (1982) Ann. 230). Rev. Biochem. 51:53). Alternatively, oligosaccharides can be identified by fluo As used herein Neutral Active refers to a shASEGP gly rescence assisted carbohydrate electrophoresis (FACE) coprotein with catalytic activity towards a glycosaminogly Callewaert et al. (2001) Glycobiology 11, 275-281. 25 can substrate in vitro at a PH between 5 and 8 under condi As used herein, the term “sialic acid refers to any member tions of salt less than 150 mM and buffering strength less than of a family of nine-carbon carboxylated Sugars. The most 50 mM. common member of the sialic acid family is N-acetyl As used herein, a stabilized solution refers to a shASEGP neuraminic acid (2-keto-5-acetamindo-3,5-dideoxy-D-glyc that retains greater than 60% of its initial activity after storage ero-D-galactononulopyranos-1-onic acid (often abbreviated 30 at room temperature for 30 days. as Neu5Ac, NeuAc, or NANA). A second member of the As used herein unless otherwise specified, a unit is family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in expressed in turbidity reducing units (TRU). One TRU is which the N-acetyl group of NeuAc is hydroxylated. A third defined as the amount of hyaluronidase activity required to sialic acid family member is 2-keto-3-deoxy-nonuloSonic reduce the turbidity of an acidified solution of hyaluronic acid acid (KDN) (Nadano et al. (1986).J. Biol. Chem. 261: 11550 35 and is equivalent to the U.S.P./National Formulary (NF XIII) 11557: Kanamori et al. (1990) J. Biol. Chem. 265: 21811 units (NFU). The ELISA-like enzyme assay described herein 21819. Also included are 9-substituted sialic acids such as a can be related to the TRU, the NFU, and U.S.P. unit through 9-O-C.sub. 1-C. Sub.6acyl-Neu5Ac like 9-O-lactyl-Neu5Ac a standard curve of a sample of hyaluronidase (e.g., USP or or 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and WHO standard) standardized through the U.S.P. Therefore, 9-azido-9-deoxy-Neu5Ac. For review of the sialic acid fam 40 the enzyme activities determined by the ELISA-like enzyme ily, see, e.g., Varki (1992) Glycobiology 2: 25-40; Sialic assay are actually relative TRU, since enzyme activity is not Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. actually measured using the turbidometric assay (Dorfman et (Springer-Verlag, N.Y. (1992)). The synthesis and use of al., 1948, J. Biol. Chem. 172: 367). sialic acid compounds in a sialation procedure is disclosed in As used herein, potency is defined by the amount of shA international application WO 92/16640, published Oct. 1, 45 SEGP protein required to degrade substrate in vitro based 1992. upon a Turbidity Reducing Unit or Relative Turbidity Reduc As used herein, PNGase refers to an Asparagine Peptide ing Unit. specific N-glycosidase F such as the Flavobacterium manin As used herein, specific activity refers to Units of activity goseptum peptide-N-glycosidase F. PNG ASE enzymes are per mg protein. The amount of shASEGP protein is defined characterized by their specificity towards N-linked rather 50 by the absorption of a solution of shASEGP at 280 nm than O-linked oligosaccharides. Characterization of assuming a molar extinction coefficient of approximately 1.7. PNGASE efficacy can be defined by both SDS PAGE elec in units of M' cm. trophoresis, or fluorescent assisted carbohydrate electro Polyethylene glycol (PEG) has been widely used in bio phoresis. materials, biotechnology and medicine primarily because As used herein substantially terminated Sialation refers to 55 PEG is a biocompatible, nontoxic, nonimmunogenic and N-linked oligosaccharides terminating with Sialic acid resi water-soluble polymer (Zhao and Harris, ACS Symposium due as a terminal Sugar. Terminal sialic acids can be identified Series 680: 458-72, 1997). In the area of drug delivery, PEG by FACE analysis of released carbohydrates following treat derivatives have been widely used in covalent attachment ment with neuraminidase. (i.e., “PEGylation') to proteins to reduce immunogenicity, The circulatory lifetime of glycoproteins in the blood is 60 proteolysis and kidney clearance and to enhance solubility highly dependent on the composition and structure of its (Zalipsky, Adv. Drug Del. Rev. 16:157-82, 1995). Similarly, N-linked carbohydrate groups. This fact is of direct relevance PEG has been attached to low molecular weight, relatively for therapeutic glycoproteins that are intended to be admin hydrophobic drugs to enhance solubility, reduce toxicity and istered parenterally. In general, maximal circulatory half-life alter biodistribution. Typically, PEGylated drugs are injected of a glycoprotein requires that its N-linked carbohydrate 65 as solutions. groups terminate in the sequence NeuAc-Gal-GlcNAc. With A closely related application is synthesis of crosslinked out the terminal sialic acid (NeuAc), the glycoprotein is rap degradable PEG networks or formulations for use in drug US 7,871,607 B2 41 42 delivery since much of the same chemistry used in design of formation. Another theory or at least a partial factor is that the degradable, soluble drug carriers can also be used in design of heavier the conjugate is, the more reduced immune response degradable gels (Sawhney et al., Macromolecules 26:581-87, is obtained. 1993). It is also known that intermacromolecular complexes In the case of human shASEGPs being used in the human can be formed by mixing solutions of two complementary 5 body, a preferred combination as described herein, the ten polymers. Such complexes are generally stabilized by elec dency for the sSHASEGP to elicitan immune response can be trostatic interactions (polyanion-polycation) and/or hydro significantly reduced as compared to, for example, non-hu gen bonds (polyacid-polybase) between the polymers man animal-derived enzymes Such as bovine or ovine hyalu involved, and/or by hydrophobic interactions between the ronidases (i.e. even if those animal-derived enzymes could be polymers in an aqueous Surrounding (Krupers et al., Eur. 10 completely purified away from other animal-derived pro Polym J. 32: 785-790, 1996). For example, mixing solutions teins, which is difficult to impossible to achieve in practice). of polyacrylic acid (PAAc) and polyethylene oxide (PEO) Although human shASEGPs thus represent a substantial and under the proper conditions results in the formation of com very important improvement over animal-derived products, plexes based mostly on hydrogen bonding. Dissociation of they can for many uses be improved still further by post 15 translational modifications (PTMs) such as PEGylation to these complexes at physiologic conditions has been used for enhance their resistance to degradation or elimination and/or delivery of free drugs (i.e., non-PEGylated). In addition, to improve other properties making them even more benefi complexes of complementary polymers have been formed cial for particular applications. from both homopolymers and copolymers. For example, although preferred compositions for humans In one aspect, the polyethylene glycol has a molecular (such as the human shASEGPs) exhibit reduced immunoge weight ranging from about 3 kD to about 50 kD, and prefer nicity compared to animal-derived hyaluronidases or non ably from about 5 kD to about 30 kD. Covalent attachment of human shASEGPs, PEGylation of such human shASEGPs the PEG to the drug (known as "PEGylation') may be accom can be used for example to extend their half life in the blood plished by known chemical synthesis techniques. For stream and/or in other tissues within the body. Without being example, in one aspect of the present invention, the PEGyla 25 restricted to a particular mechanism of action, it is generally tion of protein may be accomplished by reacting NHS-acti considered that the attachment of PEG moieties to shA vated PEG with the protein under suitable reaction condi SEGPs can be used to effectively shield the molecule, tions. decreasing its relative sensitivity to proteases and potentially also decreasing the extent and rate of its clearance and elimi While numerous reactions have been described for PEGy 30 nation from the body. lation, those that are most generally applicable confer direc The polymeric molecules coupled to the polypeptide may tionality, utilize mild reaction conditions, and do not neces be any suitable polymeric molecule with a molecular weight sitate extensive downstream processing to remove toxic as defined according to the invention, including natural and catalysts or bi-products. For instance, monomethoxy PEG synthetic homopolymers, such as polyols (i.e. poly-OH), (mPEG) has only one reactive terminal hydroxyl, and thus its 35 polyamines (i.e. poly-NH2) and polycarboxylacids (i.e. poly use limits some of the heterogeneity of the resulting PEG protein product mixture. Activation of the hydroxyl group at COOH), and further heteropolymers i.e. polymers compris the end of the polymer opposite to the terminal methoxy ing one or more different coupling groups e.g. a hydroxyl group is generally necessary to accomplish efficient protein group and amine groups. PEGylation, with the aim being to make the derivatised PEG 40 Examples of Suitable polymeric molecules include poly more Susceptible to nucleophilic attack. The attacking meric molecules selected from the group comprising poly alkylene oxides (PAO), such as polyalkylene glycols (PAG), nucleophile is usually the epsilon-amino group of a lysyl including polypropylene glycols (PEG), methoxypolyethyl residue, but other amines can also react (e.g. the N-terminal ene glycols (mPEG) and polypropylene glycols, PEG-gly alpha-amine or the ring amines of histidine) if local condi cidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI tions are favorable. A more directed attachment is possible in 45 PEG) branched polyethylene glycols (PEGs), polyvinyl proteins containing a single lysine or cysteine. The latter alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, residue can be targeted by PEG-maleimide for thiol-specific poly-D.L-amino acids, polyethylene-co-maleic acid anhy modification. Alternatively, PEG hydrazide can be reacted dride, polystyrene-co-maleic acid anhydride, dextrans with periodate oxidized shASEGP and reduced in the pres including carboxymethyl-dextrans, heparin, homologous ence of NaCNBH. More specifically, PEGylated CMP sug 50 ars can be reacted with shASEGP in the presence of appro albumin, celluloses, including methylcellulose, carboxym priate glycosyl-transferases. One technique is the ethylcellulose, ethylcellulose, hydroxyethylcellulose car "PEGylation” technique where a number of polymeric mol boxyethylcellulose and hydroxypropylcellulose, hydroly sates of chitosan, starches such as hydroxyethyl-Starches and ecules are coupled to the polypeptide in question. When using hydroxypropyl-Starches, glycogen, agaroses and derivatives this technique the immune system has difficulties in recog 55 nizing the epitopes on the polypeptide's Surface responsible thereof, guar gum, pullulan, inulin, Xanthan gum, carrag for the formation of antibodies, thereby reducing the immune eenan, pectin, alginic acid hydrolysates and bio-polymers. response. For polypeptides introduced directly into the circu Preferred polymeric molecules are non-toxic polymeric latory system of the human body to give a particular physi molecules such as (m)polyethylene glycol (mPEG) which ological effect (i.e. pharmaceuticals) the typical potential 60 further requires a relatively simple chemistry for its covalent immune response is an IgG and/or IgM response, while coupling to attachment groups on the enzyme’s Surface. polypeptides which are inhaled through the respiratory sys Generally seen polyalkylene oxides (PAO), such as poly tem (i.e. industrial polypeptide) potentially may cause an IgE ethylene oxides, such as PEG and especially MPEG, are the response (i.e. allergic response). One of the theories explain preferred polymeric molecules, as these polymeric mol ing the reduced immune response is that the polymeric mol 65 ecules, in comparison to polysaccharides such as dextran, ecule(s) shield(s) epitope(s) on the Surface of the polypeptide pullulan and the like, have few reactive groups capable of responsible for the immune response leading to antibody cross-linking, which is undesirable. US 7,871,607 B2 43 44 Improvements in PEGylation and other types of post-trans chloride in water to make 1000 mL. Enzyme Diluent Stock lational modifications (PTMs) have been extensive and there Solution 500 mL of Phosphate Buffer Solution with 500 are now a large number of PTM techniques and reagents mL of water. Enzyme Diluent Working Solution—33 mg of known in the art, and new ones are regularly being developed. hydrolyzed gelatin in 50 mL of Enzyme Diluent Stock Solu Techniques and reagents for PEGylation include, for 5 tion prepared within 2 hours of use. Sample Stabilization example: (i) specialized linkers and coupling chemistries Buffer Solution (“SSB' Soln.)—125 microliters (uL) of a (see, e.g., the reagents and technologies reviewed by Harris, J. 20% Human Serum Albumin Solution and 50 uL of a 1 M M., Adv. Drug Deliv. Rev. 54: 459-476 (2002) and the pri Calcium Chloride solution in 50 mL of Enzyme Diluent mary references reviewed therein); (ii) branched PEGs which Working Solution, and mix thoroughly. Serum Stock Solu effectively allow additional PEG groups to be attached to a 10 single conjugation site (see, e.g., Veronese, F. M. et al., tion—Dilute 1 volume of Horse Serum with 9 volumes of Bioorg. Med. Chem. Lett. 12: 177-180 (2002)); (iii) site Acetate Buffer Solution. Adjust with 4N hydrochloric acid to specific PEGylation, including site-specific monoPEGyla a pH of 3.1 and allow the solution to stand at room tempera tion (see, e.g., Chapman, A. P. et al., Nature Biotech. 17: ture for 18 to 24 hrs. Store the solution at 4°C., and use within 780-783 (1999); and (iv) site-directed enzymatic PEGylation 15 30 days. Serum Working Solution—10 mL of the Serum (e.g. using a transglutaminase reaction as described by Sato, Stock Solution in 30 mL of the Acetate Buffer Solution, H., Adv. Drug Deliv. Rev. 54: 487-504 (2002)). There are also adjusted to room temperature. Hyaluronic Acid Stock Solu additional technologies and reagents available from an tion—Sodium Hyaluronic Acid to a concentration 5.0 mg/mL increasing numbr of commercial Suppliers (see, e.g., Nektar/ in water. Hyaluronic Acid Working Solution 0.75 mL of the Shearwater (www.nektar.com), Sunbio (www.sunbio.com Hyaluronic Acid Stock Solution in 4.25 mL of the Phosphate and www.sunbio.com/peg-shop), Celares GmbH (ww Buffer Solution. Standard Stock Solution—One container of w.celares.com), and others). USP Reference Standard Hyaluronidase to a concentration B. Tissue Expression Profiles shASEGP 1000 Units/mL in water, aliquoted into 50 uL portions, and While previously thought to be testis specific, human shA stored at -20°C. Standard Working Solution 40 uL of Stan SEGP is expressed in multiple tissues in humans when using 25 dard Stock Solution in 960 uL of cold Enzyme Diluent Work more sensitive techniques such as RT-PCR. The shASEGP ing Solution to obtain a solution having a known concentra transcript is found in medulla (brain), microvascular endot tion of 40 Units/mL, prepared immediately before use in the helium, prostate, breast, retina, pooled human melanocyte, assay. fetal heart, and pregnant uterus. shASEGP is also expressed All enzyme samples are diluted in a “Low Protein Binding in germ cell tumors. RT-PCR based detection of shASEGP 30 96-well plate according to the following guidelines: transcripts is generally required to detect levels in tissues a) The range of maximum sensitivity of this assay is other than testis. between 10-30 Units/mL. To minimize the number of times C. Assays for shASEGP Enzyme Activity anassay must be repeated in order to get results that are within Turbidometric Microtiter Assay for Hyaluronidase Activ 35 range, first determine the approximate number of total units/ ity mL for the sample, and then choose a (whole number) dilu Hyaluronidase activity can be detected by way of a modi tion Such that the final concentration is approximately 20 fied turbidometric assay in acidified serum solution. The Units/ml. reagents required are as follows: b) Minimum Sample volumes needed to perform assay are 40 as follows: FPLC Fractions=50 uL., Tissue Culture Superna tants=1 mL, Purified/Concentrated/Final Step Material=10 UV sterilized 2X-deionized water or Braun RSOOO-O1 LL. sterile water for irrigation c) For samples with serial dilutions, 1: 10 dilutions in the Hylumed Medical - Sodium Genzyme Advanced 4876 Hyaluronate, High Molecular Weight Biomaterials 45 “Low Protein Binding 96-well plate are made in triplicate by HA pipetting 360 uL of the “SSB' Solution and 40 uL of Sample Hyaluronidase Reference Standard USP 312OO into each well. Potassium Acetate, Granular, USP, JTBaker 2914-O1 ACS For preparation of USPStandard prepare the USPStandard Acetic Acid, Glacial, 99-96 Sigma A-6283 Curve in the “Low Protein Binding 96-well plate as follows: Sodium Phosphate Monobasic Mallinkrodt 7774 50 Monohydrate, USP Granular Sodium Phosphate Dibasic Mallinkrodt 7771 Anhydrous, USP Sodium Chloride, Crystals, GR, EMScience SXO420-5 USP Standard Curve: ACS Enzyme Standard Final Gelatin Hydrolysate Enzymatic Sigma G-O262 55 Horse Serum, Donor Herd, cell Sigma H-1270 Diluent Working Conc. (in culture tested, Hybridoma culture Wells: Standard: Soln. (in IL): Soln. (in IL): Units/mL): tested, USA Origin A1-A3 St01 O 1OO 40 Human Serum Albumin 20% Griffols B1-B3 St02 2O 8O 32 Hydrochloric Acid, ACS Reagent Sigma H-702O C1 C3 St03 40 60 24 Calcium Chloride, Dihydrate, JTBaker 1336-O1 60 D1 D3 St04 60 40 16 Granular, USP, -FCC E1 E3 St05 8O 2O 8 F1 F3 St06 90 10 4 The following reagents are prepared: Acetate Buffer Solu G1 G3 St07 1OO O O tion—14.0 g of potassium acetate and 25.0 mL of glacial acetic acid in water to make 1000 mL. Phosphate Buffer 65 For preparation of the Hyaluronic Acid Control in columns Solution 2.5 g of sodium phosphate monobasic, 1.0 g of 1-3, prepare the H.A. Control in the “Flat Bottom' 96-well anhydrous Sodium phosphate dibasic, and 8.2 g of sodium plate is prepared as follows: US 7,871,607 B2 45 46 rapid measurement of Hyaluronidase activity from cultured cells and biological samples with an inter-assay variation of less than 10%. H.A. Controls: The specific activity of hyaluronidase is expressed in tur Hyaluronic Acid Enzyme Diluent bidity reducing units (TRU). One TRU is defined as the Working Soln. (in Working amount of hyaluronidase activity required to reduce the tur Wells: Control: |L): Soln. (in IL): bidity of an acidified solution of hyaluronic acid and is equivalent to the U.S.P./National Formulary (NF XIII) units H1 H3 CoO1 O 60 (NFU). The ELISA-like enzyme assay used for purification is 10 related to the TRU, the NFU, and U.S.P. unit through a stan The Reaction Plate: 30 uI per well of Hyaluronic Acid dard curve of a sample of hyaluronidase (e.g., USP) standard Working Solution is pipetted using a 50 uL 8-channel transfer ized through the U.S.P. Therefore, the enzyme activities pipette into a “Flat Bottom' 96-well microtiter plate leaving determined by the ELISA-like enzyme assay are actually wells H1-H3 empty. 60 uL/well of Enzyme Diluent Working relative TRU, since enzyme activity is not actually measured Solution is pipetted into wells H1-H3 of the same plate as the 15 using the turbidometric assay (Dorfman et al., 1948, J. Biol. HA control. Chem. 172: 367). Many Hyaluronidase assays have been based upon the Serum Working Solution: 40 mL of Serum Working Solu measurement of the generation of new reducing N-acety tion is dispensed into a transfer basin and next to the Heat lamino groups (Bonner and Cantey, Clin. Chim. Acta 13: Block. 746-752, 1966), or loss of viscosity (De Salegui et al., Arch. Pre-warming stage: Once both plates have been prepared, Biochem. Biophys. 121 : 548-554, 1967) or turbidity (Dorf the Low Protein Binding 96-Well plate containing the diluted man and Ott, J. Biol. Chem. 172: 367, 1948). With purified samples, standards, controls and the Flat Bottom 96-well substrates all of these methods suffice for determination of the plate containing the Hyaluronic Acid Working Solution are presence or absence of endoglucosamidic activity. placed onto a heat block and allow them to warm for 5 min. at 25 Substantially purified glycosaminoglycan Substrates can 370 C. also be used for in a Gel Shift Assay. Glycosaminoglycans are The Reaction is initiated by the addition of Enzyme to mixed with recombinant shASEGP to test for endoglucosi Substrate: 30 ul, from the enzyme plate into all of the wells in dase activity that results in a shift in substrate mobility within column #1 of the 96-Well flat bottom plate (containing the the gel. Chondroitin-4 and 6 sulfate, dermatan Sulfate, hepa substrate) using a 5-50 uL 8-channel pipette. The Enzyme? 30 ran-sulfate can be obtained from Sigma Chemical. Human Substrate reaction mixture is aspirated 5 times (drawing the umbilical cord Hyaluronan can be obtained from ICN. Each solution up and down with the transfer during the first 15 test substrate is diluted to 0.1 mg/ml in a buffer range from pH seconds to ensure complete sample mixing. After mixing the 3.5-7.5. 10 ul samples of purified shASEGP or conditioned enzyme and Substrate, the tips are ejected and a new set of tips media from sFHASEGP expressing cells as well as are mixed loaded on the transfer pipettor for the next column. A timer is 35 with 90 ul of test substrate in desired buffer and incubated for restarted, and at time (t)=0: 30, this process is repeated for 3 hours at 37 C. Following incubation samples are neutralized column 2. At the next 30 second interval (t)=1: 00, this is with sample buffer (Tris EDTA PH 8.0, Bromophenol Blue repeated process for column 3. This process is repeated mov and glycerol) followed by electrophoresis. Glycosaminogly ing from left to right across the plate, every 30 seconds until cans are detected by staining the gels in 0.5% Alcian Blue in all of the wells contain both enzyme and substrate. 40 3% Glacial Acetic Acid overnight followed by destaining in Stopping the reaction: When timer reaches 6 minutes (t)=6: 7% Glacial Acetic Acid. Degradation is determined by com 00, 240 uL of the Serum Working Solution is pippetted into parison Substrate mobility in the presence and absence of each well, using a 50-300 uL 8-channel transfer pipette, into enzyme. column 1 of the 96-well flat bottom plate from the adjacent 50 Hyaluronidase activity can also be detected by substrate mL Reagent Reservoir. The mixture is aspirated 3 times 45 gel Zymography (Guentenhoner et al., 1992, Matrix 388 (drawing the solution up and down with the transfer Pipettor) 396). In this assay a sample is applied to a SDS-PAGE gel during the first 10 seconds to ensure complete mixing. The containing hyaluronic acid and the proteins in the sample process is repeated every 30 seconds, proceeding from col separated by electrophoresis. The gel is then incubated in an umns 1 to 12. Upon completion of the last column (column enzyme assay buffer and Subsequently stained to detect the 12), the reaction plate is removed from the heat block and 50 hyaluronic acid in the gel. Hyaluronidase activity is visual place the plate onto the read tray of the plate reader at 640 nM. ized as a cleared Zone in the Substrate gel. A linear curve fit is generated from the standard curve that D. Identification and Isolation of shASEGP Polypeptide permits extrapolation of test samples. Genes Alternative Assays for Hyaluronidase The shASEGP polypeptide gene and/or domains thereof, 55 can be obtained by methods well known in the art for DNA Biotinylated Hyaluronan Microtiter Assay isolation. Any method known to those of skill in the art for The free carboxyl groups on glucuronic acid residues of identification of nucleic acids that encode desired genes can Hyaluronan are biotinylated in a one step reaction using be used. Any method available in the art can be used to obtain biotin-hydrazide (Pierce), Sulfo NHS (Pierce) and 1-Ethyl a full-length (i.e., encompassing the entire coding region) dimethylaminopropyl-carbodiimide (Sigma). This biotiny 60 cDNA or genomic DNA clone encoding a shASEGP lated HA substrate is covalently coupled to a 96 well micro polypeptide. For example, the polymerase chain reaction titer plate in a second reaction. At the completion of the (PCR) can be used to amplify a sequence that is expressed in enzyme reaction, residual Substrate is detected with an avi normal tissues, e.g., nucleic acids encoding a SEHASEGP din-peroxidase reaction that can be read in a standard ELISA polypeptide (SEQ. Nos: 1 and 2), in a genomic or cDNA plate reader. As the substrate is covalently bound to the micro 65 library. Oligonucleotide primers that hybridize to sequences titer plate, artifacts such as pH-dependent displacement of the at the 3' and 5' termini of the identified sequences can be used biotinylated substrate does not occur. The sensitivity permits as primers to amplify by PCR sequences from a nucleic acid US 7,871,607 B2 47 48 sample (RNA or DNA generally a cDNA library, from an ments then can be separated according to size by standard appropriate Source (e. testis, prostate, breast). techniques, including but not limited to, agarose and poly PCR can be carried out, e.g., by use of a Perkin-Elmer acrylamide gel electrophoresis and column chromatography. Cetus thermal cycler and Taq polymerase (Gene Amp). The Once the DNA fragments are generated, identification of DNA being amplified can include mRNA or cDNA or the specific DNA fragment containing the desired gene can be genomic DNA from any eukaryotic species. One can choose accomplished in a number of ways. to synthesize several different degenerate primers, for use in For example, a portion of the shASEGP polypeptide (of the PCR reactions. any species) gene (e.g., a PCR amplification product obtained It is also possible to vary the stringency of hybridization as described above or an oligonucleotide having a sequence conditions used in priming the PCR reactions, to amplify 10 of a portion of the known nucleotide sequence) or its specific nucleic acid homologs (e.g., to obtainsHASEGP polypeptide RNA, or a fragment thereof be purified and labeled, and the sequences from species other than humans or to obtain human generated DNA fragments can be screened by nucleic acid sequences with homology to shASEGP polypeptide) by hybridization to the labeled probe (Benton and Davis, Sci allowing for greater or lesser degrees of nucleotide sequence ence 196: 180 (1977); Grunstein and Hogness, Proc. Natl. similarity between the known nucleotide sequence and the 15 Acad. Sci. U.S.A. 72: 3961 (1975)). Those DNA fragments nucleic acid homolog being isolated. For cross-species with substantial homology to the probe will hybridize. It is hybridization, low stringency to moderate stringency condi also possible to identify the appropriate fragment by restric tions are used. For same species hybridization, moderately tion enzyme digestion (s) and comparison of fragment sizes stringent to highly stringent conditions are used. The condi with those expected according to a known restriction map if tions can be empirically determined. Such is available or by DNA sequence analysis and compari After Successful amplification of the nucleic acid contain son to the known nucleotide sequence of shASEGP polypep ing all or a portion of the identified shASEGP polypeptide tide. Further selection can be carried out on the basis of the sequence or of a nucleic acid encoding all or a portion of a properties of the gene. Alternatively, the presence of the gene sHASEGP polypeptide homolog, that segment can be can be detected by assays based on the physical, chemical, or molecularly cloned and sequenced, and used as a probe to 25 immunological properties of its expressed product. For isolate a complete cDNA or genomic clone. This, in turn, example, cDNA clones, or DNA clones which hybrid-select permits the determination of the gene’s complete nucleotide the proper mRNA can be selected which produce a protein sequence, the analysis of its expression, and the production of that, e.g., has similar or identical electrophoretic migration, its protein product for functional analysis. Once the nucle isoelectric focusing behavior, proteolytic digestion maps, otide sequence is determined, an open reading frame encod 30 antigenic properties, Hyaluronidase activity. If an anti-SHA ing the shASEGP polypeptide gene protein product can be SEGP polypeptide antibody is available, the protein can be determined by any method well known in the art for deter identified by binding of labeled antibody to the putatively mining open reading frames, for example, using publicly sHASEGP polypeptide synthesizing clones, in an ELISA available computer programs for nucleotide sequence analy (enzyme-linked immunosorbent assay)-type procedure. sis. Once an open reading frame is defined, it is routine to 35 Alternatives to isolating the shASEGP polypeptide determine the amino acid sequence of the protein encoded by genomic DNA include, but are not limited to, chemically the open reading frame. In this way, the nucleotide sequences synthesizing the gene sequence from a known sequence or of the entire shASEGP polypeptide genes as well as the making cDNA to the mRNA that encodes the shASEGP amino acid sequences of shASEGP polypeptide proteins and polypeptide. analogs can be identified. 40 For example, RNA for cDNA cloning of the shASEGP Any eukaryotic cell potentially can serve as the nucleic polypeptide gene can be isolated from cells expressing the acid source for the molecular cloning of the shASEGP protein. The identified and isolated nucleic acids then can be polypeptide gene. The nucleic acids can be isolated from inserted into an appropriate cloning vector. A large number of Vertebrate, mammalian, human, porcine, bovine, feline, vector-host systems known in the art can be used. Possible avian, equine, canine, as well as additional primate sources, 45 vectors include, but are not limited to, plasmids or modified insects, plants and other organisms. The DNA can be obtained viruses, but the vector system must be compatible with the by standard procedures known in the art from cloned DNA host cell used. Such vectors include, but are not limited to, (e.g., a DNA “library”), by chemical synthesis, by cDNA bacteriophages such as lambda derivatives, or plasmids Such cloning, or by the cloning of genomic DNA, or fragments as pBR322 or pUC plasmid derivatives or the Bluescript thereof, purified from the desired cell (see, e.g., Sambrook et 50 vector (Stratagene, LaJolla, Calif.). The insertion into a clon al. 1989, Molecular Cloning, A Laboratory Manual. 2d Ed., ing vector can, for example, be accomplished by ligating the Cold Spring Harbor Laboratory Press, Cold Spring Harbor, DNA fragment into a cloning vector that has complementary N.Y.; Glover, D. M. Ed., 1985, DNA Cloning: A Practical cohesive termini. Approach, MRL Press, Ltd., Oxford, U. K. Vol. 1, 11. Clones If the complementary restriction sites used to fragment the derived from genomic DNA can contain regulatory and intron 55 DNA are not present in the cloning vector, the ends of the DNA regions in addition to coding regions; clones derived DNA molecules can be enzymatically modified. Alterna from cDNA will contain only exon sequences. For any tively, any site desired can be produced by ligating nucleotide Source, the gene is cloned into a suitable vector for propaga sequences (linkers) onto the DNA termini; these ligated link tion thereof. ers can include specific chemically synthesized oligonucle In the molecular cloning of the gene from genomic DNA, 60 otides encoding restriction endonuclease recognition DNA fragments are generated, some of which will encode the sequences. In an alternative method, the cleaved vector and desired gene. sHASEGP polypeptide gene can be modified by homopoly The DNA can be cleaved at specific sites using various meric tailing. restriction enzymes. Recombinant molecules can be introduced into host cells Alternatively, one can use DNAse in the presence of man 65 via transformation, transfection, infection, electroporation, ganese to fragment the DNA, or the DNA can be physically calcium precipitation and other methods, so that many copies sheared, for example, by sonication. The linear DNA frag of the gene sequence are generated. US 7,871,607 B2 49 50 In specific embodiments, transformation of host cells with then cultured in chemically defined medium in the absence of recombinant DNA molecules that incorporate the isolated hypoxanthine and thymidine, followed by further gene ampli sHASEGP polypeptide gene, cDNA, or synthesized DNA fication with increasing concentrations of methotrexate. sequence enables generation of multiple copies of the gene. A variety of host-vector Systems can be used to express the Thus, the gene can be obtained in large quantities by grow- 5 protein coding sequence. These include but are not limited to ing transformants, isolating the recombinant DNA molecules mammalian cell systems infected with virus e.g. Vaccinia from the transformants and, when necessary, retrieving the virus, adenovirus, etc.); insect cell systems infected with inserted gene from the isolated recombinant DNA. virus (e.g. baculovirus); microorganisms such as yeast con E. Vectors, Plasmids and Cells that Contain Nucleic Acids taining yeast vectors; or bacteria transformed with bacte Encoding a shASEGP Polypeptide or Hyaluronidase 10 riophage, DNA, plasmid DNA, or cosmid DNA. The expres Domain Thereof and Expression of shASEGP Polypeptides sion elements of vectors vary in their strengths and Vectors and Cells. specificities. Depending on the host-vector System used, any For recombinant expression of one or more of the shA one of a number of Suitable transcription and translation SEGP polypeptides, the nucleic acid containing all or a por elements can be used. Note that bacterial expression of shA tion of the nucleotide sequence encoding the shASEGP 15 SEGPDNA will not resultina catalytically active shASEGP polypeptide can be inserted into an appropriate expression perse, but when combined with proper glycosylation machin vector i.e., a vector that contains the necessary elements for ery can be artificially glycosylated as such. the transcription and translation of the inserted protein coding Any methods known to those of skill in the art for the sequence. The necessary transcriptional and translational sig insertion of nucleic acid fragments into a vector can be used nals can also be supplied by the native promoter for shA- 20 to construct expression vectors containing a chimeric gene SEGP genes, and/or their flanking regions. containing appropriate transcriptional/translational control Also provided are vectors that contain nucleic acid encod signals and protein coding sequences. These methods can ing the shASEGPs that can be introduced into an expression include in vitro recombinant DNA and synthetic techniques system capable of producing a soluble neutral active SHA and in vivo recombinants (genetic recombination). Expres SEGP 25 sion of nucleic acid sequences encoding shASEGP polypep Cells containing the vectors are also provided. The cells tide, or domains, derivatives, fragments or homologs thereof, include eukaryotic and prokaryotic cells, and the vectors Suit can be regulated by a second nucleic acid sequence so that the able for use therein. genes or fragments thereof are expressed in a host trans Eukaryotic cells, including dihydrofolate reductase defi formed with the recombinant DNA molecule(s). For cient Chinese Hamster Ovary Cells (DG44), containing the 30 example, expression of the proteins can be controlled by any vectors are provided. Suitable cells include yeast cells, fungal promoter/enhancer known in the art. In a specific embodi cells, plant cells, insect cells and animal cells. The cells are ment, the promoter is not native to the genes for shASEGP used to produce a shASEGP polypeptide or Hyaluronidase polypeptide. Promoters which can be used include but are not domain thereof by (a) growing the above-described cells limited to the SV40 early promoter (Bernoist and Chambon, under conditions whereby the encoded shASEGP polypep- 35 Nature 290: 304-310 (1981) the promoter contained in the 3' tide or Hyaluronidase domain of the shASEGP polypeptide long terminal repeat of Rous sarcoma virus (Yamamoto et al., is expressed by the cell, and then (b) recovering the expressed Ce/22: 787-797 (1980) the herpes thymidine kinase pro Hyaluronidase domain protein. In the exemplified embodi moter (Wagner et al. Proc. Natl. Acad. Sci. USA 78: 1441 ments, the Hyaluronidase domain is secreted into the 1445 (1981) the regulatory sequences of the metallothionein medium. 40 gene (Brinster et al., Nature 296: 39-42 (1982)); prokaryotic In one embodiment, the vectors include a sequence of expression vectors such as the 13-Lactamase promoter (Villa nucleotides that encodes a polypeptide that has Hyalu Kamaroffet al., Proc. Natl. Acad. Sci. USA 75: 3727-3731 ronidase activity and contains all or a portion of only the 1978)) Or The TAC Promoter Deboer et al., Proc. Natl. Acad. Hyaluronidase domain, or multiple copies thereof, of a shA Sci. USA 80: 21-25 (1983)); see also “Useful Proteins from SEGP protein are provided. Also provided are vectors that 45 Recombinant Bacteria': in Scientific American 242: 79-94 comprise a sequence of nucleotides that encodes the Hyalu (1980)); plant expression vectors containing the opaline Syn ronidase domain and additional portions of a shASEGP pro thetase promoter (Herrar-Estrella et al., Nature 303: 209-213 tein up to and including a full-length shASEGP protein, as (1984)) or the cauliflower mosaic virus 35S RNA promoter well as multiple copies thereof, are also provided. The vectors (Garder et al., Nucleic Acids RES. 9: 2871 (1981)), and the can be selected for expression of the shASEGP protein or 50 promoter of the photosynthetic enzyme ribulose bisphos Hyaluronidase domain thereof in the cell or such that the phate carboxylase (Herrera-Estrella et AL., Nature 310: 115 sHASEGP protein is expressed as a secreted protein. Alter 120 (1984)); promoter elements from yeast and other fungi natively, the vectors can include signals necessary for secre Such as the Gala promoter, the alcohol dehydrogenase pro tion of encoded proteins. When the Hyaluronidase domain is moter, the phosphoglycerol kinase promoter, the alkaline expressed the nucleic acid is linked to nucleic acid encoding 55 phosphatase promoter, and the following animal transcrip a secretion signal. Such as the Saccharomyces cerevisiae a tional control regions that exhibit tissue specificity and have mating factor signal sequence or a portion thereof, or the been used in transgenic animals: elastase I gene control native signal sequence. region which is active in pancreatic acinar cells (Swift Et Al. In order to generate a soluble, neutral active shASEGP, Cell 38: 639-646 (1984); Ornitz Et Al., Cold Spring Harbor cells capable of introducing N-linked glycosylation are 60 Symp. Quant. Biol. 50: 399-409 (1986); Macdonald, Hepa required. In the preferred embodiment, mammalian Chinese tology 7: 425-515 (1987)); insulin gene control region which Hamster Ovary cells deficient in dihydrofolate reductase such is active in pancreatic beta cells (Hanahanet AL., Nature 315: as DG44, are electroporated with a plasmid encoding a strong 115-122 (1985)), immunoglobulin gene control region which mammalian promoter, Such as CMV, nucleic acid encoding a is active in lymphoid cells (Grosschedl et AL., Cell 38: 647 sHASEGP followed by an internal ribosomal entry site, the 65 658 (1984); Adams et al., Nature 318: 533-538 (1985); Alex mouse dihydrofolate reductase gene and the SV40 polyade ander et AL., Mol. Cell Biol. 7: 1436-1444 (1987)), mouse nylation sequence as shown in SEQID NO 51. Such cells are mammary tumor virus control region which is active in tes US 7,871,607 B2 51 52 ticular, breast, lymphoid and mast cells (Leder et AL., CELL Any number of selection systems may be used to recover 45: 485-495 (1986)), albumin gene control region which is transformed cell lines. These include, but are not limited to, active in liver (PINCKERT et AL., Genes and Devel. 1: 268 the herpes simplex virus thymidine kinase (Wigler Metal 276 (1987)), alpha-fetoprotein gene control region which is (1977) Cell 11: 223-32) and adenine phosphoribosyltrans active in liver (Krumlauf et AL., Mol. Cell. Biol. 5: 1639 ferase (Lowy I et al (1980) Cell 22: 817-23) genes which can 1648 (1985); Hammer et AL., Science 235: 53-58 1987)), be employed in TK- or APRT-cells, respectively. Also, alpha-1 antitrypsingene control region which is active in liver antimetabolite, antibiotic or herbicide resistance can be used (Kelsey et al., Genes And Devel. 1: 161-171 (1987)), beta as the basis for selection; for example, DHFR which confers globin gene control region which is active in myeloid cells resistance to methotrexate (Wigler Metal (1980) Proc Natl (Mogram et al., Nature 315: 338-340 (1985): Kollias et AL., 10 Acad Sci 77: 3567-70); mpt, which confers resistance to the CE/46: 89-94 (1986)), myelin basic protein gene control aminoglycosides neomycin and G-418 (Colbere-Garapin Fet region which is active in oligodendrocyte cells of the brain all (1981) J Mol Biol 150: 1-14) and als or pat, which confer (Readhead et al., Cell 48: 703-712 (1987)), myosin light resistance to chlorSulfuron and phosphinotricin acetyltrans chain-2 gene control region which is active in skeletal muscle ferase, respectively (Murry, supra). Additional selectable (Sani, Nature 314: 283-286 (1985)), and gonadotrophic 15 genes have been described, for example, trpE, which allows releasing hormone gene control region which is active in cells to utilize indole in place of tryptophan, or hisD, which gonadotrophs of the hypothalamus (Mason et al., Science allows cells to utilize histinol in place of histidine (Hartman S 234: 1372-1378 (1986)). Cand RC Mulligan (1988) Proc Natl AcadSci 85: 8047-51). In a specific embodiment, a vector is used that contains a Recently, the use of visible markers has gained popularity promoter operably linked to nucleic acids encoding a SHA with Such markers as anthocyanins, beta glucuronidase and SEGP polypeptide, or a domain, fragment, derivative or its substrate, GUS, and luciferase and its substrate, luciferin, homolog, thereof, one or more origins of replication, and being widely used not only to identify transformants, but also optionally, one or more selectable markers (e. G., an antibi to quantify the amount of transient or stable protein expres otic resistance gene). sion attributable to a specific vector system (Rhodes CA etal Specific initiation signals may also be required for efficient 25 (1995) Methods Mol Biol 55: 121-131). translation of a shASEGP sequence. These signals include Identification of Transformants Containing the Polynucle the ATG initiation codon and adjacent sequences. In cases otide Sequence where shASEGP, its initiation codon and upstream Although the presence/absence of marker gene expression sequences are inserted into the appropriate expression vector, Suggests that the gene of interest is also present, the presence no additional translational control signals may be needed. 30 and expression of an active shASEGP should be confirmed. However, in cases where only coding sequence, or a portion For example, if the shASEGP is inserted within a marker thereof, is inserted, exogenous transcriptional control signals gene sequence, recombinant cells containing shASEGP can including the ATG initiation codon must be provided. Fur be identified by the absence of marker gene function. Alter thermore, the initiation codon must be in the correct reading natively, a marker gene can be placed in tandem with a SHA frame to ensure transcription of the entire insert. Exogenous 35 SEGP sequence under the control of a single promoter. transcriptional elements and initiation codons can be of vari Expression of the marker gene in response to induction or ous origins, both natural and synthetic. The efficiency of selection usually indicates expression of the tandem SHA expression may be enhanced by the inclusion of enhancers SEGP as well. Detection of a properly glycosylated neutral appropriate to the cell system in use (Scharf D et al (1994) active shASEGP can be determined by way of testing the Results Probl Cell Differ 20: 125-62: Bittner et al (1987) 40 conditioned media for shASEGP enzyme activity under Methods in Enzymol 153: 516-544). appropriate conditions. In addition, a host cell strain may be chosen for its ability to Purification of SHASEGP modulate the expression of the inserted sequences or to pro Host cells transformed with a shASEGP nucleotide cess the expressed protein in the desired fashion. Such modi sequence may be cultured under conditions suitable for the fications of the polypeptide include, but are not limited to, 45 expression and recovery of the encoded protein from cell acetylation, carboxylation, glycosylation, phosphorylation, culture. The protein produced by a recombinant cell is pref lipidation and acylation. Post-translational processing which erably secreted but may be contained intracellularly depend cleaves a “prepro form of the protein may also be important ing on the sequence and/or the vector used. As will be under for correct insertion, folding and/or function. Different host stood by those of skill in the art, expression vectors containing cells such as CHO (DG44, DXB11 CHO-K1), HeLa, MDCK, 50 sHASEGP can be designed with signal sequences that facili 293, WI38, etc have specific cellular machinery and charac tate direct secretion of shASEGP through a prokaryotic or teristic mechanisms for Such post-translational activities and eukaryotic cell membrane. Other recombinant constructions may be chosen to ensure the correct modification and pro may join SHASEGP to nucleotide sequence encoding a cessing of the introduced, foreign protein. polypeptide domain which will facilitate purification of For long-term, high-yield production of recombinant pro 55 soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12: teins, stable expression is preferred. For example, cell lines 441-53; cf discussion of vectors infra containing fusion pro that stably express shASEGP may be transformed using teins). expression vectors which contain viral origins of replication sHASEGP may also be expressed as a recombinant protein or endogenous expression elements and a selectable marker with one or more additional polypeptide domains added to gene. Following the introduction of the vector, cells may be 60 facilitate protein purification. Such purification facilitating allowed to grow for 1-2 days in an enriched media before they domains include, but are not limited to, metal chelating pep are switched to selective media. The purpose of the selectable tides such as histidine-tryptophan modules that allow purifi marker is to confer resistance to selection, and its presence cation on immobilized metals, protein A domains that allow allows growth and recovery of cells that successfully express purification on immobilized immunoglobulin, and the the introduced sequences. Resistant clumps of stably trans 65 domain utilized in the FLAGS extension/affinity purification formed cells can be proliferated using tissue culture tech system (Immunex Corp, Seattle Wash.). The inclusion of a niques appropriate to the cell type. cleavable linker sequences such as Factor XA or enterokinase US 7,871,607 B2 53 54 (Invitrogen, San Diego Calif.) between the purification electrophoresis, immunoassay, cross-linking to marker-la domain and shASEGP is useful to facilitate purification. One beled product, and assays of proteolytic activity. Such expression vector provides for expression of a fusion The shASEGP polypeptides can be isolated and purified protein compromising a SEHASEGP and contains nucleic acid by standard methods known in the art (either from natural encoding 6 histidine residues followed by thioredoxin and an 5 Sources or recombinant host cells expressing the complexes enterokinase cleavage site. The histidine residues facilitate or proteins), including but not restricted to column chroma purification on IMIAC (immobilized metalion affinity chro tography (E.g., ion exchange, affinity, gel exclusion, matography as described in Porath et al (1992) Protein reversed-phase high pressure and fast protein liquid), differ Expression and Purification 3: 263-281) while the enteroki ential centrifugation, differential solubility, or by any other nase cleavage site provides a means for purifying the chemok 10 standard technique used for the purification of proteins. ine from the fusion protein. In one embodiment, a shASEGP can be purified to homo In addition to recombinant production, fragments of SHA geneity from the chemically defined conditioned media of SEGP may be produced by direct peptide synthesis using HZ24 transfected and methotrexate amplified DG44 cells by solid-phase techniques (see, e.g., Stewart et al (1969) Solid 1) tangential flow diafiltration, 2) binding and elution from Phase Peptide Synthesis, W H Freeman Co., San Francisco; 15 anion exchange chromatography, 3) flow through phenyl Merrifield.J. (1963) J Am Chem Soc 85: 2149-2154). In vitro Sepharose chromatography, 4) binding and elution from phe protein synthesis may be performed using manual techniques nylboronate chromatography and 4) binding and elution with or by automation. Automated synthesis may be achieved, for hydroxyapatite chromatography. example, using Applied Biosystems 431A Peptide Synthe Functional properties can be evaluated using any Suitable sizer (PerkinElmer, Foster City Calif.) in accordance with the assay known in the art. instructions provided by the manufacturer. Various fragments Alternatively, once a shASEGP polypeptide or its domain of shASEGP may be chemically synthesized separately and or derivative is identified, the amino acid sequence of the combined using chemical methods to produce the full-length protein can be deduced from the nucleotide sequence of the molecule. gene that encodes it. As a result, the protein or its domain or Expression vectors containing the coding sequences, or 25 derivative can be synthesized by standard chemical methods portions thereof, of a shASEGP polypeptide, is made, for known in the art (e.G. see Hunkapiller et al. Nature 310: example, by Subcloning the coding portions into the EcoR1 105-111 (1984)) followed by glycosylation in vitro. restriction site of each of the three PGEX vectors (glutathione Manipulations of shASEGP polypeptide sequences can be S—transferase expression vectors (Smith and Johnson, Gene made at the protein level. Also contemplated herein are shA 7:31-40 (1988)). This allows for the expression of products in 30 SEGP polypeptide proteins, domains thereof, derivatives or the correct reading frame. Exemplary vectors and systems for analogs or fragments thereof, which are differentially modi expression of the Hyaluronidase domains of the shASEGP fied during or after translation, e.g., by glycosylation, acety polypeptides include the well-known Pichia vectors (avail lation, phosphorylation, amidation, pegylation, derivatiza able, for example, from Invitrogen, San Diego, Calif.), par tion by known protecting/blocking groups, proteolytic ticularly those designed for secretion of the encoded proteins. 35 cleavage, linkage to an antibody molecule or other cellular The protein can also be expressed cytoplasmically, such as in ligand. the inclusion bodies. One exemplary vector is described in the Any of numerous chemical modifications can be carried examples. out by known techniques, including but not limited to specific Plasmids for transformation of E. coli cells, include, for chemical cleavage by cyanogen bromide, trypsin, chymot example, the pET expression vectors (see, U.S. Pat. No. 40 rypsin, papain, V8, NABH4, acetylation, formylation, oxida 4.952,496; available from Novagen, Madison, Wis.; see, also tion, reduction, metabolic synthesis in the presence of tuni literature published by Novagen describing the system). camycin and other Such agents. Such plasmids include pET 11a, which contains the T71ac In addition, domains, analogs and derivatives of a SEHA promoter. T7 terminator, the inducible E. coli lac operator, SEGP polypeptide can be chemically synthesized. For and the lac repressor gene; pET 12A-C, which contains the T7 45 example, a peptide corresponding to a portion of a shASEGP promoter, T7 terminator, and the E. COLIOMPT secretion polypeptide, which includes the desired domain or which signal; and pPT 15B and PET19B (Novagen, Madison, Wis.), mediates the desired activity in vitro can be synthesized by which contain a His-Tag leader sequence for use in purifica use of a peptide synthesizer. tion with a His column and a thrombin cleavage site that Furthermore, if desired, nonclassical amino acids or permits cleavage following purification over the column; the 50 chemical amino acid analogs can be introduced as a Substi T7-lac promoter region and the T7 terminator. tution or addition into the shASEGP polypeptide sequence. The vectors are introduced into host cells, such as Pichia Non-classical amino acids include but are not limited to the cells and bacterial cells, such as E. coli, and the proteins D-isomers of the common amino acids, a-amino isobutyric expressed therein. Exemplary Pichia strains include, for acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, example, GS115. Exemplary bacterial hosts contain chromo 55 E-ABU. e-AhX, 6-amino hexanoic acid, Aib, 2-amino isobu somal copies of DNA encoding T7 RNA polymerase oper tyric acid, 3-amino propionoic acid, ornithine, norleucine, ably linked to an inducible promoter, such as the LACUV norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, promoter (see, U.S. Pat. No. 4,952,496). Such hosts include, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylala but are not limited to, the lysogenic E. coli strain BL21 (DE3). nine, B-alanine, fluoro-amino acids, designer amino acids The shASEGP domains, derivatives and analogs can be 60 Such as B-methyl amino acids, ca-methyl amino acids, na produced by various methods known in the art. For example, methylamino acids, and amino acid analogs in general. Fur once a recombinant cell expressing a shASEGP polypeptide, thermore, the amino acid can bed (dextrorotary) or 1 (levoro or a domain, fragment or derivative thereof, is identified, the tary). individual gene product can be isolated and analyzed. This is In cases where natural products are suspected of being achieved by assays based on the physical and/or functional 65 mutant or are isolated from new species, the amino acid properties of the protein, including, but not limited to, radio sequence of the shASEGP polypeptide isolated from the active labeling of the product followed by analysis by gel natural source, as well as those expressed in vitro, or from US 7,871,607 B2 55 56 synthesized expression vectors in Vivo or in vitro, can be extracts or purified enzymes capable of introducing the determined from analysis of the DNA sequence, or alterna desired N-linked Sugar moieties. tively, by direct sequencing of the isolated protein. Such Selection of an Expression System analysis can be performed by manual sequencing or through Eukaryotic cell expression systems vary in the extent and use of an automated amino acid sequenator. 5 type of glycosylation they introduce into an ectopically Modifications—A variety of modifications of the shA expressed polypeptide. CHO cells are, for example, highly SEGP polypeptides and domains are contemplated herein. A efficient at the introduction of N-linked glycosylation into an sHASEGP-encoding nucleic acid molecule can be modified active shASEGP polypeptide. by any of numerous strategies known in the art (Sambrook ET Additional eukaryotic expression systems that introduce A/. (1990), Molecular Cloning, A Laboratory Manual. 2d ed., 10 N-linked glycosylation to generate a functional shASEGP Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). product can be tested by introducing a human shASEGP The sequences can be cleaved at appropriate sites with restric expression plasmid into said cells and testing for neutral tion endonuclease (s), followed by further enzymatic modi activity. Proper N-linked glycosylation can be determined by fication if desired, isolated, and ligated in vitro. In the pro way of FACE analysis of PNG ASE released oligosaccha duction of the gene encoding a domain, derivative or analog 15 rides. Glycosylation profiles of catalytically active shA ofsFHASEGP, care should be taken to ensure that the modified SEGPs are further provided herein. Verification of glycosy gene retains the original translational reading frame, uninter lation can also be made by treatment of shASEGP from said rupted by translational stop signals, in the gene region where cells with PNGASE-F or by growth of such cells in tunica the desired activity is encoded. mycin following introduction of shASEGP encoding nucleic Additionally, the shASEGP-encoding nucleic acid mol acids. ecules can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, N-glycosylation of shASEGP polypeptide in vitro. The or to create variations in coding regions and/or form new sHASEGP polypeptide can be N-glycosylated by contact of restriction endonuclease sites or destroy pre-existing ones, to sHASEGP polypeptide with cell-free extracts containing facilitate further in vitro modification. Also, as described 25 activity capable of transferring N-linked sugars to shASEGP herein muteins with primary sequence alterations, such as polypeptide Such as canine microsomal membranes or replacements of Cys residues and elimination or addition of through coupled transcription and translation as is commer glycosylation sites are contemplated; the shASEGP of SEQ cially available (Promega Madison Wis.). ID No. 1 has seven potential glycosylation sites. Such muta Oligosaccharides are considered to have a reducing end tions can be effected by any technique for mutagenesis known 30 and a non-reducing end, whether or not the Saccharide at the in the art, including, but not limited to, chemical mutagenesis reducing end is in fact a reducing Sugar. In accordance with and in vitro site-directed mutagenesis (Hutchinson et al., i. accepted nomenclature, oligosaccharides are depicted herein Biol. Chem. 253: 6551-6558 (1978)), use of TABE Linkers with the non-reducing end on the left and the reducing end on (Pharmacia). In one embodiment, for example, a shASEGP the right. All oligosaccharides described herein are described polypeptide or domain thereof is modified to include a fluo 35 with the name or abbreviation for the non-reducing saccha rescent label. In other specific embodiments, the shASEGP ride (e.g., Gal), followed by the configuration of the glyco polypeptide is modified to have a heterobifunctional reagent, sidic bond (alpha. or beta.), the ring bond, the ring position Such heterobifunctional reagents can be used to crosslink the of the reducing saccharide involved in the bond, and then the members of the complex. name or abbreviation of the reducing Saccharide (e.g., In addition, domains, analogs and derivatives of a SHA 40 GlcNAc). The linkage between two Sugars may be expressed, SEGP can be chemically synthesized. For example, a peptide for example, as 2.3.2.fw darw.3, or (2.3). Each saccharide is a corresponding to a portion of a shASEGP, which includes the pyranose. desired domain or which mediates the desired activity in vitro As used herein, N-linked Sugar moiety refers to an oli can be synthesized by use of a peptide synthesizer. Further gosaccharide attached to a shASEGP via the amide nitrogen more, if desired, nonclassical amino acids or chemical amino 45 of Asn residues. N-linked oligosaccharides fall into several acid analogs can be introduced as a Substitution or addition major types (oligomannose, complex, hybrid, Sulfated), all of into the shASEGP sequence. Non-classical amino acids which have (Man) 3-GlcNAc-GlcNAc-cores attached via the include but are not limited to the D-isomers of the common amide nitrogen of Asn residues that fall within-Asn-Xaa-Thr/ amino acids, a-amino isobutyric acid, 4-aminobutyric acid, Ser- sequences (where Xaa is not Pro). N-linked sites are Abu, 2-aminobutyric acid, S-ABU, e-Ahk, 6-amino hexanoic 50 often indirectly assigned by the appearance of a “blank” cycle acid, Aib, 2-amino isobutyric acid, 3-amino propionoic acid, during sequencing. Positive identification can be made after ornithine, norleucine, norvaline, hydroxyproline, sarcosine, release of the oligosaccharide by PNGase F, which converts citrulline, cysteic acid, t-butylglycine, t-butylalanine, phe the glycosylated Asn to Asp. After PNGase F release, nylglycine, cyclohexylalanine, B-alanine, fluoro-amino N-linked oligosaccharides can be purified using Bio-Gel P-6 acids, designer amino acids such as ti-methyl amino acids, 55 chromatography, with the oligosaccharide pool Subjected to ca-methyl amino acids, na-methyl amino acids, and amino preparative high pH anion exchange chromatography acid analogs in general. Furthermore, the amino acid can be d (HPAEC) (Townsendet al., (1989) Anal. Biochem. 182, 1-8). (dextrorotary) or 1 (levorotary). Certain oligosaccharide isomers can be resolved using F. Generation of a Functionally Active Glycosylated shA HPAEC. Fucose residues will shift elution positions earlier in SEGP with N-Linked Sugar Moieties. 60 the HPAEC chromatogram, while additional sialic acid resi Properly N-glycosylated human shASEGP is required to dues will increase the retention time. Concurrent treatment of generate a catalytically stable protein. N-linked glycosylation glycoproteins whose oligosaccharide structures are known of shASEGP can be achieved through various techniques. (e.g., bovine fetuin, a-lacid glycoprotein, ovalbumin, RNAse Glycosylation of shASEGP can be achieved by introducing B, transferrin) can facilitate assignment of the oligosaccha nucleic acids encoding shASEGP into cells of eukaryotic 65 ride peaks. The collected oligosaccharides can be character origin capable of proper N-linked glycosylation or alterna ized by a combination of compositional and methylation link tively, by contacting shASEGP polypeptide with cell free age analyses (Waeghe et al., (1983) Carbohydr Res. 123, US 7,871,607 B2 57 58 281-304.), with anomeric configurations assigned by NMR amount of available glycoprotein, as well as the potential for spectroscopy (Van Halbeek (1993) in Methods Enzymol greater sensitivity using laser induced fluorescence makes 230). this approach attractive. Alternatively, oligosaccharides can be identified by fluo Monosaccharide composition analysis of small amounts of rescence assisted carbohydrate electrophoresis (FACE) sHASEGP is best performed on PVDF (PSQ) membranes, Callewaert et al. (2001) Glycobiology 11, 275-281. after either electroblotting (Weitzhandleretal, (1993).J. Biol. G. Detection and Characterization of N-Linked Sugar Moi Chem. 268, 5121-5130.) or if smaller aliquots are to be ana eties on SHASEGP lyzed on dot blots. PVDF is an ideal matrix for carbohydrate Determining whether a protein is in fact glycosylated is the analysis since neither mono- or oligosaccharides bind to the initial step in glycoprotein glycan analysis. Polyacrylamide 10 membrane, once released by either acid or enzymatic gel electrophoresis in the presence of sodium dodecyl Sulfate hydrolysis. (SDS-PAGE) has become the method of choice as the final step prior to protein sequencing. Glycosylated proteins often FACE analysis is an efficient means of detecting glycosy lation profiles of shASEGPs. FACETMN-Linked Oligosac migrate as diffuse bands by SDS-PAGE. A marked decrease charide Profiling (Prozyme) with 30% oligosaccharide gels is in bandwidth and change in migration position after treatment 15 with peptide-N-4-(N-acetyl-D-glucosaminyl) asparagine one such mechanism. Oligosaccharides cleaved from 100 g amidase (PNGase F) is considered diagnostic of N-linked of glycoproteins by enzymatic digestion with N-Glycanase glycosylation. If the other types of glycosylation are predomi (a.k.a. PNGase), labeled using the fluorophore ANTS, and nant other approaches must be used. Lectin blotting methods separated by electrophoresis can be used for detection of provide an approach that is independent of the class of gly sHASEGP glycosylation profiles. The relative positions of cosylation (N versus O). Lectins, carbohydrate-binding pro the oligosaccharide bands are determined by running the teins from various plant tissues, have both high affinity and sample and dilutions of the sample alongside an oligosaccha narrow specificity for a wide range of defined Sugar epitopes ride standard ladder which designated the migration distance found on glycoprotein glycans (Cummings, R. D. (1994) in Degree of Polymerization (DP) units. Methods in Enzymol. 230, 66-86.). When conjugated with 25 H. Screening Methods to Identify Compounds That Modu biotin or digoxigenin, they can be easily identified on mem late shASEGP Activity. brane blots through a colorimetric reaction utilizing avidin or Several types of assays are exemplified and described anti-digoxigenin antibodies conjugated with alkaline phos herein. It is understood that the Hyaluronidase domains can phatase (Haselbeck, et al. (1993) Methods in Mol. Biol. 14, be used in other assays. It is shown here, however, that the 161-173.), analogous to secondary antibody-alkaline phos 30 Hyaluronidase domains exhibit catalytic activity. phatase reactions employed in Western blotting. Screening AS Such they are ideal for in vitro screening assays. with a panel of lectins with well-defined specificity can pro They can also be used in binding assays. vide considerable information about a glycoprotein's carbo hydrate complement. Importantly, the color development The shASEGP full length Zymogens, activated enzymes, amplification is Sufficiently high that 10-50 ng of a glycopro 35 and Hyaluronidase domains are contemplated for use in any tein can easily be seen on a membrane blot of an SDS-PAGE. screening assay known to those of skill in the art, including Although lectins exhibit very high affinity for their cognate those provided herein. Hence the following description, if ligands. Some do reveal significant avidity for structurally directed to Hyaluronidase assays is intended to apply to use of related epitopes. Thus, it is important to carefully note the a single chain Hyaluronidase domain or a catalytically active possibility of cross-reactivity when choosing a panel of lec 40 portion thereof of any Hyaluronidase, including a shASEGP. tins, and apply those with the highest probability of individu Other assays, such as binding assays are provided herein, ally distinguishing complex, hybrid and high mannose particularly for use with a shASEGP, including any variants, N-linked glycans from O-linked structures. Such as splice variants thereof. Monosaccharide analysis can also be used to determine 1. Catalytic Assays for identification of agents that modu whether shASEGP is glycosylated and as in the case of lectin 45 late the Hyaluronidase activity of a shASEGP protein. Meth analysis provides additional information on structural fea ods for identifying a modulator of the catalytic activity of a tures. Quantitative monosaccharide composition analysis i) sHASEGP, particularly a single chain Hyaluronidase domain identifies glycosylated proteins, ii) gives the molar ratio of or catalytically active portion thereof, are provided herein. individual Sugars to protein, iii) Suggests, in some cases, the The methods can be practiced by: contacting the shASEGP, presence of oligosaccharide classes, iv) is the first step in 50 a full-length Zymogen or activated form, and particularly a designing a structural elucidation strategy, and V) provides a single-chain domain thereof, with a substrate of the shA measure of production consistency for recombinant glyco SEGP in the presence of a test substance, and detecting the protein therapeutics. In recent years high-pH anion-exchange proteolysis of the substrate, whereby the activity of the shA chromatography with pulsed amperometric detection SEGP is assessed, and comparing the activity to a control. For (HPAEC-PAD) has been extensively used to determine 55 example, a control can be the activity of the shASEGP monosaccharide composition (Townsend, et al. (1995) in assessed by contacting a shASEGP, including a full-length Carbohydrate Analysis: High-performance liquid chroma Zymogen or activated form, and particularly a single-chain tography and capillary electrophoresis (Z. El Rassi ed.). pp. domain thereof, particularly a single-chain domain thereof, 181-209.). More recently, fluorophore-based labeling meth with a substrate of the shASEGP, and detecting the proteoly ods have been introduced and many are available in kit form. 60 sis of the substrate, whereby the activity of the shASEGP is A distinct advantage of fluorescent methods is an increase in assessed. The results in the presence and absence of the test sensitivity (50-fold). One potential disadvantage is that dif compounds are compared. A difference in the activity indi ferent monosaccharides may demonstrate different selectiv cates that the test substance modulates the activity of the ity for the fluorophore during the coupling reaction, either in sHASEGP. Activators of shASEGP activation cleavage are the hydrolysate or in the external standard mixture. However, 65 also contemplated; Such assays are discussed below. the increase in sensitivity and the ability to identify which In one embodiment a plurality of the test Substances are monosaccharides are present from a small portion of the total screened simultaneously in the above Screening method. In US 7,871,607 B2 59 60 another embodiment, the shASEGP is isolated from a target SEGP polypeptide. Any shASEGP polypeptide is cell as a means for then identifying agents that are potentially contemplated as target for identifying modulators of the specific for the target cell. activity thereof. In another embodiment, a test Substance is a therapeutic 2. Binding assays. Also provided herein are methods for compound, and whereby a difference of the shASEGP activ identification and isolation of agents, particularly compounds ity measured in the presence and in the absence of the test that bind to shASEGPs. The assays are designed to identify Substance indicates that the target cell responds to the thera agents that bind to the isolated Hyaluronidase domain (or a peutic compound. protein, other than a shASEGP polypeptide, that contains the Hyaluronidase domain of a shASEGP polypeptide), and to One method includes the steps of (a) contacting the shA 10 the activated form, including the activated form derived from SEGP polypeptide or Hyaluronidase domain thereof with one the full-length Zymogen or from an extended Hyaluronidase or a plurality of test compounds under conditions conducive domain. The identified compounds are candidates or leads for to interaction between the ligand and the compounds; and (b) identification of compounds for treatments of disorders and identifying one or more compounds in the plurality that spe diseases involving aberrant Hyaluronidase activity. The shA cifically binds to the ligand. 15 SEGP polypeptides used in the methods include any shA Another method provided herein includes the steps of a) SEGP polypeptide as defined herein, including the shA contacting a shASEGP polypeptide or Hyaluronidase SEGP single chain Hyaluronidase domain or proteolytically domain thereof with a substrate of the shASEGP polypep active portion thereof. tide, and detecting the degradation of Substrate, whereby the A variety of methods are provided herein. These methods activity of the shASEGP polypeptide is assessed; b) contact can be performed in Solution or in Solid phase reactions in ing the shASEGP polypeptide with a substrate of the shA which the shASEGP polypeptide (s) or Hyaluronidase SEGP polypeptide in the presence of a test substance, and domain (s) thereof are linked, either directly or indirectly via detecting the degradation of the substrate, whereby the activ a linker, to a solid Support. Screening assays are described in ity of the shASEGP polypeptide is assessed; and c) compar the Examples, and these assays have been used to identify ing the activity of the shASEGP polypeptide assessed in 25 candidate compounds. steps a) and b), whereby the activity measured in step a) For purposes herein, all binding assays described above are differs from the activity measured in stepb) indicates that the provided for shASEGP. test substance modulates the activity of the shASEGP Methods for identifying an agent, such as a compound, that polypeptide. specifically binds to a shASEGP single chain Hyaluronidase In another embodiment, a plurality of the test Substances is 30 domain, a full-length activated shASEGP or two chain screened simultaneously. In comparing the activity of a SHA Hyaluronidase domain thereof are provided herein. The SEGP polypeptide in the presence and absence of a test sub method can be practiced by (a) contacting the shASEGP with stance to assess whether the test Substance is a modulator of one or a plurality of test agents under conditions conducive to the shASEGP polypeptide, it is unnecessary to assay the binding between the shASEGP and an agent; and (b) identi activity in parallel, although Such parallel measurement is 35 fying one or more agents within the plurality that specifically typical. It is possible to measure the activity of the shASEGP binds to the SHASEGP. polypeptide at one time point and compare the measured For example, in practicing such methods the shASEGP activity to a historical value of the activity of the shASEGP polypeptide is mixed with a potential binding partner or an polypeptide. extract or fraction of a cell under conditions that allow the For instance, one can measure the activity of the shASEGP 40 association of potential binding partners with the polypep polypeptide in the presence of a test Substance and compare tide. After mixing, peptides, polypeptides, proteins or other with historical value of the activity of thesHASEGP polypep molecules that have become associated with a shASEGP are tide measured previously in the absence of the test Substance, separated from the mixture. The binding partner that bound to and vice versa. This can be accomplished, for example, by the shASEGP can then be removed and further analyzed. To providing the activity of the shASEGP polypeptide on an 45 identify and isolate a binding partner, the entire protein, for insert or pamphlet provided with a kit for conducting the instance the entire disclosed protein of SEQID No. 1 can be assay. used. Alternatively, a fragment of the protein can be used. Methods for selecting substrates for a particular shASEGP A variety of methods can be used to obtain cell extracts or are described in the EXAMPLES, and particular Hyalu 50 body fluids. Such as blood, serum, urine, Sweat, synovial fluid, ronidase assays are exemplified. CSF and other such fluids. Combinations and kits containing the combinations For example, cells can be disrupted using either physical or optionally including instructions for performing the assays chemical disruption methods. Examples of physical disrup are provided. The combinations include a shASEGP tion methods include, but are not limited to, Sonication and polypeptide and a substrate of the shASEGP polypeptide to 55 mechanical shearing. Examples of chemical lysis methods be assayed; and, optionally reagents for detecting proteolysis include, but are not limited to, detergent lysis and enzyme of the substrate. The substrates, which can be chromogenic or lysis. A skilled artisan can readily adapt methods for prepar fluorogenic molecules, including glycosaminoglycans, Sub ing cellular extracts in order to obtain extracts for use in the ject to proteolysis by a particular shASEGP polypeptide, can present methods. be identified empirically by testing the ability of the shA 60 Once an extract of a cell is prepared, the extract is mixed SEGP polypeptide to cleave the test substrate. Substrates that with the shASEGP under conditions in which association of are cleaved most effectively i.e. at the lowest concentrations the protein with the binding partner can occur. A variety of and/or fastest rate or under desirable conditions), are identi conditions can be used, including conditions that resemble fied. conditions found in the cytoplasm of a human cellor in a body Additionally provided herein is a kit containing the above 65 fluid. Such as blood. Features, such as osmolarity pH, tem described combination. The kit optionally includes instruc perature, and the concentration of cellular extract used, can be tions for identifying a modulator of the activity of a shA varied to optimize the association of the protein with the US 7,871,607 B2 61 62 binding partner. Similarly, methods for isolation of molecules transduced signal is assessed by detecting an effect on an of interest from body fluids are known. expression from a reporter gene (see, e.g., U.S. Pat. No. After mixing under appropriate conditions, the bound 5,436,128). complex is separated from the mixture. A variety of tech 4. Methods for Identifying Agents that Modulate the niques can be used to separate the mixture. For example, Expression a Nucleic Acid encoding a shASEGP. Another antibodies specific to a shASEGP can be used to immuno embodiment provides methods for identifying agents that precipitate the binding partner complex. Alternatively, stan modulate the expression of a nucleic acid encoding a SHA dard chemical separation techniques such as chromatography SEGP. Such assays use any available means of monitoring for changes in the expression level of the nucleic acids encoding and density/sediment centrifugation can be used. 10 a SHASEGP. After removing the non-associated cellular constituents in Assay formats can be used to monitor the ability of the the extract, the binding partner can be dissociated from the agent to modulate the expression of a nucleic acid encoding a complex using conventional methods. For example, dissocia sHASEGP. For instance, mRNA expression can be monitored tion can be accomplished by altering the salt concentration or directly by hybridization to the nucleic acids. Also enzyme pH of the mixture. 15 assays as described can be used to detect agents that modulate To aid in separating associated binding partner pairs from the expression of shASEGP. the mixed extract, the shASEGP can be immobilized on a Cell lines are exposed to the agent to be tested under Solid Support. For example, the protein can be attached to a appropriate conditions and time and total RNA or mRNA is nitrocellulose matrix or acrylic beads. Attachment of the pro isolated by Standard procedures (see, e.g. Sambrook et al tein or a fragment thereof to a solid Support aids in separating (1989) MOLECULAR CLONING: A LABORATORY peptide/binding partner pairs from other constituents found in MANUAL, 2nd Ed. Cold Spring Harbor Laboratory Press). the extract. The identified binding partners can be either a Probes to detect differences in RNA expression levels single protein or a complex made up of two or more proteins. between cells exposed to the agent and control cells can be Alternatively, the nucleic acid molecules encoding the prepared from the nucleic acids. It is typical, but not neces single chain Hyaluronidases can be used in a yeast two-hybrid 25 sary, to design probes which hybridize only with target system. The yeast two-hybrid system has been used to iden nucleic acids under conditions of high Stringency. Only tify other protein partner pairs and can readily be adapted to highly complementary nucleic acid hybrids form under con employ the nucleic acid molecules herein described. ditions of high Stringency. Accordingly, the stringency of the Another in vitro binding assay, particularly for a SEIA assay conditions determines the amount of complementarity SEGP, uses a mixture of a polypeptide that contains at least 30 that should exist between two nucleic acid strands in order to the catalytic domain of one of these proteins and one or more form a hybrid. Stringency should be chosen to maximize the candidate binding targets or Substrates. After incubating the difference in stability between the probe: target hybrid and mixture under appropriate conditions, the ability of the shA potential probe: non-target hybrids. SEGP or a polypeptide fragment thereof containing the cata For example, N- and C-terminal fragments of the shA lytic domain to bind to or interact with the candidate substrate 35 SEGP can be expressed in bacteria and used to search for is assessed. For cell-free binding assays, one of the compo proteins that bind to these fragments. Fusion proteins, such as nents includes or is coupled to a detectable label. The label His-tag or GST fusion to the N- or C-terminal regions of the can provide for direct detection, Such as radioactivity, lumi sHASEGP can be prepared for use as a substrate. These nescence, optical or electron density, etc., or indirect detec fusion proteins can be coupled to, for example, Glutathione tion Such as an epitope tag, an enzyme, etc. A variety of 40 Sepharose beads and then probed with cell lysates or body methods can be employed to detect the label depending on the fluids. Prior to lysis, the cells or body fluids can be treated nature of the label and other assay components. For example, with a candidate agent that can modulate a shASEGP or the label can be detected bound to the solid substrate or a proteins that interact with domains thereon. Lysate proteins portion of the bound complex containing the label can be binding to the fusion proteins can be resolved by SDS-PAGE, separated from the solid substrate, and the label thereafter 45 isolated and identified by protein sequencing or mass spec detected. troscopy, as is known in the art. 3. Detection of signal transduction shASEGP, which is a Antibody probes are prepared by immunizing Suitable membrane anchored protein, can be involved directly or indi mammalian hosts in appropriate immunization protocols rectly in signal transduction directly as a cell Surface receptor 50 using the peptides, polypeptides or proteins if they are of or indirectly by activating proteins, such as pro-growth fac sufficient length (e.g., 4, 5, 6,7,8,9, 10, 11, 12, 13, 14, 15, 20, tors that can initiate signal transduction. 25, 30, 35, 40 or more consecutive amino acids the shASEGP In addition, secreted sPIASEGP, such as the soluble polypeptide or if required to enhance immunogenicity, con domain of shASEGP as described in SEQID NO. 4, can be jugated to Suitable carriers. Methods for preparing immuno involved in signal transduction either directly by binding to or 55 genic conjugates with carriers, such as bovine serum albumin interacting with a cell Surface receptor or indirectly by acti (BSA), keyhole limpet hemocyanin (KLH), or other carrier Vating proteins, such as pro-growth factors that can initiate proteins are well known in the art. In some circumstances, signal transduction. Assays for assessing signal transduction direct conjugation using, for example, carbodiimide reagents are well known to those of skill in the art, and can be adapted can be effective; in other instances linking reagents such as for use with the shASEGP polypeptide. 60 those supplied by Pierce Chemical Co., Rockford, Ill., can be Assays for identifying agents that affect or alter signal desirable to provide accessibility to the hapten. Hapten pep transduction mediated directly or indirectly, Such as via acti tides can be extended at either the amino or carboxy terminus vation of a pro-growth factor, by a shASEGP, particularly the with a Cys residue or interspersed with cysteine residues, for full length or a sufficient portion to anchor the extracellular example, to facilitate linking to a carrier. domain or a functional portion thereof of a shASEGP on the 65 Administration of the immunogens is conducted generally Surface of a cell are provided. Such assays, include, for by injection over a suitable time period and with use of example, transcription based assays in which modulation of a Suitable adjuvants, as is generally understood in the art. Dur US 7,871,607 B2 63 64 ing the immunization schedule, titers of antibodies are taken refers to a nucleic acid capable of hybridizing to a portion of to determine adequacy of antibody formation. a shASEGP polypeptide RNA (generally mRNA) by virtue Anti-peptide antibodies can be generated using synthetic of some sequence complementarity, and generally under high peptides corresponding to, for example, the carboxy terminal stringency conditions. The antisense nucleic acid can be amino acids of the shASEGP. 5 complementary to a coding and/or noncoding region of a Synthetic peptides can be as Small as 1-3 amino acids in sHASEGP polypeptide mRNA. Such antisense nucleic acids length, generally at least 4 or more amino acid residues long. have utility as therapeutics that reduce or inhibit shASEGP The peptides can be coupled to KLH using standard methods polypeptide function, and can be used in the treatment or and can be immunized into animals, such as rabbits or ungu prevention of disorders as described Supra. lates. Polyclonal antibodies can then be purified, for example 10 The shASEGP polypeptide antisense nucleic acids are of using Actigel beads containing the covalently bound peptide. at least six nucleotides and are generally oligonucleotides While the polyclonal antisera produced in this way can be (ranging from 6 to about 150 nucleotides including 6 to 50 satisfactory for some applications, for pharmaceutical com nucleotides). The antisense molecule can be complementary positions, use of monoclonal preparations are generally used. to all or a portion of the Hyaluronidase domain. For example, Immortalized cell lines which secrete the desired monoclonal 15 the oligonucleotide is at least 10 nucleotides, at least 15 antibodies can be prepared using the standard method of nucleotides, preferably at least 20, 40, 80 to at least 100 Kohler et al., (Nature 256: 495-7 (1975)) or modifications nucleotides, or at least 125 nucleotides. The oligonucleotides which effect immortalization of lymphocytes or spleen cells, can be DNA or RNA or chimeric mixtures or derivatives or as is generally known. The immortalized cell lines secreting modified versions thereof, single-stranded or double the desired antibodies are screened by immunoassay in which stranded. The oligonucleotide can be modified at the base the antigen is the peptide hapten, polypeptide or protein. moiety, Sugar moiety, or phosphate backbone. The oligo When the appropriate immortalized cell culture secreting nucleotide can include other appending groups such as pep the desired antibody is identified, the cells can be cultured tides, or agents facilitating transport across the cell membrane either in vitro or by production in vivo via ascites fluid. Of (see e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 86: particular interest, are monoclonal antibodies that recognize 25 6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. the catalytic domain or activation cleavage site (region) of a USA 84: 648-652 (1987); PCT Publication No. WO SHASEGP. 88/09810, published Dec. 15, 1988) or blood-brain barrier The antibodies or fragments can also be produced. Regions (see e.g., PCT Publication No. WO 89/10134, published Apr. that bind specifically to the desired regions of receptor also 25, 1988), hybridization-triggered cleavage agents (see e.g., can be produced in the context of chimeras with multiple 30 Kroletal. BioTechniques 6:958-976 (1988)) or intercalating species origin. agents (see e.g., Zon. Pharm. Res. 5: 539-549 (1988)). Agents that are assayed in the above method can be ran The sIASEGP polypeptide antisense nucleic acid gener domly selected or rationally selected or designed. ally is an oligo-nucleotide, typically single-stranded DNA or The agents can be, as examples, peptides, Small molecules, RNA or an analog thereof or mixtures thereof. For example, and carbohydrates. A skilled artisan can readily recognize 35 the oligonucleotide includes a sequence antisense to a portion that there is no limit as to the structural nature of the agents. of a nucleic acid that encodes a human shASEGP polypep The peptide agents can be prepared using standard Solid tide. The oligonucleotide can be modified at any position on phase (or Solution phase) peptide synthesis methods, as is its structure with Substituents generally known in the art. known in the art. In addition, the DNA encoding these pep The shASEGP polypeptide antisense oligonucleotide can tides can be synthesized using commercially available oligo 40 include at least one modified base moiety which is selected nucleotide synthesis instrumentation and produced recombi from the group including, but not limited to 5-fluorouracil, nantly using standard recombinant production systems. The 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, production using Solid phase peptide synthesis is necessitated Xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) if non-gene-encoded amino acids are to be included. uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-car I. Methods of Treatment 45 boxymethylaminomethyluracil, dihydrouracil, beta-D-galac sHASEGPs identified by the methods herein are used for tosylqueosine, inosine, N6-isopentenyladenine, 1-meth treating or preventing abnormal accumulations of shASEGP ylguanine, 1-methylinosine, 2,2-dimethylguanine, Substrates in an animal, particularly a mammal, including a 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-me human. In one embodiment, the method includes administer thylcytosine, N6-adenine, 7-methylguanine, 5-methylami ing to a mammal an effective amount of a shASEGP glyco 50 nomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta protein, whereby the disease or disorder is treated or pre D-mannosylqueosine, 5-apos-methoxycarboxymethyluracil, vented. 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, In another embodiment, a shASEGP inhibitor can be used uracil-5-oxyacetic acid (v), Wybutoxosine, pseudouracil, in the treatment of an excess amount of neutral hyaluronidase queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, activity. The mammal treated can be a human. The inhibitors 55 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl provided herein are those identified by the Screening assays. ester, uracil-5-oxyacetic acid (V), 5-methyl-2-thiouracil, In addition, antibodies and antisense nucleic acids or double 3-(3-amino-3-n-2-carboxypropyl) uracil, (ACP3) w, and 2.6- stranded RNA (dsRNA), such as RNAi are contemplated. diaminopurine. 1. Antisense treatment: In a specific embodiment, as In another embodiment, the oligonucleotide includes at described hereinabove, shASEGP polypeptide function is 60 least one modified Sugar moiety selected from the group reduced or inhibited by shASEGP polypeptide antisense including but not limited to arabinose, 2-fluoroarabinose, nucleic acids, to treat or prevent excessive chondroitinase Xylulose, and hexose. The oligonucleotide can include at least activity. The therapeutic or prophylactic use of nucleic acids one modified phosphate backbone selected from a phospho of at least six nucleotides, generally up to about 150 nucle rothioate, a phosphorodithioate, a phosphoramidothioate, a otides, that are antisense to a gene or cDNA encoding SHA 65 phosphoramidate, a phosphordiamidate, a methylphospho SEGP polypeptide or a portion thereof is provided. A shA nate, an alkyl phosphotriester, and a formacetal or analog SEGP polypeptide “antisense' nucleic acid as used herein thereof. US 7,871,607 B2 65 66 The oligonucleotide can be ana-anomeric oligonucleotide. a shASEGP Interfering RNA (RNAi) fragments, particularly An a-anomeric oligonucleotide forms specific double double-stranded (ds) RNAi can be used to generate loss-of stranded hybrids with complementary RNA in which the sHASEGP function. Methods relating to the use of RNAi to strands run parallel to each other (Gautier et al., Nucl. Acids silence genes in organisms including, mammals, C. elegans, Res. 15:6625-6641 (1987)). 5 Drosophila and plants, and humans are known (see, e.g., Fire The oligonucleotide can be conjugated to another mol et al. (1998) Nature 391:806-811; Fire (1999) Trends Genet. ecule. Such as, but are not limited to, a peptide; hybridization 15: 358-363; Sharp (2001) Genes Dev. 15: 485-490; Ham triggered cross-linking agent, transport agent or a hybridiza mond et al. (2001) Nature Rev. Genet. 2: 110-119: Tuschl tion-triggered cleavage agent. The oligonucleotides can be (2001) Chem. Biochem. 2: 239-245; Hamilton et al. (1999) synthesized by Standard methods known in the art, e.g. by use 10 Science 286: 950-952; Hammond et al. (2000) Nature 404: of an automated DNA synthesizer (such as are commercially 293-296: Zamore et al. (2000) Cell 101: 25-33; Bernstein et available from Biosearch, Applied Biosystems, etc.). As al. (2001) Nature 409:363-366; Elbashir et al. (2001) Genes examples, phosphorothioate oligonucleotides can be synthe Dev. 15: 188-200; Elbashiretal. (2001)Nature 411: 494-498: sized by the method of Stein et al., Nucl. Acids Res. 16:3209 International PCT application No. WO 01/29058: Interna (1988)), methylphosphonate oligonucleotides can be pre 15 tional PCT application No. WO99/32619). pared by use of controlled pore glass polymer Supports (Sarin Double-stranded RNA (dsRNA)-expressing constructs are et al., Proc. Natl. Acad. Sci. USA 85: 7448-7451 (1988)), etc. introduced into a host, such as an animal or plant using, a In a specific embodiment, the shASEGP polypeptide anti replicable vector that remains episomal or integrates into the sense oligonucleotide includes catalytic RNA or a ribozyme genome. By selecting appropriate sequences, expression of (see, e.g., PCT International Publication WO 90/11364, pub dsRNA can interfere with accumulation of endogenous lished Oct. 4, 1990; Sarver et al., Science 247: 1222-1225 mRNA encoding a shASEGP. RNAi also can be used to (1990)). In another embodiment, the oligonucleotide is a inhibit expression in vitro. 2'-O-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15: Regions include at least about 21 (or 21) nucleotides that 6131-6148 (1987)), or a chimeric RNA-DNA analogue Inoue are selective (i.e. unique) for shASEGP are used to prepare et al., FEBS Lett. 215: 327-330 (1987)). 25 the RNAi. Smaller fragments of about 21 nucleotides can be Alternatively, the oligonucleotide can be double-stranded transformed directly (i.e., in vitro or in vivo) into cells; larger RNA (dsRNA) such as RNAi. RNAi dsRNA molecules are generally introduced using vec In an alternative embodiment, the shASEGP polypeptide tors that encode them. dsRNA molecules are at least about 21 antisense nucleic acid is produced intracellularly by tran bp long or longer, such as 50, 100, 150, 200 and longer. Scription from an exogenous sequence. 30 Methods, reagents and protocols for introducing nucleic acid For example, a vector can be introduced in vivo such that it molecules into cells in vitro and in vivo are known to those of is taken up by a cell, within which cell the vector or a portion skill in the art. thereof is transcribed, producing an antisense nucleic acid 3. Gene Therapy in an exemplary embodiment, nucleic (RNA). Such a vector would contain a sequence encoding the acids that include a sequence of nucleotides encoding a SHA sHASEGP polypeptide antisense nucleic acid. Such a vector 35 SEGP polypeptide or functional domains or derivative can remain episomal or become chromosomally integrated, thereof, are administered to promote shASEGP polypeptide as long as it can be transcribed to produce the desired anti function, by way of gene therapy. Gene therapy refers to sense RNA. Such vectors can be constructed by recombinant therapy performed by the administration of a nucleic acid to DNA technology methods standard in the art. Vectors can be a subject. In this embodiment, the nucleic acid produces its plasmid, viral, or others known in the art, used for replication 40 encoded protein that mediates a therapeutic effect by promot and expression in mammalian cells. Expression of the ing shASEGP polypeptide function. Any of the methods for sequence encoding the shASEGP polypeptide antisense genetherapy available in the art can be used (see, Goldspiel et RNA can be by any promoter known in the art to act in al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Bio mammalian, including human, cells. Such promoters can be therapy 3: 87-95 (1991); Tolstoshev, An. Rev. Pharmacol. inducible or constitutive. Such promoters include but are not 45 Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926 limited to: the SV40 early promoter region (Bernoist and 932 (1993); and Morgan and Anderson, An. Rev. Biochem. Chambon, Nature 290: 304-310 (1981), the promoter con 62: 191-217 (1993); TIBTECH 115: 155-215 (1993). For tained in the 3' long terminal repeat of Rous sarcoma virus example, one therapeutic composition for gene therapy (Yamamoto et al., Ce/22: 787-797 (1980), the herpes thymi includes a shASEGP polypeptide-encoding nucleic acid that dine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. 50 is part of an expression vector that expresses a shASEGP USA 78: 1441-1445 (1981), the regulatory sequences of the polypeptide or domain, fragment or chimeric protein thereof metallothionein gene (Brinster et al., Nature 296: 39-42 in a Suitable host. In particular, such a nucleic acid has a (1982), etc. promoter operably linked to the shASEGP polypeptide cod The antisense nucleic acids include sequence complemen ing region, the promoter being inducible or constitutive, and, tary to at least a portion of an RNA transcript of a shASEGP 55 optionally, tissue-specific. In another particular embodiment, polypeptide gene, including a human SHASEGP polypeptide a nucleic acid molecule is used in which the shASEGP gene. Absolute complementarity is not required. The amount polypeptide coding sequences and any other desired of shASEGP polypeptide antisense nucleic acid that is effec sequences are flanked by regions that promote homologous tive in the treatment or prevention of neoplastic disease recombination at a desired site in the genome, thus providing depends on the nature of the disease, and can be determined 60 for intrachromosomal expression of the shASEGP protein empirically by standard clinical techniques. nucleic acid (Koller and Smithies, Proc. Natl. Acad. Sci. USA Where possible, it is desirable to determine the antisense 86: 8932-8935 (1989); Zijlstra et al., Nature 342:435-438 cytotoxicity in cells in vitro, and then in useful animal model (1989)). systems prior to testing and use in humans. Delivery of the nucleic acid into a patient can be either 2. RNA interference RNA interference (RNAi) (see, e.g. 65 direct, in which case the patient is directly exposed to the Chuang et al. (2000) Proc. Natl. Acad. Sci. USA 97: 4985) nucleic acid or nucleic acid-carrying vector, or indirect, in can be employed to inhibit the expression of a gene encoding which case, cells are first transformed with the nucleic acid in US 7,871,607 B2 67 68 vitro, then transplanted into the patient. These two adenoviruses in gene therapy can be found in Rosenfeld et al., approaches are known, respectively, as in vivo or ex vivo gene Science 252: 431-434 (1991); Rosenfeld et al., Cell 68: 143 therapy. 155 (1992); and Mastrangeli et al., J. Clin. Invest.91: 225-234 In a specific embodiment, the nucleic acid is directly (1993). administered in vivo, where it is expressed to produce the Adeno-associated virus (AAV) has also been proposed for encoded product. This can be accomplished by any of numer use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. ous methods known in the art, e.g., by constructing it as part 204: 289-300 (1993). of an appropriate nucleic acid expression vector and admin Another approach to gene therapy involves transferring a istering it so that it becomes intracellular, e.g., by infection gene to cells in tissue culture by Such methods as electropo using a defective or attenuated retroviral or other viral vector 10 ration, lipofection, calcium phosphate mediated transfection, (see U.S. Pat. No. 4,980.286), or by direct injection of naked or viral infection. Usually, the method of transfer includes the DNA, or by use of microparticle bombardment (e.g., a gene transfer of a selectable marker to the cells. The cells are then gun; Biolistic, Dupont), or coating with lipids or cell-surface placed under selection to isolate those cells that have taken up receptors or transfecting agents, encapsulation in liposomes, and are expressing the transferred gene. Those cells are then microparticles, or microcapsules, or by administering it in 15 delivered to a patient. linkage to a peptide which is known to enter the nucleus, by In this embodiment, the nucleic acid is introduced into a administering it in linkage to a ligand Subject to receptor cell prior to administration in vivo of the resulting recombi mediated endocytosis (see e.g., Wu and Wu, J. Biol. Chem, nant cell. Such introduction can be carried out by any method 262: 4429-4432 (1987)) (which can be used to target cell known in the art, including but not limited to transfection, types specifically expressing the receptors), etc. In another electroporation, microinjection, infection with a viral or bac embodiment, a nucleic acid-ligand complex can be formed in teriophage vector containing the nucleic acid sequences, cell which the ligand is a fusogenic viral peptide to disrupt endo fusion, chromosome-mediated gene transfer, microcell-me Somes, allowing the nucleic acid to avoid lysosomal degra diated gene transfer, spheroplast fusion, etc. Numerous tech dation. In yet another embodiment, the nucleic acid can be niques are known in the art for the introduction of foreign targeted in vivo for cell specific uptake and expression, by 25 genes into cells (see e.g., Loeffler and Behr, Meth. Enzymol. targeting a specific receptor (see, e.g., PCT Publications WO 217: 599-618 (1993); Cohen et al., Meth. Enzymol. 217: 92/06180 dated Apr. 16, 1992 (Wu et al.); WO92/22635 dated 618-644 (1993); Cline, Pharmac. Ther. 29: 69-92 (1985)) and Dec. 23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, can be used, provided that the necessary developmental and 1992 (Findeis et al.); WO 93/14188 dated Jul. 22, 1993 physiological functions of the recipient cells are not dis (Clarke et al.), WO 93/20221 dated Oct. 14, 1993 (Young)). 30 rupted. The technique should provide for the stable transfer of Alternatively, the nucleic acid can be introduced intracellu the nucleic acid to the cell, so that the nucleic acid is express larly and incorporated within host cell DNA for expression, ible by the cell and generally heritable and expressible by its by homologous recombination (Koller and Smithies, Proc. cell progeny. Natl. Acad. Sci. USA 86: 8932-8935 (1989); Zijistra et al., The resulting recombinant cells can be delivered to a Nature 342:435-438 (1989)). 35 patient by various methods known in the art. In an embodi In a specific embodiment, a viral vector that contains the ment, epithelial cells are injected, e.g., Subcutaneously. In sHASEGP polypeptide nucleic acid is used. For example, a another embodiment, recombinant skin cells can be applied retroviral vector can be used (see Miller et al., Meth. Enzy as a skin graft onto the patient. Recombinant blood cells (e.g., mol. 217: 581-599 (1993)). These retroviral vectors have hematopoietic stem or progenitor cells) can be administered been modified to delete retroviral sequences that are not nec 40 intravenously. The amount of cells envisioned for use essary for packaging of the viral genome and integration into depends on the desired effect, patient state, etc., and can be host cell DNA. The shASEGP polypeptide nucleic acid to be determined by one skilled in the art. used in gene therapy is cloned into the vector, which facili Cells into which a nucleic acid can be introduced for pur tates delivery of the gene into a patient. More detail about poses of gene therapy encompass any desired, available cell retroviral vectors can be found in Boesen et al., Biotherapy 6: 45 type, and include but are not limited to epithelial cells, endot 29.1-302 (1994), which describes the use of a retroviral vector helial cells, keratinocytes, fibroblasts, muscle cells, hepato to deliver the mdr1 gene to hematopoietic stem cells in order cytes; blood cells such as T lymphocytes, B lymphocytes, to make the stem cells more resistant to chemotherapy. monocytes, macrophages, neutrophils, eosinophils, mega Other references illustrating the use of retroviral vectors in karyocytes, granulocytes; various stem or progenitor cells, in gene therapy are: Clowes et al., J. Clin. Invest. 93: 644-651 50 particular hematopoietic stem or progenitor cells, e.g., Such (1994); Kiem et al., Blood 83: 1467-1473 (1994); Salmons as stem cells obtained from bone marrow, umbilical cord and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and blood, peripheral blood, fetal liver, and other sources thereof. Grossman and Wilson, Curr. Opin. In Genetics And Devel. 3: For example, a cell used for gene therapy is autologous to 110-114 (1993). the patient. In an embodiment in which recombinant cells are Adenoviruses are other viral vectors that can be used in 55 used in genetherapy, a shASEGP polypeptide nucleic acid is gene therapy. Adenoviruses are especially attractive vehicles introduced into the cells such that it is expressible by the cells for delivering genes to respiratory epithelia. Adenoviruses or their progeny, and the recombinant cells are then adminis naturally infect respiratory epithelia where they cause a mild tered in vivo for therapeutic effect. In a specific embodiment, disease. Other targets for adenovirus-based delivery systems stem or progenitor cells are used. Any stem and/or progenitor are liver, the central nervous system, endothelial cells, and 60 cells that can be isolated and maintained in vitro can poten muscle. Adenoviruses have the advantage of being capable of tially be used in accordance with this embodiment. infecting non-dividing cells. Kozarsky and Wilson, Current Such stem cells include but are not limited to hematopoi Opinion in Genetics and Development 3: 499-503 (1993) etic stem cells (HSC), stem cells of epithelial tissues such as present a review of adenovirus-based gene therapy. Bout et the skin and the lining of the gut, embryonic heart muscle al., Human Gene Therapy 5: 3-10 (1994) demonstrated the 65 cells, liver stem cells (PCT Publication WO94/08598, dated use of adenovirus vectors to transfer genes to the respiratory Apr. 28, 1994), and neural stem cells (Stemple and Anderson, epithelia of rhesus monkeys. Other instances of the use of Cell 71:973-985 (1992)). US 7,871,607 B2 69 70 Epithelial stem cells (ESC) or keratinocytes can be The shASEGP polypeptide and a second agent can be obtained from tissues such as the skin and the lining of the gut packaged as separate compositions for administration by known procedures (Rheinwald, Meth. Cell Bio. 21A: 229 together or sequentially or intermittently. Alternatively, they (1980)). In stratified epithelial tissue such as the skin, renewal can be provided as a single composition for administration or occurs by mitosis of stem cells within the germinal layer, the 5 as two compositions for administration as a single composi layer closest to the basal lamina. Stem cells within the lining tion. The combinations can be packaged as kits. of the gut provide for a rapid renewal rate of this tissue. ESC 2. Formulations and Route of Administration or keratinocytes obtained from the skin or lining of the gut of The shASEGP polypeptides and soluble human hyalu a patient or donor can be grown in tissue culture (Rheinwald, ronidase domain thereof provided herein can be formulated Meth. Cell Bio. 21A: 229 (1980); Pittelkow and Scott, Cano. 10 as pharmaceutical compositions, typically for single dosage Clinic Proc. 61: 771 (1986)). If the ESC are provided by a administration. The concentrations of the polypeptides in the donor, a method for Suppression of host versus graft reactivity formulations are effective for delivery of an amount, upon (e.g., irradiation, drug orantibody administration to promote administration, that is effective for the intended treatment. moderate immunosuppression) also can be used. Typically, the compositions are formulated for single dosage With respect to hematopoietic stem cells (HSC), any tech 15 administration. To formulate a composition, the weight frac nique which provides for the isolation, propagation, and tion of a shASEGP polypeptide, soluble human hyalu maintenance in vitro of HSC can be used in this embodiment. ronidase domains thereof or mixture thereof is dissolved, Techniques by which this can be accomplished include (a) the Suspended, dispersed or otherwise mixed in a selected vehicle isolation and establishment of HSC cultures from bone mar at an effective concentration such that the treated condition is row cells isolated from the future host, or a donor, or (b) the relieved or ameliorated. use of previously established long-term HSC cultures, which Pharmaceutical carriers or vehicles suitable for adminis can be allergenic or Xenogenic. tration of the shASEGP or soluble human hyaluronidase Non-autologous HSC generally are used with a method of domains thereof provided herein include any Such carriers Suppressing transplantation immune reactions of the future known to those skilled in the art to be suitable for the particu host/patient. In a particular embodiment, human bone mar 25 row cells can be obtained from the posterior iliac crest by lar mode of administration. needle aspiration (see, e.g., Kodo et al., J. Clin. Invest. 73: In addition, the polypeptides can be formulated as the sole 1377-1384 (1984)). For example, the HSC can be made pharmaceutically active ingredient in the composition or can highly enriched or in substantially pure form. This enrich be combined with other active ingredients. Liposomal Sus ment can be accomplished before, during, or after long-term 30 pensions, including tissue-targeted liposomes, can also be culturing, and can be done by any techniques known in the art. Suitable as pharmaceutically acceptable carriers. These can Long-term cultures of bone marrow cells can be established be prepared according to methods known to those skilled in and maintained by using, for example, modified Dexter cell the art. For example, liposome formulations can be prepared culture techniques (Dexter et al., J. Cell Physiol. 91: 335 as described in U.S. Pat. No. 4,522,811. (1977)) or Witlock-Witte culture techniques (Witlock and 35 The active shASEGP or soluble human hyaluronidase Witte, Proc. Natl. Acad. Sci. USA 79: 3608-3612 (1982)). domain thereof is included in the pharmaceutically accept In a specific embodiment, the nucleic acid to be introduced able carrier in an amount Sufficient to exert a therapeutically for purposes of gene therapy includes an inducible promoter useful effect in the absence of undesirable side effects on the operably linked to the coding region, such that expression of patient treated. The therapeutically effective concentration the nucleic acid is controllable by controlling the presence or 40 can be determined empirically by testing the polypeptides in absence of the appropriate inducer of transcription. known in vitro and in Vivo systems such as by using the assays 3. Prodrugs—A method for treating tumors is provided. provided herein or see, e.g., Taliani et al. (1996) Anal. Bio The method is practiced by administering a prodrug that is chem. 240: 60-67, Filocamo et al. (1997) J. Virology 71: cleaved at a specific site by a HASEGP to release an active 1417-1427, Sudo et al. (1996) Antiviral Res. 32:9-18, Buf drug or precursor that can be converted to active drug in vivo. 45 fard et al. (1995) Virology 209:52-59, Bianchi et al. (1996) Upon contact with a cell that expresses shASEGP activity, Anal. Biochem. 237: 239-244, Hamatake et al. (1996) Inter the prodrug is converted into an active drug. The prodrug can virology 39:249-258, Steinkühler et al. (1998) Biochem. 37: be a conjugate that contains the active agent, Such as an 8899-8905, D'Souza et al. (1995) J. Gen. Virol. 76: 1729 anti-tumor drug, such as a cytotoxic agent, or other therapeu 1736, Takeshita et al. (1997) Anal. Biochem. 247: 242-246; tic agent (TA), linked to a substrate for the targeted shA 50 see also, e.g., Shimizu et al. (1994).J. Virol. 68: 8406-8408; SEGP, such that the drug or agent is inactive or unable to enter Mizutani et al. (1996).J. Virol. 70: 7219-7223, Mizutani et al. a cell, in the conjugate, but is activated upon cleavage. The (1996) Biochem. Biophys. Res. Commun. 227: 822-826, Luet prodrug, for example, can contain anchondroitin Sulfate mol al. (1996) Proc. Natl. Acad. Sci. (USA)93: 1412-1417, Hahm ecule, typically a relatively short, less than about 20 disac et al. (1996) Virology 226: 318-326, Ito et al. (1996).J. Gen. charide units, that is catalytically cleaved by the targeted 55 Virol. 77: 1043-1054, Mizutani et al. (1995) Biochem. Bio sHASEGP. Cytotoxic agents, include, but are not limited to, phys. Res. Commun. 212: 906-911, Cho et al. (1997).J. Virol. alkylating agents, antiproliferative agents and tubulin binding Meth. 65: 201-207 and then extrapolated therefrom for dos agents. Others include, Vinca drugs, mitomycins, bleomycins ages for humans. and taxanes. Typically a therapeutically effective dosage is contem J. Pharmaceutical Compositions and Modes of Adminis 60 plated. The amounts administered can be on the order of tration 0.001 to 1 mg/ml, including about 0.005-0.05 mg/ml and 1. Components of the compositions. Pharmaceutical com about 0.01 mg/ml, of blood volume. Pharmaceutical dosage positions containing an active shASEGP are provided herein. unit forms are prepared to provide from about 1 mg to about Also provided are combinations of compounds that modulate 1000 mg, including from about 10 to about 500 mg, and the activity of a shASEGP polypeptide and another treatment 65 including about 25-75 mg of the essential active ingredient or or compound for treatment of a hyaluronidase disorder. Such a combination of essential ingredients per dosage unit form. as an antibody compound. The precise dosage can be empirically determined. US 7,871,607 B2 71 72 In some instances, a high Unit dose of human shASEGP is and can be determined empirically using known testing pro preferable. For example, with intravenous administration of tocols or by extrapolation from in vivo or in vitro test data. It sHASEGP concentrations of SHASEGP from 500-100,000 is to be noted that concentrations and dosage values can also Units per ml are preferable. Lyophilized formulations of vary with the severity of the condition to be alleviated. It is to sHASEGP are also ideal for storage of large Unit doses of 5 be further understood that for any particular subject, specific sHASEGP 200,000 Unit lyophilized vials of shASEGP are dosage regimens should be adjusted over time according to contemplated for intravenous delivery. the individual need and the professional judgment of the High concentration doses are also contemplated for the person administering or Supervising the administration of the delivery of small volumes of shASEGP. Administration of compositions, and that the concentration ranges set forth 10-100 ul of 5000 Units/ml shASEGP is contemplated for 10 herein are exemplary only and are not intended to limit the injection in the anterior chamber to dissolve preadministered Scope or use of the claimed compositions and combinations Viscoelastic Substances during cataract and phakic intraocular containing them. lens implantation Surgeries. Small Volume injections of Pharmaceutically acceptable derivatives include acids, 50-200 U/ml doses are also contemplated for intravitreal salts, esters, hydrates, Solvates and prodrug forms. The procedures such as the treatment of vitreous hemorrhage or 15 derivative is typically selected Such that its pharmacokinetic vitro-retinal detachment in diabetic retinopathy. properties are Superior to the corresponding neutral SHA Somewhat larger Volume injections (e.g. between about SEGP or soluble human hyaluronidase domain thereof. one to several ml, or more) are contemplated for somewhat Thus, effective concentrations or amounts of one or more larger and/or more readily drained spaces within the body. For of the polypeptides herein or pharmaceutically acceptable example, as illustrated herein, periorbital injections of one to derivatives thereof are mixed with a suitable pharmaceutical several ml or more can be made into one or more spaces carrier or vehicle for systemic, topical or local administration Surrounding the eye, e.g. by injection into the Sub-Tenon's or to form pharmaceutical compositions. SHASEGP polypep episcleral space, in order to promote the transcleral delivery tides or soluble human hyaluronidase domains thereof are of pharmacologic agents to tissues at the back of the eye Such included in an amount effective for ameliorating or treating as the choroid plexus and/or retina. As will be appreciated by 25 the disorder for which treatment is contemplated. The con those of skill in the art, some spaces within the body can centration of active polypeptide in the composition depends readily accommodate larger Volumes. By way of illustration, on absorption, inactivation, excretion rates of the active the episcleral or Sub-Tenon's space around the eye can readily polypeptide, the dosage schedule, amount administered, par accommodate one to several ml or even larger Volumes (e.g. ticular formulation as well as other factors known to those of up to or greater than 5 to 10 ml, depending for example on the 30 skill in the art. characteristics of the particular tissue and the rate at which the The therapeutic agents for use in the methods can be volume is delivered). administered by any route known to those of skill in the art, In addition, as described and illustrated herein, it appears Such as, but are not limited to, topically, intraarticularly, intra that the introduction of a shASEGP (potentially in combina cisternally, intraocularly, peri-orbitally, intraventricularly, tion with one or more additional pharmacologic or other 35 intrathecally, intravenously, intraarterially, intramuscularly, agents) in a larger Volume can be used to further drive delivery intraperitoneally, intradermally, Subcutaneously, intratrache of agents to desired target sites within the body. Without being ally, as well as by any combination of any two or more restricted to a particular mechanism of action, it is believed thereof. Dry powder pulmonary formulations can be envi that such larger Volume injections can further drive perme Sioned as well. ation or spreading of both shASEGP as well as other agent(s) 40 The most suitable route for administration will vary present in the treated tissue by a process of convective trans depending upon the proposed use. Such as, for example, use port that is promoted by an increased pressure differential. as a delivery agent to facilitate subcutaneous delivery of flu Again, without wishing to be restricted to a particular mecha ids, use to reduce intraocular pressure in the eyes of glaucoma nism of action, it is believed that Such an increased pressure patients receiving Viscoelastics or use as a “spreading agent' differential can be brought about in the context of the present 45 to enhance the activity of chemotherapeutics, and the location invention by, inter alia, one or both of the following: (i) an of interest, Such as a particularinternal organ, a tumor growth, elevated driving pressure or elevated “pressure head (e.g. intraocular cavity and the epidermis. Modes of administration caused by increased hydrostatic pressure associated with an include, but are not limited to, topically, locally, intraarticu injection that largely fills or somewhat overfills or distends a larly, intracisternally, intraocularly, peri-orbitally, intraven space or site of injection); and (ii) a reduced downstream 50 tricularly, intrathecally, intravenously, intramuscularly, pressure or reduced “impedance' (e.g. caused by the opening intratracheally, intraperitoneally, intradermally, Subcutane of channels in the interstitial matrix associated with localized ously, and by a combination of any two or more thereof. For hyaluronidase activity and consequent degradation of rela example, for treatment of various cancers, such as squamous tively hydrated hyaluronan). Preferred hyaluronidases for use cell carcinoma, breast cancer, urinary bladder cancer and in this and other applications of the present invention (such as 55 gastrointestinal cancer, local administration, including facilitating the delivery pharmacologic and other agents) are administration to the site of the tumor growth (e.g., intrathe sHASEGPs, particularly human shASEGPs in the context of cally, intraventricularly, or intracisternally) provides the delivery of agents to one or more tissues in the human body. advantage that the therapeutic agent can be administered in a However, these and other approaches to the use and applica high concentration without risk of the complications that can tion of hyaluronidases can also be applied using non-human 60 accompany systemic administration of a therapeutic agent. sHASEGPs, such as mammalian sEASEGPs, as well other As an exemplary class of pharmacologic agents that can be human or mammalian hyaluronidases, preferably soluble combined with shASEGPs (or other glycosaminoglyca forms of Such hyaluronidases. nases) (e.g. combined by co-formulation with one or more The active ingredient can be administered at once, or can be sHASEGPs or by co-administration with one or more shA divided into a number of smaller doses to be administered at 65 SEGPs (which may be administered prior to, coincident with intervals of time. It is understood that the precise dosage and or following administration of the agent)), a number of agents duration of treatment is a function of the disease being treated having effectiveness or potential effectiveness in the treat US 7,871,607 B2 73 74 ment of various cancers have been described in the art, and VERLUMATM); Oprelvekins (e.g. NEUMEGATM); Oxalipl.- novel anti-cancer agents are regularly being developed which atins (e.g. ELOXATINTM); Paclitaxels (e.g. PAXENETM, may likewise be combined with one or more shASEGPs (e.g. TAXOLTM); Pamidronates (e.g. AREDIATM); Pegademases provided by co-formulation or by co-administration before, (e.g. ADAGENTM); Pegaspargases (e.g. ONCASPARTM); coincident with and/or following administration of the agent) 5 Pegfilgrastims (e.g. NEULASTATM); Pentostatins (e.g. to improve the pharmacokinetics, pharacodynamics or other NIPENTTM); Pipobromans (e.g. VERCYTETM); Plicamycin/ useful properties of the agent. Mithramycins (e.g. MITHRACINTM); Porfimer sodiums (e.g. By way of illustration (and without limitation) a number of PHOTOFRINTM); Procarbazines (e.g. MATULANETM); anti-cancer or chemotherapeutic agents have now been Quinacrines (e.g. ATABRINETM); Rasburicases (e.g. approved for the treatment of various cancers which can be 10 ELITEKTM); Rituximabs (e.g. RITUXANTM); Sargra combined with shASEGPs (or other glycosaminoglyca mostims (e.g. PROKINETM); Streptozocins (e.g. nases) by co-formulation or co-administration (before, coin ZANOSARTM); Talcs (e.g. SCLEROSOLTM); Tamoxifens cident with or after administration of the anti-cancer agent). (e.g. NOLVADEXTM); Temozolomides (e.g. TEMODARTM); Such agents include, for example, Aldesleukins (e.g. PRO Teniposides/VM-26s (e.g. VUMONTM); Testolactones (e.g. LEUKINTM); Alemtuzumabs (e.g. CAMPATHTM); Alitretin 15 TESLACTM); Thioguanines incl. 6-TG: Thiotepas (e.g. THI oins (e.g. PANRETINTM); Allopurinols (e.g. OPLEXTM); Topotecans (e.g. HYCAMTINTM); Toremifenes ZYLOPRIMTM); Altretamines (e.g. HEXALENTM); Amifos (e.g. FARESTONTM); Tositumomabs (e.g. BEXXARTM); tines (e.g. ETHYOLTM); Anastrozoles (e.g. ARIMIDEXTM); Trastuzumabs (e.g. HERCEPTINTM); Tretinoins/ATRA (e.g. Arsenic Trioxides (e.g. TRISENOXTM); Asparaginases (e.g. VESANOIDTM); Uracil Mustards; Valrubicins (e.g. VAIL ELSPARTM); BCG Live (e.g. TICETM BCG); Bexarotenes STARTM); Vinblastines (e.g. VELBANTM); Vincristines (e.g. (e.g. TARGRETINTM); Bleomycins (e.g. BLENOXANETM); ONCOVINTM); Vinorelbines (e.g. NAVELBINETM); Zoledr Busulfan intravenous (e.g. BUSULFEXTM); Busulfan orals onates (e.g. ZOMETATM); as well as other agents described (e.g. MYLERANTM); Calusterones (e.g. METHOSARBTM); herein and in the art. Capecitabines (e.g. XELODATM); Carboplatins (e.g. PARA As with other classes of pharmacologic agents described PLATINTM); Carmustines (e.g. BCNUTM, BiCNUTM); Car 25 herein, a SEIASEGP (or other glycosaminoglycanase) may be mustines with Polifeprosans (e.g. GLIADELTM Wafer); Cele used in conjunction with the administration of (or co-formu coxibs (e.g. CELEBREXTM); Chlorambucils (e.g. lated with) an individual pharmacologic agent (Such as an LEUKERANTM); Cisplatins (e.g. PLATINOLTM); Cladrib antineoplastic for purposes of illustration, or with a member ines (e.g. LEUSTATINTM, 2-CdATM): Cyclophosphamides of another class of agents as described herein and/or known in (e.g. CYTOXANTM, NEOSARTM); Cytarabines (e.g. 30 the art). Alternatively, a shASEGP (or other glycosaminogly CYTOSAR-UTM); Cytarabine liposomals (e.g. DepoCytTM); canase) may be used in conjunction with the administration of Dacarbazines (e.g. DTIC-DomeTM); Dactinomycins (e.g. (or co-formulated with) more than one pharmacologic agent COSMEGENTM); Darbepoetin Alfas (e.g. ARANESPTM); of a given class (such as multiple antineoplastic agents for Daunorubicin liposomals (e.g. DANUOXOMETM); Dauno purposes of illustration, or multiple members of other classes rubicins/Daunomycins (e.g. CERUBIDINETM); Denileukin 35 of agents as described herein and/or known in the art). In the Diftitoxes (e.g. ONTAKTM); Dexrazoxanes (e.g. ZIN case of combinations with two or more agents of a given class, ECARDTM); Docetaxels (e.g. TAXOTERETM); Doxorubicins it is often preferred that the two or more agents act by two or (e.g. ADRIAMYCINTM, RUBEXTM); Doxorubicin liposo more different mechanisms of action. A shASEGP (or other mals (e.g. DOXILTM); Dromostanolone propionates (e.g. glycosaminoglycanase) may also be used in conjunction with DROMOSTANOLONETM and MASTERONETM Injection); 40 the administration of (or co-formulated with) one pharmaco Elliott’s B Solutions (e.g. Elliott's B SolutionTM); Epirubicins logic agent of a first given class (such as an antineoplastic for (e.g. ELLENCETM); Epoetin alfas (e.g. EPOGENTM); Estra purposes of illustration, or a member of one of the other mustines (e.g. EMCYTTM); Etoposide phosphates (e.g. ETO classes of agents as described herein and/or known in the art), POPHOSTM); Etoposide VP-16s (e.g. VEPESIDTM); and one or more additional pharmacologic agents of one or Exemestanes (e.g. AROMASINTM); Filgrastims (e.g. 45 more different classes that are useful for treating any of a NEUPOGENTM); Floxuridines (e.g. FUDRTM); Fludarabines variety of conditions. While certain principles are thus (e.g. FLUDARATM); Fluorouracils incl. 5-FUs (e.g. ADRU referred to here in the case of anticancer agents for purposes CILTM); Fulvestrants (e.g. FASLODEXTM); Gemcitabines of illustration, these principles are also applicable to, for (e.g. GEMZARTM); Gemtuzumabs/Ozogamicins (e.g. example, anti-infectives and the numerous members of other MYLOTARGTM); Goserelin acetates (e.g. ZOLADEXTM); 50 classes of agents that are described and illustrated herein, as Hydroxyureas (e.g. HYDREATM); Ibritumomabs/Tiuxetans well as additional agents that are already known in the art or (e.g. ZEVALINTM); Idarubicins (e.g. IDAMYCINTM): Ifos are newly developed. famides (e.g. IFEXTM); Imatinib mesylates (e.g. In the case of any of the combinations and/or co-formula GLEEVECTM); Interferon alfa-2as (e.g. ROFERON-ATM); tions described herein (comprising at least one shASEGP (or Interferon alfa-2bs (e.g. INTRON ATM; Irinotecans (e.g. 55 other glycosaminoglycanase) and at least one other agent CAMPTOSARTM); Letrozoles (e.g. FEMARATM); Leucov (such as a pharmacologic agent, e.g., a member of one of the orins (e.g. WELLCOVORINTM, LEUCOVORINTM); classes of agents illustrated herein)), it is also contemplated Levamisoles (e.g. ERG AMISOLTM); Lomustines/CCNUs that Such combination and/or co-formulation can further (e.g. CeeBUTM); Meclorethamines/Nitrogen mustards (e.g. comprise one or more agents (such as various stabilizers, MUSTARGENTM); Megestrol acetates (e.g. MEGACETM); 60 excipients and the like) which do not themselves exert sub Melphalans/L-PAMs (e.g. ALKERANTM); Mercaptopurine stantial pharmacodynamic effects but which promote the use incl. 6-MPs (e.g. PURINETHOLTM); Mesnas (e.g. fulness of the combination and/or co-formulation (e.g. by MESNEXTM); Methotrexates: Methoxsalens (e.g. UVA stabilizing one or more of the components or an entire for DEXTM); Mitomycin Cs (e.g. MUTAMYCINTM, mulation, by helping to promote or target the activity of one or MITOZYTREXTM); Mitotanes (e.g. LYSODRENTM); 65 more of the agents or of the shASEGP, and/or by reducing Mitoxantrones (e.g. NOVANTRONETM); Nandrolone Phen toxicity or otherwise improving the safety profile of the com propionates (e.g. DURABOLIN-50TM); Nofetumomabs (e.g. bination or co-formulation. US 7,871,607 B2 75 76 As will be appreciated by those of skill in the art, new Etoposides; Etoprines; Exisulinds: Fadrozoles; Fazarabines: versions and formulations of these and other anti-cancer Fenretinides; Floxuridines; Fludarabines; Fluorouracils; Flu agents (as well as members of other classes of agents as oxymesterones; Flurocitabines; Fosquidones; Fostriecins; described herein) are regularly being developed that can like Fostriecins; Fotretamines; Galarubicins; Galocitabines; wise be combined with shASEGPs (or other glycosami- 5 Gemcitabines; Geroquinols; Gimatecans; Gimeracils; Glox noglycanases). Furthermore, since Such enzymes can be used azones: Glufosfamides; Hydroxyureas; Idarubicins: Ifosfa to improve pharmacokinetic and/or pharmacodynamic pro mides; Ilmofosines; Ilomastats; Imexons; ImproSulfans; files of a large variety of agents, and allow agents to be IndiSulams; Inproduones; Interferons; Interferon Alfas; Inter delivered by different (and potentially more targeted) routes, feron Betas; Interferon Gammas; Interleukin-2s and other for example, shASEGPs (or other glycosaminoglycanases) 10 Interleukins (including recombinant Interleukins); Intopli can be used to enhance the PK and/or PD profiles of a number cines; Iobenguanes 131-I; proplatins; Irinotecans; of agents previously considered to have sub-optimal profiles. IrSogladines; Ixabepilones; Ketotrexates; L-Alanosines; Lan Testing of agents in appropriate animal models with and reotides; Lapatinibs; Ledoxantrones; Letrozoles; Leupro without concomitant shASEGP can be used to assess the lides; Leuprorelins (Leuprorelides); Lexacalcitols; LiaroZo relative effectiveness and toxicity of particular agents. 15 les; Lobaplatins; Lometrexols; Lomustines; Lonafamibs; As illustrated and exemplified herein, a number of large LoSoxantrones; Lurtotecans; Mafosfamides; Mannosulfans; molecule agents (such as antibodies and other proteins, par Marimastats: Masoprocols; Maytansines: Mechlore ticularly those administered by non-IV parenteral injection), thamines; Megestrols; Melengestrols; Melphalans; Menog exhibit relatively poor pharmacokinetic profiles and can be arils; Mepitiostanes; Mercaptopurines; Metesinds; Methotr particularly enhanced by co-formulation or co-administra- 20 eXates; Metomidates; Metoprines: Meturedepas; tion with shASEGPs (or other glycosaminoglycanases). Milboplatins; Miproxifenes; Misonidazoles; Mitindomides: Without being restricted to a particular mechanism of action, Mitocarcins; Mitocromins; Mitoflaxones; Mitogillins: it is generally considered that Small molecule agents exhibit Mitoguazones; Mitomalcins; Mitomycins; Mitonafides: ing relatively low hydrophobicity are more effectively dis Mitocquidones; Mitospers; Mitotanes; Mitoxantrones; Mito persable by, e.g., convective transport within interstitial fluids 25 Zolomides; Mivobulins; Mizoribines; Mofarotenes; Mopi as can be promoted by shASEGPs (or other glycosaminogly damols; Mubritinibs; Mycophenolic Acids; Nedaplatins: canases). A commonly used method for predicting Such Nelzarabines; Nemorubicins; Nitracrines; Nocodazoles; attributes is to assess the calculated and/or estimate octanol: Nogalamycins; Nolatrexeds; Nortopixantrones; Octreotides: water partition coefficients (K) which have been reported Ormaplatins; Ortataxels; Oteracils; Oxisurans; Oxophenars for a number of compounds. In that regard, agents exhibiting 30 ines; Paclitaxels; Pamidronates; Patubilones; Pegaspargases; log K of <3, more preferably <2 or <1, more preferably <0. Peldesines: Peliomycins; Pelitrexols: Pemetrexeds; Pentam still more preferably <(-1). As will be appreciated by those of ustines: Peplomycins: Perfosfamides; Perifosines: Picopl skill in the art, many agents can also be modified covalently atins; Pinafides; Pipobromans; Piposulfans; Pirfenidones: and/or non-covalently to increase their hydrophilicity, and/or Piroxantrones: Pixantrones; Plevitrexeds; Plicamycins: to encompass such agents within Vessicles or macromolecu- 35 Plomestanes; Plomestanes; Porfimers; Porfiromycins; Pred lar complexes to facilitate their permeation and/or delivery. nimustines; Procarbazines: Propamidines; Prospidiums; For purposes of further illustration (and without limita Pumitepas; Puromycins; Pyrazofurins; Ranimustines; Ribo tion), examplary anti-cancer or chemotherapeutic pharmaco prines; Ritrosulfans; Rogletimides; Roquinimex's; Rufocro logic agents that can be combined with shASEGPs (or other momycins; Sabarubicins; Safingols; Satraplatins; Sebripl glycosaminoglycanases) are included in the following list 40 atins; Semustines; SimtraZenes; Sizofirans; Sobuzoxanes; (and others are known in the art): Acivicins; Aclarubicins; Sorafenibs; Sparfosates; Sparfosic Acids; Sparsomycins; Acodazoles: Acronines; AdoZelesins; Aldesleukins; Alitreni Spirogermaniums; Spiromustines; Spiroplatins; Spiropl noins (9-Cis-Retinoic Acids); Altretamines; Alvocidibs: atins; Squalamines; Streptonigrins; Streptovarycins; Strepto Ambazones; Ambomycins; Ametantrones; Aminoglutethim Zocins; Sufosfamides; Sulofenurs; Tacedinalines; Talisomy ides; Amsacrines; AnastroZoles; Anaxirones; Ancitabines; 45 cins; Tallimustines; Tariquidars; Tauromustines; Tecogalans; Anthramycins; Apaziquones; Argimesnas; Asparaginases; Tegafurs; Teloxantrones; Temoporfins; Teniposides; TeroX Asperlins; Atrimustines; AZacitidines; AZetepas, Azotomy irones; Testolactones; Thiamiprines; Thioguanines; Thiote cins; Banoxantrones; Batabulins; Batimastats; Benaxibines; pas; Tiamiprines; Tiazofurins; Tilomisoles; Tilorones; Tim Bendamustines; BenZodepas, Bexarotenes; Bicalutamides; codars; Timonacics; Tirapazamines; Topixantrones; Bietaserpines: Biricodars; Bisantrenes; Bisantrenes; Bisna- 50 Toremifenes; Trabectedins (Ecteinascidin 743); Trastuzum fide Dimesylates; Bizelesins; Bleomycins; Bortezomibs: abs; Trestolones; Triciribines; Triciribines; Trilostanes; Tri Brequinars; Bropirimines; Budotitanes; Busulfans; Cactino metrexates; Triplatin Tetranitrates; Triptorelins; Trofosfa mycins; Calusterones; Canertinibs; Capecitabines; Carace mides; Tubulozoles: Ubenimexs; Uracil Mustards; Uredepas; mides; Carbetimers; Carboplatins; Carboquones; Carmofurs; Valspodars; Vapreotides; Verteporfins; Vinblastines; Vincris Carmustines; Carubicins; Carzelesins; Cedefingols; Cema- 55 tines; Vindesines; Vinepidines; Vinflunines; Vinformides: dotins; Chlorambucils; Cioteronels; Cirolemycins; Cispl Vinglycinates; Vinleucinols: Vinleurosines; Vinorelbines: atins; Cladribines; Clanfenurs; Clofarabines; Crisinatols; Vinrosidines; Vintriptols; Vinzolidines; Vorozoles: Xantho Cyclophosphamides; Cytarabines; Dacarbazines; Dactino mycin A's (Guamecyclines); Zeniplatins; Zilascorbs 2-H; mycins; Daunorubicins; Decitabines; Dexniguldipines; Dex Zinostatins; Zorubicins; ZoSuquidars; and the like. ormaplatins; DeZaguanines; Diaziquones; Dibrospidiums; 60 Pharmaceutical and cosmetic carriers or vehicles suitable Dienogests; Dinalins; Disermolides; Docetaxels; Dofequi for administration of the shASEGP polypeptides or soluble dars; Doxifluridines; Doxorubicins; Droloxifenes; Dromo human hyaluronidase domain thereof provided herein stanolone Propionates; DuaZomycins; Ecomustines; Edatr include any such carriers known to those skilled in the art to exates; Edotecarins; Eflornithines; Elacridars; Elinafides; be suitable for the particular mode of administration. In addi Elsamitrucins; Emitefurs; Enloplatins: Enpromates: Enzas- 65 tion, the polypeptides can be formulated as the sole pharma taurins; Epipropidines; Epirubicins; Eptaloprosts; Erbulozo ceutically active ingredient in the composition or can be com les; Esorubicins; Estramustines; Etanidazoles; Etoglucids; bined with other active ingredients that do not impair the US 7,871,607 B2 77 78 desired action, or with materials that supplement the desired intramuscular or intravenous routes for example), SHA action known to those of skill in the art. For example, the SEGPs (or other glycosaminoglycanases) can be readily co sHASEGP polypeptides provided herein can be used as a formulated with or co-administered with any of a varety of delivery or "spreading agent in combination with a second Such pharmacologic agents to enhance their delivery to active compound. Such as a therapeutically effective agent, desired sites within the body. For example, depending on the including, but not limited to a drug or a prodrug, to facilitate particular combination of pharmacologic agent and route of delivery of or to enhance the activity of the second active administration, delivery can be enhanced by increasing the ingredient. In a particular embodiment, a shASEGP polypep extent (and/or rate) at which the agent diffuses through a tide or a soluble human hyaluronidase domain thereof can be tissue at or near the site of delivery (e.g. following its injec co-formulated or co-administered with an anesthetic agent, 10 Such as Lignocaine, Bupivicaine or a mixture of the two, and, tion) and/or the extent (and/or rate) at which the agent dif optionally, a hormonal agent, such as epinephrine, to decrease fuses and/or is carried by convective transport through a tar or stop blood uptake during ophthalmic Surgery. In another get tissue (e.g. following its release from the bloodstream, particular embodiment, a shASEGP polypeptide or a soluble lymph, cerebrospinal fluid or other fluid system). The extent human hyaluronidase domain thereof can be co-formulated 15 by which delivery is enhanced can be readily evaluated, for or co-administered with a pharmacologic agent such as an example, in animal model systems in which the addition of a anti-inflammatory agent to enhance the delivery of the phar sHASEGP results in a measurable increase in the bioavail macologic to mammalian tissues in vivo (e.g. to portions of ability of the agent in tissues of interest within the body. the eye) where such agent is desired for treating a particular Without wishing to be bound by theory, it is believed that in condition. By co-administration is intended the administra the case of non-intravenous parenterals (such as Subcutane tion of shASEGP (or other glycosaminoglycanase) together ous, intradermal and intramuscular injectables), the presence with or in close temporal proximity to the administration of a of shASEGP (or other glycosaminoglycanase) in the vicinity pharmacologic agent. Typically it is preferred that the SHA of an injection site and in “downstream” sites such as those SEGP (or other glycosaminoglycanase) be administered between an injection site and a distal vascular bed, can shortly before (e.g. 5 to 60 minutes before) or, for maximal 25 enhance the diffusion and/or convective transport of the agent convenience, together with the pharmacologic agent. As will into a delivery system such as the vasculature. Enhanced be appreciated by those of skill in the art, the desired prox delivery in Such a situation can be associated with an increase imity of co-administration depends in significant part on the in the amount of the agent that effectively passes through and effective half lives of the agents in the particular tissue setting, out of the initial delivery site (potentially by both increasing and the particular disease being treated, and can be readily 30 the volume and rate of bulk fluid flow). optimized by testing the effects of administering the agents at In the case of many pharmacologic agents, which are Sub varying times in suitable models, such as in suitable animal ject to degradation, removal or other forms of inactivation in models. In some situations, the optimal timing of administra tissues, increasing the rate of its passage through a tissue can tion of the shASEGP (or other glycosaminoglycanase) will in turn Substantially increase the amount of effective agent exceed 60 minutes. By way of illustration and without wish 35 that transits through the tissue, further increasing its bioavail ing to be bound by theory, in some tumor situations for ability. For both non-intravenously-administered parenterals example the maximal entry of chemotherapeutic agent into that Subsequently enter the bloodstream or other conduit, as the tumor may occur after the degradation of hyaluronan in well agents delivered directly into the conduit vessels (such as the tumor is Substantially complete so as to reduce tumor by intravenous administration, IV), the ability of the agent to interstitial volume, which is followed by a hyaluronan-regen 40 diffuse through a tissue Supplied by the conduit can also be erative phase during which fluid is drawn back into the tumor enhanced by the presence of shASEGP in the tissue. Whether resulting in increased adsorption of a chemotherapeutic agent sHASEGP is present in the “delivery tissue' such as in the from plasma. In the latter case, optimal administration of former case, and/or in the “target tissue’ as in the latter case, sHASEGP (or other glycosaminoglycanase) may be obtained the presence of shASEGP can thus enhance the effective between about one to twenty hours prior to administration of 45 extent and/or rate of delivery of the pharmacologic to target the pharmacologic agent. In order to optimize the timing of tissues within the body. In either case, the shASEGP can be pre-administration relative to pharmacologic agent for a par used to increase the effective bioavailability of a pharmaco ticular combination of disease condition and pharmacologic logic agent with which it has been co-formulated or co-ad agent, one of skill can conduct a routine time course of pre ministered. As a result, the shASEGP can be used to achieve administration, typically comparing one or several time 50 elevated and/or more rapidly achieved concentrations of the points of an hour or less with one or several timepoints of agent to provide, for example, a more potent and/or more greater than hour (but typically not exceeding a day) in order rapid response for a given dose. Alternatively, the shASEGP to optimize the timing of administration. A shASEGP can be used to allow a given response to be achieved with a polypeptide or a soluble human hyaluronidase domain lower dose of administered agent, for example. The ability of thereof can also be co-formulated or co-administered with 55 a SFIASEGP (or other glycosaminoglycanase) to enhance various chemotherapeutics, such as a toxin and a tumor bulk fluid flow at and near a site of injection can also improve necrosis factor, to enhance the activity of the chemotherapeu other aspects of associated pharmacologic delivery. For tic and/or the accessibility of the target tumors to the chemo example, the increase in bulk fluid flow can help to allow the therapeutic. The active compound is included in the carrier in volume offluid injected to be more readily dispersed from the an amount sufficient to exert a therapeutically useful effect in 60 site of injection (reducing potentially painful or other adverse the absence of serious toxic effects on the treated individual. consequences of injection). Indeed, it has long been recog The effective concentration can be determined empirically by nized that Such adverse sensations associated with non-IV testing the compounds using in vitro and in Vivo systems, parenteral injections are a key reason that patients may resist including the animal models described herein. or avoid non-IV parenteral administration (which may effec In the case of pharmacologic agents that are delivered by 65 tively require a product to be administered intravenously parenteral administration (i.e. by routes other than through when it might otherwise be given more conveniently and/or the digestive tract, Such as by Subcutaneous, intradermal, safely through a non-IV route). US 7,871,607 B2 79 80 In using a SEIASEGP (or other glycosaminoglycanase) of tis A Vaccines (e.g. HAVRIXTM, TWINRIXTM and the present invention to enhance delivery of one or more VAQTATM), Hepatitis B Vaccines (e.g. recombinants COM pharmacologic or other agents in the body (a use that is VAXTM, ENGERIXTM-B, RECOMBIVAXTM-HB, TWIN sometimes referred to as “spreading'), the shASEGP (or RIXTM, and non-recombinants BAYHEPTM-B and NABITM other glycosaminoglycanase) itself may be administered by HB), Ibritumomab Tiuxetans (e.g. ZEVALINTM), the same route as is the pharmacologic agent (in which case it Immunoglobulins, Influenza Virus Vaccines (e.g. FLU may be co-formulated or otherwise co-administered together MISTM), Infliximabs (e.g. REMICADETM), Insulins (e.g. with the pharmacologic agent or it may be administered sepa HUMALOGTM, HUMALOGTM MIX 75/25TM, HUMU rately, preferably in advance of the pharmacologic agent as LINTM products (incl. 50/50, 70/30, Regular, NPH, Ultra and discussed above. Alternatively, or in addition, the shASEGP 10 Ultralente), and NOVOLINTM), Insulin Glargines (e.g. LAN (or other glycosaminoglycanase) may be administered by a TUSTM), Interferon Alfa-2a's (ROFERANTM-A), Interferon different route of administration. For example, the pharma Alfa-2b's (e.g. INTRON-ATM), Interferon Alfacon-1s (e.g. cologic agent may be introduced locally by non-IV parenteral INFERGENTM), Interferon Alfa-n3s (e.g. ALFERON NTM), injection and the shASEGP may be introduced intrave Interferon Betas (e.g. BETASERONTM And nously. As another illustrative and non-limiting example, the 15 BETAFERONTM), Interferon Beta-1a's (e.g. AVONEXTM sHASEGP may be introduced locally by non-IV parenteral And REBIFTM), Interferon Gammas (e.g. ACTIMMUNETM), injection (in a tissue that is desired to be targeted by the Iodixanols (e.g. VISIPAQUETM), Iohexols (OM pharmacologic agent) and the pharmacologic agent itselfmay NIPAQUETM), Iopamidols, loversols (e.g. OPTIRAYTM), be introduced by another route. Such as intravenous, non Ketorolacs (e.g. TOR ADOLTM), Laronidases (e.g. ALDU parenteral (e.g. oral) or other route. As will thus be appreci RAZYMETM), Levofloxacins (e.g. LEVAQUINTM), ated by those of skill in the art, shASEGPs of the present Lidocaines, Linezolids (e.g. ZYVOXTM), Lorazepams (e.g. invention can be used to enhance the delivery and bioavail ATIVANTM), Measles Vaccines (e.g. ATTENUVAXTM), ability of a variety of pharmacologic and other agents includ Measles-Mumps-Rubella Virus Vaccines (e.g. M-M-RTM II), ing agents that are typically delivered by parenteral as well as Medroxyprogesterones (e.g. DEPO-PROVERATM), Mero non-parental routes. 25 penems (e.g. MERREMTM IV), Methylprednisolones (e.g. For example, shASEGPs (or other glycosaminoglyca SOLU-MEDROLTM), Midazolams (e.g. VERSEDTM), Mor nases) can be co-formulated or co-administered with a variety phines (e.g. ASTRAMORPH/PFTM), Octreotides (e.g. SAN of pharmacologic agents that are typically administered by DOSTATINTM), Omalizumabs (e.g. XOLAIRTM), parenteral delivery, e.g. to enhance their effectiveness and/or Ondansetrons (e.g. ZOFRANTM), Palivizumabs (e.g. SYN to improve their risk/benefit profile in treating disease. 30 AGISTM), Pantoprazoles (e.g. PROTONIXTM), Pegaspar By way of illustration of the variety of pharmacologic gases (e.g. ONCASPARTM), Pegfilgrastims (e.g. NEU agents that can be parenterally-administered and their deliv LASTATM), Peg-Interferon Alfa-2a's (e.g. PEGASYSTM), ery and/or bioavailability enhanced through co-formulation Peg-Interferon Alfa-2b's (e.g. PEG-INTRONTM), Pegviso and/or co-administration with shASEGPs (or other gly mants (e.g. SOMAVERTTM), Pertussis vaccines, Piperacil cosaminoglycanases) of the present invention, the following 35 lins (e.g. ZOSYNTM), Pneumococcal Vaccines (e.g. PNEU list is not exhaustive (and is thus not intended to be limiting) MOVAXTM 23) and Pneumococcal Conjugate Vaccines (e.g. but simply to provide exemplary agents: Adalimumabs (e.g. PREVNARTM), Promethazines (e.g. PHENERGANTM), HUMIRATM), Agallsidase Betas (e.g. FABRAZYMETM), Reteplases (e.g. RETAVASETM). Somatropins (e.g. Aldesleukins (PROLEUKINTM IL-2), Alefacepts (e.g. GENOTROPINTM, HUMATROPETM, NORDITROPINTM, AMEVIVETM), Ampicillins (e.g. UNASYNTM Injection), 40 NUTROPINTM, SAIZENTM, SEROSTIMTM, ZORB Anakinras (e.g. KINERETM), Antipoliomyelitic Vaccines TIVETM), Sulbactams, Sumatriptans (e.g. IMITREXTM), (e.g. PEDIARIXTM), Anti-Thymocytes (e.g. THYMOGLO Tazobactams, Tenecteplases (e.g. TNKASETM). Tetanus BULINTM), Azithromycins (e.g. ZITHROMAXTM IV), Beca Purified Toxoids, Ticarcillins (e.g. TIMENTINTM), Tositu plermins (e.g. REGRANEXTM), Caspofungins (e.g. CANCI momabs (e.g. BEXXARTM), Triamcinolone Acetonides,Van DASTM), Cefazolins (e.g. ANCEFTM and CEFAZOLINTM), 45 comycins (e.g. VANCOCINTM). Varicella vaccines (e.g. Cefepimes (e.g. MAXIPIMETM), Cefotetans (e.g. CEFO VARIVAXTM) as well as other vaccines; and the like. TANTM), Ceftazidimes (e.g. FORTAZTM), Ceftriaxones (e.g. As described and illustrated herein, pharmacologic agents ROCEFINTM), Cetuximabs (e.g. ERBITUXTM), Cilastatins of a number of different classes and routes of administration (e.g. PRIMAXINTM IV), Clavulanic Acids (e.g. in conjunc can be benefited by co-administration and/or co-formulation tion with Amoxicillins such as in AUGMENTINTM), Clinda 50 with shASEGPs (or other glycosaminoglycanases) to mycins (e.g. CLEOCINTM), Darbepoetin Alfas (e.g. improve one or more of their aspects of use. Such improve ARANESPTM), Deaclizumabs (e.g. ZENAPAXTM), Diphthe ments can comprise, for example, one or any combination of ria Toxoids (typically in combinations e.g. DAPTACELTM, the following: (i) improving the extent and/or the rate of INFANRIXTM and PEDIARIXTM), Efalizumabs (e.g. RAP absorption and thus bioavailability of an agent (for example TIVATM), Epinephrines (e.g. EPIPENTM), Erythropoietin 55 by causing more of it to reach the bloodstream after non-IV Alphas (e.g. EPOGENTM And PROCRITTM), Etanercepts parenteral administration, and/or less of it to be degraded (e.g. ENBRELTM), Filgrastims (e.g. NEUPOGENTM), Flu after Such local administration by more rapid permeation), conazoles (e.g. DIFLUCANTM Injection). Follicle-Stimulat which can be brought about for example by accelerating ing Hormones such as Follitropin Alphas (e.g. GONAL-FTM) interstitial flow and potentially convective transport follow and Follitropin Betas (e.g. FOLLISTIMTM), Fosphenyloins 60 ing non-IV parenteral administration (e.g. by applying hydro (e.g. CEREBYXTM), Fluconazoles (e.g. DIFLUCANTM), static pressure associated with the Volume of injection com Gadodiamides (e.g. OMNISCANTM), Gadopentetates (e.g. bined with a reduction in impedance to flow associated with MAGNEVISTTM), Gatifloxacins (e.g. TEQUINTM), Glati degradation of hyaluronan); (ii) providing for a safer or more ramers (e.g. COPAXONETM). GM-CSF’s (e.g. LEUK convenient route of administration (for example allowing for INETM), Goserelins (e.g. ZOLADEXTM), Granisetrons (e.g. 65 non-IV parenteral administration in place of IV, or for KYTRILTM), Haemophilus Influenza B's (e.g. COMVAXTM improved or more convenient non-IV parenteral administra and HibTITERTM), Haloperidols (e.g. HALDOLTM), Hepati tions such as intradermal or Subcutaneous in place of intra US 7,871,607 B2 81 82 muscular); (iii) more favorable dosing regimens (for example that can provide for, e.g., timed or other triggered release of less frequent dosing, or a more frequent but lower dose non agents, as well as variably controlled dosing and the like. A IV route as opposed to a less frequent but higher dose IV); (iv) large number of Such pharmacologic agents (that can be co reducing a side effect or otherwise decreasing toxicity (for formulated with or used in combination with a shASEGP) example an effect associated with the initially high concen are regularly applied in medical practice and aspects of their tration (high Cmax) generated by IV dosing, or from a high use well described (see, e.g., the Physicians Desk Reference, local dose generated by slow and/or incomplete absorption Thomson 2003, 2004, 2005), and many other such agents are following non-IV parenteral administration); (v) otherwise known in the art. In the latter regard, as will be appreciated by improving the pharmacokinetics and/or pharmacodynamics those of skill in the art, shASEGPs can be employed in (for example by allowing more of an agent to reach a target 10 combination with agents currently used (to e.g. improve their site (e.g. by increased interstitial fluid flow and convective applicability as described herein), with agents that are not transport due to the presence of shASEGP at or near a deliv currently used (to e.g. improve their pharacokinetics and/or ery site such as an injection site), and/or by enhancing the pharmacodynamics and thus make them useful), or with permeation within a target site (e.g. by increased interstitial agents that are newly developed. fluid flow and convective transport due to the presence of 15 Thus for pharmacologic agents (such as anticancer agents, sHASEGP within the target site), or by delivering an agent blood modifers or members of other classes of agents as preferentially to a target site (e.g. by local administration to a described herein) that are typically introduced parenterally target site in connection with local or systemic administration (e.g. by IV injection), shASEGPs (or other glycosaminogly of a shASEGP to promote permeation of the target, or by canases) can be used for example to allow Such agents to be using vehicles or macromolecular complexes to preferen administered by other potentially more convenient and/or tially target an agent to a particular site combined with local safe routes Such as by non-IV parenteral adminstration (e.g. or systemic administration of shASEGP to promote perme by intramuscular, Subcutaneous, intradermal or transdermal ation of the target site); Agents that have been delivered orally routes). For agents that are already deliverable by a non-IV can also be benefited by reformulations for parenteral deliv parenteral route, shASEGPs may be used to enhance its ery to, e.g., avoid loss of agent to the gastrointestinal tract or 25 extent and/or rate of uptake and delivery (e.g. by promoting Subsequent degradation or toxicity, allow for more effective its dispersion and Subsequent entry into the bloodstream, targeting to a desired site of action and therefore potentially which can likely be aided by convective transport as greater effectiveness and/or less toxicity, or otherwise pro described herein). Advantages of such effects include having vide for more favorable pharmacokinetic and/or pharmaco a greater portion of the agent reach the bloodstream, having dynamic profiles. In addition, shASEGPs can be used to 30 reduced local exposure to agent (in terms of concentration improve the pharmacodynamics of agents that are adminis and/or time) (e.g. at or near the injection site, which often tered orally (for example, by enhancing the permeation of an limits dosing or results in local toxicity issues), increasing the orally administered agent within its desired target site (e.g. by speed at which an agent can be delivered into the blood increased interstitial fluid flow and convective transport due stream, other improved pharmacokinetic and/or pharmady to the presence of shASEGP within the target site following 35 namic properties, and convenience of administration. In the systemic administration of shASEGP and/or following local last regard, non-IV parenteral administrations can allow administration of shASEGP to the target site). many agents to be administered out-of-hospital that would Even in situations in which the desired target site for an otherwise require hospitalization or other medical attention agent such as pharmacologic is the bloodstream itself. Such typically required for intravenous administrations. agents can be enhanced by co-administration and/or co-for 40 Illustrative examples of blood modifiers include agents mulation with shASEGPs (or other glycosaminoglycanases). Such as antihemophilic agents and blood factor deficiency For example, there are a number of agents that act as blood agents (including, for example, antihemophilic factors (e.g. modifiers, which may be administered by one of several ADVATETM, HEMOFILTM, KOATETM-DVI, KOGE routes including oral dosing as well as parenteral administra NATETM-FS, RECOMBINATETM and REFACTOTM), anti tion (such as IV injection). A number of agents that are or 45 inhibitor coagulent complexes (e.g. FEIBATM VH), anti could be administered orally nevertheless exhibit relatively thrombin IIIs (e.g. THROMBATETM III), coagulations Factor limited or slow uptake and/or are subject to undesirable deg VIIs (e.g. NOVOSEVENTM), coagulation Factor VIIs (e.g. radation (and there are other agents that are generally safe and ADVATETM, KOGENATETM-FS and RECOMBINATETM), effective once introduced into the bloodstream but are not coagulation Factor IXs (e.g. BEBULINTM, BENEFIXTM and administered orally for that reason). Parenteral administra 50 PROPLEXTM T), plasma protein fractions (e.g. PLAS tion of Such agents (e.g. by intramuscular, Subcutaneous, MANATETM), von Willebrand factors)); antiplatelet agents intradermal or transdermal routes in combination with (including, for example, abciximabs (e.g. REOPROTM), administration of shASEGP preferably at or near the site of anagrelides (e.g. AGRYLINTM), cilostazols (e.g. PLE parenteral administration) can thus be employed to provide TALTM), clopidogrel bisulfates (e.g. PLAVIXTM), dipy for introduction into the bloodstream. In addition, there are a 55 ridamoles (e.g. AGGRENOXTM and PERSANTINETM), epo number of urgent medical situations in which rapid delivery prostenols (e.g. FLOLANTM), eptifibatides (e.g. of an agent to the bloodstream is essential but oral dosing is INTEGRILINTM), tirofibans (e.g. AGGRASTATTM)); colony too slow and intravenous dosing is either inconvenient, unsafe stimulating factors (CSFs) (including, for example, Granulo or unavailable. These urgent situations include those in which cyte CSFs (e.g. NEULASTATM and NEUPOGENTM) and the blood is the target as well as those in which the blood 60 Granulocyte Macrophage CSFs (e.g. LEUKINETM)); eryth stream is simply the route of delivery to another target tissue. ropoiesis stimulators (including, for example, erythropoi In such situations, the ability to combine administration with etins such as darbepoetin alfas (e.g. ARANESPTM) and epo a shASEGP (or apply a ready-prepared co-formulation with etin alfas (e.g. EPOGENTM and PROCRITTM); hemostatics a shASEGP) can allow for rapid delivery of the agent via and albumins (including, for example, aprotinins (e.g. TRA non-IV parenteral administration. As will also be appreciated 65 SYLOLTM), combinations of antihemophilic factors and by those of skill in the art, there are a number of formulations plasma (e.g. ADVATETM). Desmopressin Acetates (e.g. and devices designed for non-IV parenteral administrations DDAVPTM), and albumins (e.g. BUMINATETM and PLAS US 7,871,607 B2 83 84 BUMINTM)); immune globulins (e.g. BAYGAMTM, CAR dorelins; Gonadotropin chorionics; Haemophilus B polysac IMUNETM NF IV, FLEBOGAMMATM, GAMIMUNETM, carides; Haloperidols; Hemins; Hemophilus influenza vac GAMMAGARD S/DTM, GAMUNEXTM, IVEEGAMTM, cines; Hepatitis vaccines; Herbals. Histamines; IVEEGAMTM EN IGIV, MICRHOGAMTM, RHOGAMTM, Hydralazines; Hydrocortisones; Hydromorphones: PANGLOBULINTM and THYMOGLOBULINTM, as well as Hydroxocobalamins; Hydroxyzines; Hyoscyamines; Ibutil hepatitis B immune globulins (e.g. BAYHEPTM B, NABI ides; Idarubicins; Ifosfamides; Imiglucerases. Immunoglo HBTM, and NOVAPLUSTM NABI-HB); thrombin inhibitors bulins; Indigo carmines; Indomethacins; Interferon alfa (including for example direct thrombin inhibitors (e.g. conls; Interferon alfa-2As; Interferon alfa-N3s; Interferon ARGATROBANTM) and lepirudin (e.g. REFLUDANTM), and beta-1 AS. Iodides; Iopromides; Iothalamic acids; Ioxaglic drotecogin alfas (e.g. XIGRISTM); anticoagulants (including, 10 acids; Ioxilans; Iron dextran injections; Isoniazids; Isoprot for example, dalteparins, enoxaperins and other heparins (e.g. erenols; Japanese encephalitis vaccines; Kanamycins; Ket FRAGMINTM and LOVENOXTM), warfarins (e.g. COUMA amines; Ketorolacs; Labetalols; Lepirudins; Leucovorins; DINTM and JANTOVENTM)); and the like. Leuprolides; Levobupivacaines; Levothyroxines; By way of further illustration, examples of various types of Lidocaines; Lincomycins; Linezolids; Liothyronines; agents that have been administered paraneterally and which 15 Lorazepams; Luteinising hormones; Lyme disease vaccines; can be co-administered and/or co-formulated with sEIA Mangafodipirs; Mannitols; Measles virus; Mechlore SEGPs (or other glycosaminoglycanases) are included in the thamines; Melphalans; Meningococcal polysaccharide vac following list: Acetazolamides; Acyclovirs; Adipiodones; cines; Meperidines; Mepivacaines; Meropenems; Mesnas; Alatrofloxacins; Aldesleukins; Alemtuzumabs; Alfentanils; Mesoridazines; Metaraminols; Methadones; Methocarbam Allergenic extracts; Allopurinols; Alpha 1-proteinase inhibi ols; Methohexitals; Methotrexates; Methyldopates; Methyl tors: Alprostadils; Amifostines; Amikacins; Aminoacids; ergonovines; Metoclopramides; Metoprolols; Metronida Aminocaproic acids; Aminophyllines; Amitriptylines; Zoles; Midazolams; Minocyclines; Mithramycins: Amobarbitals; Amrinones; Anakinras; Analgesics; Anti-po Mitomycins; Mitoxantrones; Mivacuriums; Morrhuic acids: liomyelitic vaccines; Anti-rabic serums; Anti-tetanus immu Moxifloxacins; Mumps viruses: Muromonab-CD3s; Myco noglobulins; Antithrombin IIIs; Antivenom serums; Arga 25 phenolate mofetils: Nafcillins; Nalbuphines; Nalmefenes: trobans; Arginines; Arsenics; Ascorbic acids; Asparaginases; Naloxones; Nandrolones; Neostigmines; Niacinamides: Atenolols; Atracuriums; Atropines; Aurothioglucoses; AZa Nicardipines; Nitroglycerins; Nitroprussides; Norepineph thioprines: Azithromycins; Aztreonams; Bacillus Calmette rines; Oprelvekins; Orphenadrines; Oxacillins; Oxaliplatins: Guerin (BCG) vaccines: Bacitracins; Baclofens; Basilix Oxymorphones; Oxytetracyclines; Oxytocins; Pancuroni imabs; Benzoic acids; Benztropines; Betamethasones; 30 ums; Panthenols; Pantothenic acids; Papaverines; Pegaspar Biotins; Bivalirudins; Bleomycins: Botulism antitoxins: gases; Peginterferon alfa 2As; Penicillin Gs; Pentamidines: Bretyliums; Bumetanides; Bupivacaines; Buprenorphines: Pentazocines; Pentobarbitals; Pentostatins: Perflutrens; Per Busulfans; Butorphanols; Calcitonins; Calcitriols; Calciums; phenazines; Pertussis vaccines; Phenobarbitals; Phentola Capreomycins; Carboprosts; Carmustines; Carnitines; mines; Phenylephrines; Phenyloins; Physostigmines; Phy Caspofungins; Cefamandoles; Cefazolins; CefoperaZones; 35 tonadiones; Pneumococcal vaccines; Polymyxin bs; Cefotaximes; Cefotetans; Cefoxitins; Ceftazidimes; Cefti Porfimers; Pralidoximes; Prilocalnes; Procainamides; ZOXimes; Cefuroximes; Chloramphenicols; Chloroprocaines; Procaines; Prochlorperazines; Progesterones; Promethaz Chloroquines; Chlorothiazides; Chlorpromazines; Chon ines; Propranolols; Pyridostigmine hydroxides; Pyridoxines: droitinSulfuric acids; Choriogonadotropinalfas; Chromiums; Quinidines; Quinupristins; Rabies immunoglobulins; Rabies Cidofovirs; Cimetidines: Ciprofloxacins; Cisatracuriums; 40 vaccines; Ranitidines; Rasburicases; Remifentanils; Ribofla Cisplatins; Cladribines; Clavulanic acids; Clindamycins: Vins; Rifampins; Ropivacaines; Rubella Vaccines; Samari Clonidines; Codeines; Colchicines; Colistins; Collagens: ums; Scopolamines; Seleniums; Sermorelins. Sincalides; Corticorelin ovine triflutates; Corticotrophins; Cosyntropins; Somatrems; Spectinomycins; Streptokinases; Streptomy Cyanocobalamins; Cyclophosphamides; Cyclosporines; cins; Streptozocins; Succinylcholines; Sufentanils; Sulbac Cysteines; Cytarabines; Dacarbazines; Dacliximabs; Dacti 45 tams; Sulfamethoxazoles; Sumatriptans; Tacrolimuss; Teni nomycins; Dalfopristins; Dalteparins; Danaparoids; Dant posides; Terbutalines; Teriparatides; Testosterones; Tetanus rolenes; Daunorubicins; Deferoxamines; Denileukin difti antitoxins; Tetracaines; Tetradecyl sulfates: Theophyllines: toXS: Desmopressins; Dexamethasones; Dexmedetomidines; Thiamines; Thiethylperazines; Thiopentals; Thyroid stimu Dexpanthenols; DexraZoxanes; Dextrans; Diatrizoic acids; lating hormones; Ticarcillins; Tinzaparins; Tirofibans; Tobra Diazepams; Diazoxides; Dicyclomines; Digibinds; Digox 50 mycins; TolaZolines; Tolbutamides; Topotecans; Torsemides; ins; Dihydroergotamines; Diltiazems; Diphenhydramines; Tranexamic acids; Treprostinils; Triamcinolone acetonides; Diphteria, tetanus and pertussis vaccines; Diptheria antitox Triamcinolone hexacetonides; Triamcinolones; Triethyl ins; Dipyridamoles; Dobutamines; Dopamines; Doxacuri enethiophosphoramides (Thiotepa); Trifluoperazines; Tri ums; Doxaprams; Doxercalciferols; Doxycyclines; Droperi methobenzamides; Trimethoprims; Trimetrexates; Triptore dols; Drotrecogin alfas; Dyphyllines; Edetic acids: 55 lins; Tromethamines; Tuberculins; Typhoid vaccines: Edrophoniums; Enalaprilats; Ephedrines; Epoprostenols; Urofollitropins; Urokinases; Valproic acids; Valrubicins: Ergocalciferols: Ergonovines; Ertapenems; Erythromycins; Varicella Zoster immunoglobulins; Vasopressins; Vecuroni Esmolols; Estradiols; Estrogenics; Ethacrynic acids; Ethano ums; Verapamils; Vinblastines; Vincristines; Voriconazoles: lamines; Ethanols: Ethiodized oils: Etidronic acids; Etomi Warfarins; Yellow fever vaccines: Zidovudines; Zincs; dates; Etoposides; Factor VIIs; Famotidines: Fenoldopams: 60 Ziprasidone hydrochlorides; and agents related to the forego Fentanyls: Floxuridines: Fludarabines: Flumazenils: Fluo ing or in the same or similar classes as the foregoing. resceins: Fluorouracils: Fluphenazines; Folic acids; Follicle Solutions or Suspensions used for parenteral administra stimulating hormones; Fomepizoles; FomivirSens; Fonda tion (including by intramuscular, intradermal, Subcutaneous, parinuxS.; Foscamets; Fosphenyloins; Fulvestrants; topical application and other non-oral routes) can include any Furosemides; Gadoteridols; Gadoversetamides; Ganciclov 65 of the following components: a sterile diluent, such as water irs; Gatifloxacins; Gemtuzumab ozogamicins; Gentamicins; for injection, saline Solution, fixed oil, polyethylene glycol, Glucagons; Glucoses; Glycines; Glycopyrrolates; Gona glycerine, propylene glycol or other synthetic solvent; anti US 7,871,607 B2 85 86 microbial agents, such as benzyl alcohol and methyl para undesirable side effects are tolerated when treating life bens; antioxidants, such as ascorbic acid and Sodium bisulfite: threatening illnesses that would not be tolerated when treat cheating agents, such as ethylenediaminetetraacetic acid ing disorders of lesser consequence. Amounts effective for (EDTA); buffers, such as acetates, citrates and phosphates; therapeutic use will, of course, depend on the severity of the and agents for the adjustment of tonicity, including, but not disease and the weight and general state of the Subject as well limited to Sodium chloride, calcium chloride, magnesium as the route of administration. Local administration of the chloride, dextrose, glycerol or boric acid. Parenteral prepara therapeutic agent will typically require a smaller dosage than tions can be enclosed in ampoules, disposable syringes or any mode of systemic administration, although the local con single or multiple dose vials made of glass, plastic or other centration of the therapeutic agent can, in some cases, be suitable material. 10 higher following local administration than can be achieved The shASEGP polypeptides or soluble human hyalu with safety upon systemic administration. ronidase domains thereof can be suspended in micronized or Since individual Subjects can present a wide variation in other suitable form or can be derivatized to produce a more severity of symptoms and each therapeutic agent has its soluble active product or to produce a prodrug. The form of unique therapeutic characteristics, it is up to the practitioner the resulting mixture depends upon a number of factors, 15 to determine the response of a Subject to treatment and vary including the intended mode of administration and the Solu the dosages accordingly. Dosages used in vitro can provide bility of the polypeptide in the selected carrier or vehicle. The useful guidance in the amounts useful for in situ administra effective concentration is sufficient for ameliorating the tar tion of the pharmaceutical composition, and animal models geted condition and can be empirically determined using can in some cases be used to determine effective dosages for methods known to those of skill in the art. To formulate a treatment of particular disorders. In general, however, for composition, the weight fraction of polypeptide is dissolved, local administration, it is contemplated that an effective Suspended, dispersed, or otherwise mixed in a selected amount of the therapeutic agent will be an amount within the vehicle at an effective concentration such that the targeted range from about 0.1 picograms (pg) up to about 1 ng per kg condition is relieved or ameliorated. body weight. Various considerations in arriving at an effective In instances in which the shASEGP polypeptides or 25 amount are knownto those of skill in the art and are described soluble human hyaluronidase domain thereof exhibit insuffi (see, e.g., Goodman And Gilman's: The Pharmacological cient solubility, methods for solubilizing polypeptides can be Bases of Therapeutics, 8th ed., Pergamon Press, 1990; Rem used. Such methods are known to those of skill in this art, and ington's Pharmaceutical Sciences, 17th ed., Mack Publishing include, but are not limited to, using cosolvents, such as Co., Easton, Pa., 1990; and Mantyh et al., (Science, 278: dimethylsulfoxide (DMSO), using surfactants, such as 30 275-79, 1997) involving the intrathecal injection of a neu TWEENTM and PluronicTM F68; or dissolution in aqueous ronal specific ligand-toxin, each of which is herein incorpo sodium bicarbonate. Derivatives of the polypeptides, such as rated by reference in its entirety). prodrugs of the polypeptides can also be used in formulating The formulations of the shASEGP polypeptides or soluble effective pharmaceutical compositions. For ophthalmic indi human hyaluronidase domains thereof for use herein include cations, the compositions are formulated in an ophthalmi 35 those Suitable for oral, rectal, topical, inhalational, buccal, cally acceptable carrier. For the ophthalmic uses herein, local Sublingual, other parenteral (e.g., Subcutaneous, intramuscu administration, either by topical administration or by injec lar, intradermal, transdermal or intravenous), administration tion is contemplated. Time-release formulations are also or any route. The most Suitable route in any given case desirable. Typically, the compositions are formulated for depends on the nature and severity of the condition being single dosage administration, so that a single dose adminis 40 treated and on the nature of the particular active compound ters an effective amount. that is being used. The formulations are provided for admin Upon mixing or addition of the polypeptide with the istration to humans and animals in unit dosage forms, such as vehicle, the resulting mixture can be a solution, Suspension, tablets, capsules, pills, powders, granules, sterile parenteral emulsion or other composition and can be formulated as an Solutions or Suspensions, and oral Solutions or Suspensions, aqueous mixtures, a creams, gels, ointments, emulsions, Solu 45 and oil-water emulsions containing Suitable quantities of the tions, elixirs, lotions, Suspensions, tinctures, pastes, foams, polypeptides and/or other agents orpharmaceutically accept aerosols, irrigations, sprays, Suppositories, bandages, or any able derivatives thereof. The pharmaceutical therapeutically other formulation suitable for systemic, topical or local active polypeptides and/or other agents and derivatives administration. thereof are typically formulated and administered in unit The form of the resulting mixture depends upon a number 50 dosage forms or multiple-dosage forms. A unit-dose form as of factors, including the intended mode of administration and used herein refers to physically discrete units suitable for the solubility of the compound in the selected carrier or human and animal Subjects and packaged individually as is vehicle. If necessary, pharmaceutically acceptable salts or known in the art. other derivatives of the compounds are prepared. For local The pharmaceutical compositions are provided for admin internal administration, such as, intramuscular, parenteral or 55 istration to humans and animals in unit dosage forms, such as intra-articular administration, the compounds are preferably tablets, capsules, pills, powders, granules, sterile parenteral formulated as a solution Suspension in an aqueous-based Solutions or Suspensions, and oral Solutions or Suspensions, medium, Such as isotonically buffered saline or are combined and oil-water emulsions containing Suitable quantities of the with a biocompatible support or bioadhesive intended for sHASEGP polypeptide or soluble human hyaluronidase internal administration. 60 domain thereof and, optionally, another agent or pharmaceu The shASEGP polypeptide or soluble human hyalu tically acceptable derivatives thereof. The pharmaceutically ronidase domain thereof is included in the pharmaceutically therapeutically active compounds and derivatives thereof are acceptable carrier in an amount Sufficient to exert a therapeu typically formulated and administered in unit-dosage forms tically useful effect in the absence of undesirable side effects or multiple-dosage forms. A unit-dose form as used herein on the patient treated. It is understood that number and degree 65 refers to physically discrete units suitable for human and of side effects depends upon the condition for which the animal Subjects and packaged individually as is known in the compounds are administered. For example, certain toxic and art. Each unit-dose contains a predetermined quantity of the US 7,871,607 B2 87 88 therapeutically active compound Sufficient to produce the from non-toxic carrier can be prepared. For oral administra desired therapeutic effect, in association with the required tion, the pharmaceutical compositions can take the form of pharmaceutical carrier, vehicle or diluent. Examples of unit for example, tablets or capsules prepared by conventional dose forms include, but are not limited to, ampoules, Syringes means with pharmaceutically acceptable excipients such as and individually packaged tablets or capsules. For example, a binding agents (e.g., pregelatinized maize starch, polyvinyl Small Volume formulation containing a stabilized solution pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., with 1 to 5000 Units of shASEGP in a small volume, such as lactose, microcrystalline cellulose or calcium hydrogen phos 5 to 50 ul, can be prepackaged in a syringe for use. Such as phate); lubricants (e.g., magnesium Stearate, talc or silica); after viscoelastic injection. Unit-dose forms can be adminis disintegrants (e.g., potato starch or sodium starch glycolate); tered in fractions or multiples thereof. A multiple-dose form 10 or wetting agents (e.g., Sodium lauryl Sulphate). The tablets is a plurality of identical unit-dosage forms packaged in a can be coated by methods well known in the art. single container to be administered in segregated unit-dose The shASEGPs or a soluble human hyaluronidase domain form. Examples of multiple-dose forms include vials, bottles thereof or pharmaceutically acceptable derivatives can be of tablets or capsules or bottles of pints or gallons. Hence, prepared with carriers that protect the soluble glycoprotein multiple dose form is a multiple of unit-doses that are not 15 against rapid elimination from the body, such as time release segregated in packaging. formulations or coatings. The compositions can include other The composition can contain along with the active ingre pharmaceutically effective agents known in the general art to dient, such as a shASEGP polypeptide: a diluent, such as be of value in treating one or more of the diseases or medical lactose, Sucrose, dicalcium phosphate, or carboxymethylcel conditions, including, but not limited to, a chemotherapeutic lulose; a lubricant, such as magnesium Stearate, calcium agent, an analgesic agent, an anti-inflammatory agent, an Stearate and talc, and a binder Such as starch, natural gums, antimicrobial agent, an amoebicidal agent, a trichomonocidal Such as gum acaciagelatin, glucose, molasses, polyvinylpyr agent, an anti-parkinson agent, an anti-malarial agent, an rolidine, celluloses and derivatives thereof, povidone, anticonvulsant agent, an anti-depressant agent, and antiar crospovidones and other such binders known to those of skill thritics agent, an anti-fungal agent, an antihypertensive agent, in the art. Liquid pharmaceutically administrable composi 25 antipyretic agent, an anti-parasite agent, an antihistamine tions can, for example, be prepared by dissolving, dispersing, agent, an alpha-adrenargic agonist agent, an alpha blocker or otherwise mixing an active compound as defined above and agent, an anesthetic agent, a bronchi dilator agent, a biocide optional pharmaceutical adjuvants in a carrier, such as, for agent, a bactericide agent, a bacteriostat agent, a betadrener example, water, Saline, aqueous dextrose, glycerol, glycols, gic blocker agent, a calcium channel blocker agent, a cardio ethanol, and the like, to thereby form a solution or Suspension. 30 Vascular drug agent, a contraceptive agent, a decongestant If desired, the pharmaceutical composition to be adminis agent, a diuretic agent, a depressant agent, a diagnostic agent, tered can also contain minor amounts of nontoxic auxiliary a electrolyte agent, a hypnotic agent, a hormone agent, a Substances such as wetting agents, emulsifying agents, or hyperglycemic agent, a muscle relaxant agent, a muscle con solubilizing agents, pH buffering agents and the like, for tractant agent, an ophthalmic agent, a parasympathomimetic example, acetate, sodium citrate, cyclodextrine derivatives, 35 agent, a psychic energizer agent, ophthalmic agent, a para Sorbitan monolaurate, triethanolamine sodium acetate, tri sympathomimetic agent, a psychic energizeragent, a sedative ethanolamine oleate, and other Such agents. Methods of pre agent, a sympathomimetic agent, a tranquilizer agent, an uri paring Such dosage forms are known, or will be apparent, to nary agent, a vaginal agent, a viricide agent, a Vitamin agent, those skilled in this art (see e.g., Remington's Pharmaceutical a non-steroidal anti-inflammatory agent, an angiotensin con Sciences, Mack Publishing Company, Easton, Pa., 15th Edi 40 Verting enzyme inhibitor agent, a polypeptide, a protein, a tion, 1975). The composition or formulation to be adminis nucleic acid, a drug, a prodrug, a organic molecule and a sleep tered contains a quantity of the active compound in an amount inducer, to obtain desired combinations of properties. It is to sufficient to alleviate the symptoms of the treated subject. For be understood that Such combination therapy constitutes a example, a standard stabilized formulation of shASEGP or a further aspect of the compositions and methods of treatment soluble human hyaluronidase domain thereof as provided 45 provided herein. herein includes 150 Units/ml of the soluble glycoprotein for mulated in EDTA, NaCl and CaCl. Additionally, an anti 1. Compositions for Oral Administration bacterial or anti-fungal agent, including, but not limited to Oral pharmaceutical dosage forms are solid, gel or liquid. thiomersal, can be present in the formulation. Another for The Solid dosage forms are tablets, capsules, granules, and mulation provided herein is a stabilized solution or lyo 50 bulk powders. Types of oral tablets include compressed, philized form of shASEGP or a soluble human hyaluronidase chewable lozenges and tablets, which can be enteric-coated, domain thereof in EDTA, NaCl and CaCl, containing an Sugarcoated or film-coated. Capsules can be hard or soft effective active amount of the soluble glycoprotein, Such as gelatin capsules, while granules and powders can be provided 150 Unit/ml, with the addition of lactose, such as 13 mg/ml. in non-effervescent or effervescent form with the combina Also provided herein is a formulation containing a stabilized 55 tion of other ingredients known to those skilled in the art. solution or lyophilized form of shASEGP or a soluble human The pharmaceutical compositions containing a shASEGP hyaluronidase domain thereof in EDTA, NaCl and CaCl or a soluble human hyaluronidase domain thereof can be in containing an effective active amount of the soluble glyco liquid form, for example, solutions, syrups or Suspensions, or protein, such as 150 Unit/ml, with the addition of lactose, can be presented as a drug product for reconstitution with such as 13 mg/ml, and Albumin, Pluronic(R) F68, TWEENR) 60 water or other suitable vehicle before use. Such liquid prepa and/or other detergent. Another formulation provided herein, rations can be prepared by conventional means with pharma either lyophilized or as a stabilized solution, contains an ceutically acceptable additives such as Suspending agents effect amount of shASEGP or a soluble human hyalu (e.g., Sorbitol syrup, cellulose derivatives or hydrogenated ronidase domain thereof, such as 1 to 300 Units/ml, in EDTA, edible fats); emulsifying agents (e.g., lecithin or acacia); non NaCl and CaCl. 65 aqueous vehicles (e.g., almond oil, oily esters, or fractionated Dosage forms or compositions containing active ingredi Vegetable oils); and preservatives (e.g., methyl or propyl-p- ent in the range of 0.005% to 100% with the balance made up hydroxybenzoates or Sorbic acid). US 7,871,607 B2 89 90 In certain embodiments, the formulations are solid dosage able coating. Multiple compressed tablets are compressed forms, preferably capsules or tablets. The tablets, pills, cap tablets made by more than one compression cycle utilizing Sules, troches and the like can contain any of the following the pharmaceutically acceptable Substances previously men ingredients, or compounds of a similar nature: a binder; a tioned. Coloring agents can also be used in the above dosage diluent; a disintegrating agent; a lubricant; a glidant; a Sweet forms. Flavoring and Sweetening agents are used in com ening agent; and a flavoring agent. pressed tablets, Sugar-coated, multiple compressed and Examples of binders include microcrystalline cellulose, chewable tablets. Flavoring and Sweetening agents are espe gum tragacanth, glucose solution, acacia mucilage, gelatin cially useful in the formation of chewable tablets and loz Solution, Sucrose and starch paste. Lubricants include talc, engeS. starch, magnesium or calcium Stearate, lycopodium and 10 Liquid oral dosage forms include aqueous Solutions, emul Stearic acid. Diluents include, for example, lactose, Sucrose, sions, Suspensions, Solutions and/or Suspensions reconsti starch, kaolin, salt, mannitol and dicalcium phosphate. tuted from non-effervescent granules and effervescent prepa Glidants include, but are not limited to, colloidal silicon diox rations reconstituted from effervescent granules. Aqueous ide. Disintegrating agents include crosscarmellose Sodium, Solutions include, for example, elixirs and syrups. Emulsions Sodium starch glycolate, alginic acid, corn starch, potato 15 are either oil-in-water or water-in-oil. starch, bentonite, methylcellulose, agar and carboxymethyl Elixirs are clear, Sweetened, hydroalcoholic preparations. cellulose. Coloring agents include, for example, any of the Pharmaceutically acceptable carriers used in elixirs include approved certified water soluble FD and C dyes, mixtures Solvents. Syrups are concentrated aqueous Solutions of a thereof; and water insoluble FD and C dyes suspended on Sugar, for example, Sucrose, and can contain a preservative. alumina hydrate. Sweetening agents include Sucrose, lactose, An emulsion is a two-phase system in which one liquid is mannitol and artificial Sweetening agents such as Saccharin, dispersed in the form of small globules throughout another and any number of spray dried flavors. Flavoring agents liquid. Pharmaceutically acceptable carriers used in emul include natural flavors extracted from plants such as fruits and sions are non-aqueous liquids, emulsifying agents and pre synthetic blends of compounds which produce a pleasant servatives. Suspensions use pharmaceutically acceptable Sus sensation, Such as, but not limited to peppermint and methyl 25 pending agents and preservatives. Pharmaceutically salicylate. Wetting agents include propylene glycol acceptable Substances used in non-effervescent granules, to monostearate, Sorbitan monooleate, diethylene glycol mono be reconstituted into a liquid oral dosage form, include dilu laurate and polyoxyethylene laural ether. Emetic-coatings ents, Sweeteners and wetting agents. Pharmaceutically include fatty acids, fats, waxes, shellac, ammoniated shellac acceptable Substances used in effervescent granules, to be and cellulose acetate phthalates. Film coatings include 30 reconstituted into a liquid oral dosage form, include organic hydroxyethylcellulose, sodium carboxymethylcellulose, acids and a source of carbon dioxide. Coloring and flavoring polyethylene glycol 4000 and cellulose acetate phthalate. agents are used in all of the above dosage forms. Iforal administration is desired, the shASEGP or a soluble Solvents include glycerin, sorbitol, ethyl alcohol and human hyaluronidase domain thereof could be provided in a syrup. Examples of preservatives include glycerin, methyl composition that protects it from the acidic environment of 35 and propylparaben, benzoic add, Sodium benzoate and alco the stomach. For example, the composition can beformulated hol. Examples of non-aqueous liquids utilized in emulsions in an enteric coating that maintains its integrity in the stomach include mineral oil and cottonseed oil. Examples of emulsi and releases the active compound in the intestine. The com fying agents include gelatin, acacia, tragacanth, bentonite, position can also be formulated in combination with an ant and Surfactants such as polyoxyethylene Sorbitan acid or other Such ingredient. 40 monooleate. Suspending agents include Sodium carboxym When the dosage unit form is a capsule, it can contain, in ethylcellulose, pectin, tragacanth, Veegum and acacia. Dilu addition to material of the above type, a liquid carrier Such as ents include lactose and Sucrose. Sweetening agents include a fatty oil. In addition, dosage unit forms can contain various Sucrose, syrups, glycerin and artificial Sweetening agents other materials which modify the physical form of the dosage Such as saccharin. Wetting agents include propylene glycol unit, for example, coatings of Sugar and other enteric agents. 45 monostearate, Sorbitan monooleate, diethylene glycol mono The compounds can also be administered as a component of laurate and polyoxyethylene lauryl ether. Organic additives an elixir, Suspension, syrup, wafer, sprinkle, chewing gum or include citric and tartaric acid. Sources of carbon dioxide the like. A syrup can contain, in addition to the active com include Sodium bicarbonate and sodium carbonate. Coloring pounds, Sucrose as a Sweetening agent and certain preserva agents include any of the approved certified water soluble FD tives, dyes and colorings and flavors. 50 and C dyes, and mixtures thereof. Flavoring agents include The shASEGP or a soluble human hyaluronidase domain natural flavors extracted from plants such fruits, and synthetic thereof can also be mixed with other active materials which blends of compounds that produce a pleasant taste sensation. do not impair the desired action, or with materials that Supple For a solid dosage form, the solution or Suspension, in for ment the desired action, Such as antacids, H2 blockers, and example propylene carbonate, vegetable oils or triglycerides, diuretics. The active ingredient is a compound or pharmaceu 55 is encapsulated in a gelatin capsule. Such solutions, and the tically acceptable derivative thereof as described herein. preparation and encapsulation thereof, are disclosed in U.S. Higher concentrations, up to about 98% by weight of the Pat. Nos. 4.328,245; 4,409.239; and 4,410,545. For a liquid active ingredient can be included. dosage form, the Solution, e.g., for example, in a polyethylene Pharmaceutically acceptable carriers included in tablets glycol, can be diluted with a Sufficient quantity of a pharma are binders, lubricants, diluents, disintegrating agents, color 60 ceutically acceptable liquid carrier, e.g., water, to be easily ing agents, flavoring agents, and Wetting agents. Enteric measured for administration. coated tablets, because of the enteric-coating, resist the action Alternatively, liquid or semi-solid oral formulations can be of stomach acid and dissolve or disintegrate in the neutral or prepared by dissolving or dispersing the shASEGP or a alkaline intestines. Sugar-coated tablets are compressed tab soluble human hyaluronidase domain thereof in vegetable lets to which different layers of pharmaceutically acceptable 65 oils, glycols, triglycerides, propylene glycol esters (e.g., pro Substances are applied. Film-coated tablets are compressed pylene carbonate) and other Such carriers, and encapsulating tablets which have been coated with a polymer or other suit these solutions or Suspensions in hard or softgelatin capsule US 7,871,607 B2 91 92 shells. Other useful formulations include those set forth in Ikonium chloride and benzethonium chloride. Isotonic agents U.S. Pat. Nos. Re 28,819 and 4,358,603. include sodium chloride and dextrose. Buffers include phos Formulations suitable for buccal (sublingual) administra phate and citrate. Antioxidants include Sodium bisulfate. tion include, for example, lozenges containing the shASEGP Local anesthetics include procaine hydrochloride. Suspend or a soluble human hyaluronidase domain thereof in a fla ing and dispersing agents include Sodium carboxymethylcel Vored base, usually Sucrose and acacia or tragacanth; and luose, hydroxypropyl methylcellulose and polyvinylpyrroli pastilles containing the compound in an inert base Such as done. Emulsifying agents include Polysorbate 80 gelatin and glycerin or Sucrose and acacia. (TWEENTM 80). A sequestering or chelating agent of metal In all embodiments, tablets and capsules formulations can ions includes EDTA. Pharmaceutical carriers also include be coated as known by those of skill in the art in order to 10 ethyl alcohol, polyethylene glycol and propylene glycol for modify or Sustain dissolution of the active ingredient. Thus, water miscible vehicles and sodium hydroxide, hydrochloric for example, they can be coated with a conventional enteri acid, citric acid or lactic acid for pH adjustment. cally digestible coating, Such as phenylsalicylate, waxes and The concentration of the pharmaceutically active com cellulose acetate phthalate. pound is adjusted so that an injection provides an effective 2. Injectables, Solutions and Emulsions 15 amount to produce the desired pharmacological effect. The Parenteral administration of the shASEGP or a soluble exact dose depends on the age, weight and condition of the human hyaluronidase domain thereof, generally character patient or animal as is known in the art. ized by injection, either Subcutaneously, intramuscularly or The unit-dose parenteral preparations are packaged in an intravenously is also contemplated herein. Injectables can be ampoule, a vial or a syringe with a needle. All preparations for prepared in conventional forms, either as liquid solutions or parenteral administration must be sterile, as is known and Suspensions; Solid forms Suitable for solution or Suspension practiced in the art. in liquid prior to injection, or as emulsions. Suitable excipi Illustratively, intravenous or intraarterial infusion of a ster ents are, for example, water, Saline, dextrose, glycerol or ile aqueous solution containing an active compound is an ethanol. In addition, if desired, the pharmaceutical composi effective mode of administration. Another embodiment is a tions to be administered can also contain minor amounts of 25 sterile aqueous or oily solution or Suspension containing an non-toxic auxiliary Substances such as wetting or emulsifying active material injected as necessary to produce the desired agents, pH buffering agents, stabilizers, Solubility enhancers, pharmacological effect. and other such agents, such as, for example, Sodium acetate, Injectables are designed for local and systemic administra Sorbitan monolaurate, triethanolamine oleate and cyclodex tion. Typically a therapeutically effective dosage is formu trins. Implantation of a slow-release or Sustained-release sys 30 lated to contain a concentration of at least about 0.1% w/w up tem, Such that a constant level of dosage is maintained (see, to about 90% w/w or more, preferably more than 1% w/w of e.g., U.S. Pat. No. 3,710,795) is also contemplated herein. the active compound to the treated tissue(s). The active ingre The percentage of the shASEGP or a soluble human hyalu dient, such as a shASEGP or a soluble human hyaluronidase ronidase domain thereof contained in Such parenteral com domain thereof, can be administered at once, or can be positions is dependent on the specific nature thereof, as well 35 divided into a number of smaller doses to be administered at as the activity of the compound and the needs of the subject. intervals of time. It is understood that the precise dosage and Parenteral administration of the compositions includes duration of treatment is a function of the tissue being treated intravenous, Subcutaneous and intramuscular administra and can be determined empirically using known testing pro tions. Preparations for parenteral administration include ster tocols or by extrapolation from in vivo or in vitro test data. It ile solutions ready for injection, sterile dry soluble products, 40 is to be noted that concentrations and dosage values can also such as lyophilized powders, ready to be combined with a vary with the age of the individual treated. It is to be further Solvent or sterile solution just prior to use, including hypo understood that for any particular Subject, specific dosage dermic tablets, sterile Suspensions ready for injection, sterile regimens should be adjusted over time according to the indi dry insoluble products ready to be combined with a vehicle vidual need and the professional judgment of the person just prior to use and sterile emulsions. The Solutions can be 45 administering or Supervising the administration of the formu either aqueous or nonaqueous. lations, and that the concentration ranges set forth herein are If administered intravenously, suitable carriers include exemplary only and are not intended to limit the scope or physiological saline or phosphate buffered saline (PBS), and practice of the claimed formulations. Solutions containing thickening and solubilizing agents, such The compounds provided herein can be formulated for as glucose, polyethylene glycol, and polypropylene glycol 50 parenteral administration by injection, e.g., by bolus injection and mixtures thereof. or continuous infusion. Formulations for injection can be Pharmaceutically acceptable carriers used in parenteral presented in unit dosage form, e.g., in ampoules or in multi preparations include aqueous vehicles, nonaqueous vehicles, dose containers, with an added preservative. The composi antimicrobial agents, isotonic agents, buffers, antioxidants, tions can be suspensions, Solutions or emulsions in oily or local anesthetics, Suspending and dispersing agents, emulsi 55 aqueous vehicles, and can contain formulatory agents such as fying agents, sequestering orchelating agents and other phar Suspending, stabilizing and/or dispersing agents. Alterna maceutically acceptable Substances. tively, the active ingredient can be in powder form for recon Examples of aqueous vehicles include Sodium Chloride stitution with a suitable vehicle, e.g., Sterile pyrogen-free Injection, Ringers Injection, Isotonic Dextrose Injection, water or other solvents, before use. For example, provided Sterile Water Injection, Dextrose and Lactated Ringers Injec 60 herein are parenteral formulations containing an effective tion. Nonaqueous parenteral vehicles include fixed oils of amount of shASEGP or a soluble human hyaluronidase Vegetable origin, cottonseed oil, corn oil, Sesame oil and domain thereof, such as 500 to 500,000 Units, in a stabilized peanut oil. Antimicrobial agents in bacteriostatic or fungi solution or a lyophilized from. static concentrations must be added to parenteral preparations The compound can be suspended in micronized or other packaged in multiple-dose containers which include phenols 65 suitable form or can be derivatized to produce a more soluble or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl active product or to produce a prodrug. The form of the and propyl p-hydroxybenzoic acid esters, thiomersal, benza resulting mixture depends upon a number of factors, includ US 7,871,607 B2 93 94 ing the intended mode of administration and the solubility of aerosols, irrigations, sprays, Suppositories, bandages, dermal the compound in the selected carrier or vehicle. The effective patches or any other formulations Suitable for topical admin concentration is sufficient for ameliorating the symptoms of istration. the condition and can be empirically determined. The compositions of shASEGP or a soluble human hyalu 3. Lyophilized Powders ronidase domain thereof or pharmaceutically acceptable Also provided herein are lyophilized powders containing derivatives thereof can be formulated as aerosols for topical sHASEGP or a soluble human hyaluronidase domain thereof, application, Such as by inhalation (see, e.g., U.S. Pat. Nos. which can be reconstituted for administration as Solutions, 4,044,126, 4,414,209, and 4,364,923, which describe aero emulsions and other mixtures. These formulations can also be sols for delivery of a steroid useful for treatment inflamma reconstituted and formulated as Solids or gels. 10 tory diseases, particularly asthma). These formulations for The sterile, lyophilized powder is prepared by dissolving a administration to the respiratory tract can be in the form of an Solid portion of or mixing an aliquot of a solution containing aerosol or Solution for a nebulizer, or as a microfine powder a shASEGP or a soluble human hyaluronidase domain for insufflation, alone or in combination with an inert carrier thereof in a suitable solvent. The solvent can contain an Such as lactose. In such a case, the particles of the formulation excipient that improves the stability or other pharmacological 15 will typically have diameters of less than 50 microns, such as component of the powder or reconstituted Solution, prepared less than 10 microns. from the powder. Excipients that can be used include, but are For administration by inhalation, the compositions for use not limited to, dextrose, Sorbitol, fructose, corn syrup, Xylitol, herein can be delivered in the form of an aerosol spray pre glycerin, glucose, Sucrose, lactose or other Suitable agent. sentation from pressurized packs or a nebulizer, with the use The solvent can also contain a buffer. Such as citrate, sodium of a Suitable propellant, including, but not limited to, dichlo or potassium phosphate or other such buffer known to those of rodifluoromethane, trichlorofluoromethane, dichlorotet skill in the art at, typically, about neutral pH. Subsequent rafluoroethane, carbon dioxide and other Suitable gases. In sterile filtration of the solution followed by lyophilization the case of a pressurized aerosol, the dosage unit can be under standard conditions known to those of skill in the art determined by providing a valve to deliver a metered amount. provides the lyophilized formulation. Generally, the solution 25 Capsules and cartridges of, e.g., gelatin, for use in an inhaler resulting from the sterile filtration is apportioned into vials for or insufflator can be formulated containing a powder mix of lyophilization. Each vial can contain a single dosage, Such as the compound and a suitable powder base Such as lactose or 10-1000 mg or 100-500 mg. or multiple dosages of the com starch. pound. The compositions can be formulated for local or topical Briefly, the lyophilized powder is prepared by dissolving 30 application, Such as for topical application to the skin and dextrose, Sorbitol, fructose, corn syrup, Xylitol, glycerin, glu mucous membranes, such as in the eye, in the form of gels, cose, sucrose, lactose or other suitable agent, about 1-20%, in creams, and lotions and for application to the eye or for a Suitable buffer, such as citrate, Sodium or potassium phos intracisternal or intraspinal application. Topical administra phate or other such buffer known to those of skill in the art at tion is contemplated for transdermal delivery and also for about neutral pH. Then, a selected salt, Such as, for example, 35 administration to the eyes or mucosa, or for inhalation thera the sodium salt of the shASEGP (about 1 gm of the salt per pies. Nasal Solutions of the active compound alone or in 10-100 gms of the buffer solution, typically about 1 gm/30 combination with other pharmaceutically acceptable excipi gms), is added to the resulting mixture above room tempera ents can also be administered. ture, such as at about 30-35°C., and stirred until it dissolves. For example, formulations Suitable for topical application The resulting mixture is diluted by adding more buffer, to 40 to the skin or to the eye generally are formulated as an oint decrease the resulting concentration of the salt by about ment, cream, lotion, paste, gel, spray, aerosol and oil. Carriers 10-50%, typically about 15-25%). The resulting mixture is that can be used include Vaseline, lanoline, polyethylene gly sterile filtered or treated to remove particulates and to insure cols, alcohols, and combinations of two or more thereof. The sterility, and apportioned into vials for lyophilization. The topical formulations can further advantageously contain 0.05 lyophilized powder can be stored under appropriate condi 45 to 15 percent by weight of thickeners, including, but not tions, such as at about 4 C to room temperature. limited to, hydroxypropylmethylcellulose, methylcellulose, Reconstitution of this lyophilized powder with water for polyvinylpyrrolidone, polyvinyl alcohol, poly (alkylene gly injection provides a formulation for use in parenteral admin cols), poly/hydroxyalkyl, (meth) acrylates or poly (meth) istration. For reconstitution, a therapeutically effective acrylamides. A topical formulation is often applied by instil amount of the lyophilized powder containing a shASEGP or 50 lation orasanointment into the conjunctival sac. It also can be a soluble human hyaluronidase domain thereof is added per used for irrigation or lubrication of the eye, facial sinuses, and milliliter of sterile water or other suitable carrier. The precise external auditory meatus. The topical formulations in the amount depends upon the selected compound and can be liquid state can be also present in a hydrophilic three-dimen empirically determined by methods known to those of skill in sional polymer matrix in the form of a strip, contact lens, and the art. 55 the like from which the active components are released. It can 4. Topical Administration also be injected into the anterior eye chamber, into the epis Topical mixtures are prepared as described for the local and cleral space, and other places. For example, provided herein is systemic administration. Topical administration, which is a formulation for intraocular use after viscoelastic injecting Sometimes referred to in the art as percutaneous administra containing a stabilized solution of an effective amount of a tion, typically involves applications designed for absorption 60 sHASEGP or a soluble human hyaluronidase domain thereof, across skin, mucous membranes or other Surfaces of the body such as 1 to 5000 Units of the soluble glycoprotein with 30 to (e.g., applications applied directly onto the skin, under the 150,000 Units/mg of specific activity, in a small volume, such tongue (Sublingual), against the cheek (buccal), to the eyes, as 5 to 50 ul. As another example, provided herein is a for nose or ears, or to the lungs (inhalation)). The resulting mix mulation for intraocular use to enhance delivery of one or ture can be a solution, Suspension, emulsions or the like and 65 more pharmacologic agents to tissues within the eye follow are formulated as creams, gels, ointments, emulsions, Solu ing injection by routes of administration Such as a Sub-Ten tions, elixirs, lotions, Suspensions, tinctures, pastes, foams, on's injection into the episcleral space Surrounding the Sclera, US 7,871,607 B2 95 96 comprising a stabilized solution of an effective amount of a with carriers that protect the compound against rapid elimi sHASEGP or a soluble human hyaluronidase domain thereof, nation from the body, such as time-release formulations or such as 50 to 5,000 Units of the soluble glycoprotein with 30 coatings. to 150,000 Units/mg of specific activity, in a volume such as In one embodiment of the compositions and methods pro 0.05 to 10 ml. vided herein, the therapeutic agent is administered locally in These solutions, particularly those intended for ophthalmic a slow release delivery vehicle, for example, encapsulated in use, can be formulated as 0.01%-10% isotonic solutions, pH a colloidal dispersion system or in polymer Stabilized crys about 5-7, with appropriate salts. tals. Useful colloidal dispersion systems include nanocap Co-Applications with Iontophoresis Sules, microspheres, beads, and lipid-based systems, includ 10 ing oil-in-water emulsions, micelles, mixed micelles, and In the case of topical administrations and other applica liposomes. For example, the colloidal dispersion system can tions to tissues that are susceptible to iontophoresis, the use of be a liposome or microsphere. Liposomes are artificial mem one or more shASEGPs (potentially co-formulted or co brane vesicles that are useful as slow release delivery vehicles administered with another pharmacologic or other agent) can when injected or implanted. Some examples of lipid-polymer be combined with iontophoretic technqiues in order to further 15 conjugates and liposomes are disclosed in U.S. Pat. No. drive permeation of a tissue. In these regards, iontophoresis 5,631,018, which are incorporated herein by reference in its and shASEGPs can function cooperatively in that ionto entirety. Other examples of slow release delivery vehicles are phoresis can promote delivery of shASEGPs into a tissue or biodegradable hydrogel matrices (U.S. Pat. No. 5,041.292), other location within the body, and the shASEGP so deliv dendritic polymer conjugates (U.S. Pat. No. 5,714,166), and ered can further drive permeation of the enzyme, as well as multivesicular liposomes (DepofoamTM Depotech, San co-formulated or co-administered or other molecules present Diego, Calif.) (U.S. Pat. Nos. 5,723,147 and 5,766,627). One in the tissue, i.e. by enzymatically opening channels that type of microspheres Suitable for encapsulating therapeutic facilitate diffusion as well as promoting bulk fluid flow and agents for local injection (e.g., into Subdermal tissue) is poly thus convective transport of solutes such as pharmacologics. (D.L)lactide microspheres, as described in D. Fletcher, 5. Compositions for Other Routes of Administration 25 Anesth. Analg. 84:90-94, (1997). For example, a slow release Other routes of administration, Such as topical application, formulation containing an effective amount of shASEGP or transdermal patches, and rectal administration are also con a soluble human hyaluronidase domain thereof. Such as 1 to templated herein. 5000 Units/ml, can be employed for various uses or to treat For example, pharmaceutical dosage forms for rectal various conditions, including, but not limited to, cosmetic administration are rectal Suppositories, capsules and tablets 30 formulations, treatment of spinal cord injuries and other ner for systemic effect. Rectal Suppositories are used herein mean Vous system disorders, certain opthalmic disorders, inflam solid bodies for insertion into the rectum that melt or soften at matory or autoimmune conditions, cancers, and other chronic body temperature releasing one or more pharmacologically disease conditions, in which prolonged delivery of the shA or therapeutically active ingredients. Pharmaceutically SEGP is beneficial. acceptable Substances utilized in rectal Suppositories are 35 Desirable blood levels can be maintained by a continuous bases or vehicles and agents to raise the melting point. infusion of the active agent as ascertained by plasma levels. It Examples of bases include cocoa butter (theobroma oil), should be noted that the attending physician would know how glycerin-gelatin, carbowax (polyoxyethylene glycol) and to and when to terminate, interrupt or adjust therapy to lower appropriate mixtures of mono-, di- and triglycerides of fatty dosage due to toxicity, or bone marrow, liver or kidney dys acids. Combinations of the various bases can be used. Agents 40 functions. Conversely, the attending physician would also to raise the melting point of suppositories include spermaceti know how to and when to adjust treatment to higher levels if and wax. Rectal Suppositories can be prepared either by the the clinical response is not adequate (precluding toxic side compressed method or by molding. The typical weight of a effects). rectal Suppository is about 2 to 3 gm. The efficacy and/or toxicity of the shASEGP polypeptide 45 and/or its inhibitor (s), alone or in combination with other Tablets and capsules for rectal administration are manufac agents, such as therapeutically effective agents, also can be tured using the same pharmaceutically acceptable Substance assessed by the methods known in the art (see, e.g., O & Apos; and by the same methods as for formulations for oral admin Reilly, Investigational New Drugs 15: 5-13 (1997)). istration. 6. Articles of Manufacture Formulations suitable for transdermal administration can 50 The shASEGP polypeptides or soluble human hyalu be presented as discrete patches adapted to remain in intimate ronidase domains thereof or compositions containing any of contact with the epidermis of the recipient for a prolonged the preceding agents can be packaged as articles of manufac period of time. Such patches suitably contain the active com ture containing packaging material, a compound or Suitable pound as an optionally buffered aqueous solution of, for derivative thereof provided herein, which is effective for example, 0.1 to 0.2M concentration with respect to the active 55 treatment of a diseases or disorders contemplated herein, compound. Formulations suitable for transdermal adminis within the packaging material, and a label that indicates that tration can also be delivered by iontophoresis (see e.g., Phar the compound or a suitable derivative thereof is for treating maceutical Research 3 (6): 318 (1986)) and typically take the the diseases or disorders contemplated herein. The label can form of an optionally buffered aqueous solution of the active optionally include the disorders for which the therapy is war compound. 60 ranted. The pharmaceutical compositions can also be administered The articles of manufacture provided herein contain pack by controlled release means and/or delivery devices (see e.g., aging materials. Packaging materials for use in packaging in U.S. Pat. Nos. 3,536,809; 3,598,123; 3,630,200; 3,845, pharmaceutical products are well known to those of skill in 770; 3,847,770; 3,916,899;4,008,719; 4,687,610; 4,769,027; the art (see, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,059,595; 5,073,543; 5,120,548; 5,354,566; 5,591,767; 65 5,033.352). Examples of pharmaceutical packaging materials 5,639,476; 5,674,533 and 5,733,566). The active compounds include, but are not limited to, blister packs, bottles, tubes, or pharmaceutically acceptable derivatives can be prepared inhalers, pumps, bags, vials, containers, Syringes, bottles, and US 7,871,607 B2 97 98 any packaging material Suitable for a selected formulation bred to produce homozygous knockout animals, which can and intended mode of administration and treatment. A wide then be used to produce additional knockout animals. Knock array of formulations of the compounds and compositions out animals include, but are not limited to, mice, hamsters, provided herein are contemplated, as are variety treatments sheep, pigs, cattle, and other non-human mammals. For for any disorder in which HCV infection is implicated as a example, a knockout mouse is produced. The resulting ani mediator or contributor to the symptoms or cause. mals can serve as models of specific diseases, such as cancers, Kits containing the compositions and/or the combinations that exhibit under-expression of a shASEGP polypeptide. with instructions for administration thereofare also provided Such knockout animals can be used as animal models of Such herein. The kit can further include a needle or Syringe, typi diseases e.g., to screen for or test molecules for the ability to cally packaged in sterile form, for injecting the complex, 10 treat or prevent such diseases or disorders. and/or a packaged alcohol pad. Instructions are optionally Other types of transgenic animals also can be produced, included for administration of the active agent by a clinician including those that over-express the shASEGP polypeptide. or by the patient. For example, provided herein is a kit con Such animals include “knock-in animals that are animals in taining a small Volume Syringe with an effective amount of which the normal gene is replaced by a variant, such as a sHASEGP or a soluble human hyaluronidase domain thereof, 15 mutant, an over-expressed form, or other form. For example, such as 1 to 5000 Units of the soluble glycoprotein, in a 5 to one species. Such as a rodent’s endogenous gene can be 50 ul Volume, optionally containing a second Syringe contain replaced by the gene from another species, such as from a ing a viscoelastic. Also provided herein is a kit containing a human. Animals also can be produced by non-homologous Small Volume Syringe containing an effective amount of SHA recombination into other sites in a chromosome; including SEGP or a soluble human hyaluronidase domain thereof, animals that have a plurality of integration events. such as 1 to 500 Units of the soluble glycoprotein, and a After production of the first generation transgenic animal, therapeutic amount of a second active ingredient, such as a a chimeric animal can be bred to produce additional animals drug, a small molecule, a protein or a nucleic acid. with over-expressed or mis-expressed shASEGP polypep K. Animal Models tides. Such animals include, but are not limited to, mice, Transgenic animal models and animals, such as rodents, 25 hamsters, sheep, pigs, cattle and other non-human mammals. including mice and rats, cows, chickens, pigs, goats, sheep, The resulting animals can serve as models of specific dis monkeys, including gorillas, and other primates, are provided eases, such as cancers, that are exhibit over-expression or herein. In particular, transgenic non-human animals that con mis-expression of a shASEGP polypeptide. Such animals tain heterologous nucleic acid encoding a shASEGP can be used as animal models of Such diseases e.g., to Screen polypeptide or a transgenic animal in which expression of the 30 for or test molecules for the ability to treat or prevent such polypeptide has been altered. Such as by replacing or modi diseases or disorders. In a specific embodiment, amouse with fying the promoter region or other regulatory region of the over-expressed or mis-expressed sPASEGP polypeptide is endogenous gene are provided. Such an animal can by pro produced. duced by promoting recombination between endogenous The following examples are included for illustrative pur nucleic acid and an exogenous shASEGP gene that could be 35 poses only and are not intended to limit the scope of the over-expressed or mis-expressed. Such as by expression invention. under a strong promoter, via homologous or other recombi L. Therapeutic Uses of shASEGP nation event. Naturally occurring hyaluronidase enzymes from slaugh Transgenic animals can be produced by introducing the terhouses have been the principle source of clinical enzyme nucleic acid using any know method of delivery, including, 40 preparations for over forty years. Bovine and Ovine testicles but not limited to, microinjection, lipofection and other are the principle source of this material. These clinical modes of gene delivery into a germline cell or somatic cells, enzyme preparations however are very crude, sold in prepa Such as an embryonic stem cell. Typically the nucleic acid is rations ranging from 0.5-5% purity based upon known spe introduced into a cell. Such as an embryonic stem cell (ES), cific activities between 30-100,000 Units/mg. Thus their lack followed by injecting the ES cells into a blastocyst, and 45 of purity combined with their slaughterhouse origin, leave implanting the blastocyst into a foster mother, which is fol them as both immunogenic to humans and as potential source lowed by the birth of a transgenic animal. Generally intro of Jacob Creutzfeld disease and other bovine and ovine patho duction of a heterologous nucleic acid molecule into a chro gens. Anaphylactic reactions to bovine and Ovine hyalu mosome of the animal occurs by a recombination between the ronidase preparations are known to occur. heterologous SHASEGP-encoding nucleic acid and endog 50 Cattle or bacterially derived hyaluronidase have been used enous nucleic acid. The heterologous nucleic acid can be in the treatment of diseases associated with excess hyaluronic targeted to a specific chromosome. In some instances, knock acid and to enhance the circulation of physiological fluids out animals can be produced. Such an animal can be initially and/or therapeutic agents. For example, bovine hyalu produced by promoting homologous recombination between ronidase can be co injected with anesthesia in peribulbar, a shASEGP polypeptide gene in its chromosome and an 55 retrobulbar and sub-Tenon's blocks for ophthalmic surgical exogenous shASEGP polypeptide gene that has been ren procedures. Moreover, increased Surgical complications dered biologically inactive (typically by insertion of a heter occur in its absence (Brown S M et al. J. Cataract Refract ologous sequence, e.g., an antibiotic resistance gene). In one Surg. 1999 September; 25(9): 1245-9.). Bovine hyalu embodiment, this homologous recombination is performed ronidase is also used as an antidote to local necrosis from by transforming embryo-derived stem (ES) cells with a vector 60 paravenous injection of necrotic Substances such as Vinka containing the insertionally inactivated shASEGP polypep alkaloids (Few, B.J. (1987) Amer: J. Matern. Child Nurs. 12, tide gene. Such that homologous recombination occurs, fol 23-26). Bovine testes hyaluronidase is also useful for the lowed by injecting the ES cells into a blastocyst, and implant treatment of ganglion cysts (Paul et al. J Hand Surg 1997 ing the blastocyst into a foster mother, followed by the birth of April; 22 (2): 219-21). Hyaluronidase can also be used to the chimeric animal ("knockout animal') in which a shA 65 facilitate subcutaneous delivery of fluids in hypodermoclysis SEGP polypeptide gene has been inactivated (see Capecchi, (Berger EY. Am Geriatr Soc 1984 March; 32(3): 199-203). Science 244: 1288-1292 (1989)). The chimeric animal can be Hyaluronidase has also been utilized to reduce intraocular US 7,871,607 B2 99 100 pressure in the eyes of glaucoma patients and cataract patients Geller, 1999). In addition the expression of CSPG increases receiving viscoelastics (U.S. Pat. No. 4,820,516 issued Apr. following injury of CNS (Mckeon et al., 1991; Davies et al., 11, 1989). 1997). Cattle or bacterially derived hyaluronidases have also been Studies indicate that the inhibitory effects of CSPGs are used as a “spreading agent' to enhance the activity of chemo 5 principally due to the chondroitin Sulfate (CS) glycosami therapeutics and/or the accessibility of tumors to chemothera noglycan (GAG) sugar chain (Snow et al., 1990; Cole and peutics (Schuller et al., 1991, Proc. Amer. Assoc. Cancer Res. McCable, 1991; Geisert and Bidanset, 1993). This is sup 32:173, abstract no. 1034; Czeka et al., 1990, Pharmazie 45: ported by the finding that administration of bacterial chon H.9). Combination chemotherapy with hyaluronidase is droitinase in fact promote axon regeneration when adminis effective in the treatment of a variety of cancers including 10 tered intrathecally. Moreover, electrophysiological urinary bladder cancer (Hornet al., 1985, J. Surg. Oncol. 28: experiments determined that regenerated CST axons estab 304-307), squamous cell carcinoma (Kohno et al., 94, J. Can lished functional connections (Bradbury, et al 2002). In addi cer Res. Oncol. 120: 293-297), breast cancer (Beckenlehner tion to their direct inhibitory effects, CSPGs could also inter et al., 1992, J. Cancer Res. Oncol. 118: 591-596), and gas act with cell adhesion molecules or neurotrophic factors to trointestinal cancer (Scheithauer et al., 1988, Anticancer Res. 15 influence neurite outgrowth (Roberts et al., 1988: Ruoslahti 8:391-396). Hyaluronidase is effective as the sole therapeutic and Yamaguchi, 1991; Milev et al., 1994). Recombinant agent in the treatment of brain cancer (gliomas) (PCT pub mammalian Hyaluronidases are thus useful to reverse the lished application no. WO88/02261, published Apr. 7, 1988). inhibition of CSPG's in the glial scar and to promote axon Administration of hyaluronidase also induces responsiveness regeneration following injury. of previously chemotherapy-resistant tumors of the pancreas, The amount of shASEGP required to sufficiently degrade stomach, colon, ovaries, and breast (Baumgartneret al., 1988, CSPG's in the glial scar will vary. In some cases repeated Reg. Cancer Treat. 1:55-58; Zanker et al., 1986, Proc. Amer. administration of 10-5000 Units of shASEGP by intrathecal Assoc. Cancer Res. 27: 390). Unfortunately, the contami delivery will be required to remove the CSPG's in the scar. In nants and nonhuman nature of such hyaluronidases result in other cases, sustained release of shASEGP through use of a anaphylactic reactions. 25 slow release formulation may be preferred. Alternatively, In addition to its indirect anticancer effects, cattle derived administration of gene therapy vectors encoding shASEGP hyaluronidase has direct anticarcinogenic effects. Hyalu may be effective to enhance clearance of CSPG's. ronidase prevents growth of tumors transplanted into mice sHASEGPs can also be utilized for the treatment of herni (De Maeyer et al., 1992, Int. J. Cancer 51: 657-660) and ated disks in a process known as chemonucleolysis. Chon inhibits tumor formation upon exposure to carcinogens 30 droitinase ABC, and enzyme cleaving similar Substrates as (Pawlowski et al., 1979, Int. J. Cancer 23: 105-109; Haber sHASEGP can induce the reduction of intradiscal pressure in man et al., 1981, Proceedings of the 17th Annual Meeting of the lumbar spine. (Sasaki et al., 2001, Ishikawa et al., 1999). the American Society of Clinical Oncology, Washington, There are three types of disk injuries. A protruded disk is one D.C., 22: 105, abstract no. 415). that is intact but bulging. In an extruded disk, the fibrous 35 wrapper has torn and the NP has oozed out, but is still con Given the value of cattle-derived hyaluronidases as a thera nected to the disk. In a sequestered disk, a fragment of the NP peutic, particularly in chemotherapy in conjunction with con has broken loose from the disk and is free in the spinal canal. ventional chemotherapeutics or as a chemotherapeutic in and Chemonucleolysis is effective on protruded and extruded of itself, there is a need in the field for substantially pure disks, but not on sequestered disk injuries. In the United preparations of hyaluronidase of human origin. There is also 40 States, chemonucleolysis is approved only for use in the a need for efficient, cost-effective methods of making hyalu lumbar (lower) spine. In other countries, it has also been used ronidase to provide commercially significant quantities of the Successfully to treat cervical (upper spine) hernias. Chemo enzyme. The present invention addresses these problems. nucleolysis is thus a conservative alternative to disk Surgery Hyaluronic acid is an essential component of the extracel when it is preferable to reduce disk pressure. lular matrix. Hyaluronic acid is found in the connective tissue 45 The precise composition and structure of the carbohydrate of mammals and is the main constituent of the vitreous of the chain(s) on a glycoprotein can directly influence its serum eye. In connective tissue, the water of hydration associated lifetime, since cells in the liver and reticulo-endothelial sys with hyaluronic acid creates spaces between tissues, thus tem can bind and internalize circulating glycoproteins with creating an environment conducive to cell movement and specific carbohydrates. Hepatocytes have receptors on their proliferation. Hyaluronic acid plays a key role in biological 50 Surfaces that recognize oligosaccharide chains with terminal phenomena associated with cell motility including rapid (i.e., at the outermost end(s) of glycans relative to the development, regeneration, repair, embryogenesis, embryo polypeptide) Gal residues, macrophages contain receptors for logical development, wound healing, angiogenesis, and tum terminal Man or GlcNAc residues, and hepatocytes and lym origenesis (Toole, 1991, Cell Biol. Extracell. Matrix, Hay phocytes have receptors for exposed fucose residues. No (ed), Plenum Press, New York, 1384-1386; Bertrand et al., 55 sialic acid-specific receptors have been found, however. 1992, Int. J. Cancer 52: 1-6: Knudson et al., 1993, FASEB J. Although somewhat dependent on the spatial arrangement of 7: 1233-1241). In addition, hyaluronic acid levels correlate the oligosaccharides, as a general rule, the greater the number with tumor aggressiveness (Ozello et al., 1960, Cancer Res. of exposed Sugar residues recognized by cell Surface recep 20: 600-604; Takeuchi et al., 1976, Cancer Res. 36: 2133 tors in the liver and reticulo-endothelial system, the more 2139; Kimata et al., 1983, Cancer Res. 43: 1347-1354). 60 rapidly a glycoprotein will be cleared from the serum. Following spinal cord injury, glial scars are produced by Because of the absence ofsialic acid-specific receptors, how astrocytes and contain chondroitin Sulfate proteoglycans ever, oligosaccharides with all branches terminated, or (CSPGs). CSPGs play a crucial role in the inhibition of axon “capped with sialic acid will not promote the clearance of growth (Levine, 1994; Powell et al., 1997). For example, the protein to which they are attached. during fetal development, CSPGs repel axons and inhibit 65 The presence and nature of the oligosaccharide chain(s) on neural cell adhesion. CSPG's also play an important role in a glycoprotein can also affect important biochemical proper boundary formation (Snow et al., 1990, 1992; Powell and ties in addition to its recognition by Sugar-specific receptors US 7,871,607 B2 101 102 on liver and reticulo-endothelial cells. Removal of the carbo other defect associated with decreased hyaluronan catabo hydrate from a glycoprotein will usually decrease its solubil lism. SHASEGP is preferable for the treatment of malignan ity, and it may also increase its Susceptibility to proteolytic cies associated with deficient HA catabolism as it does not degradation by destabilizing the correct polypeptide folding require cellular participation for degradation to occur. patternand/or unmasking protease-sensitive sites. For similar 5 The specific dosage appropriate for administration can be reasons, the glycosylation status of a protein can affect its readily determined by one of ordinary skill in the art accord recognition by the immune system. ing to the factors discussed above (see, for example, Harri sHASEGPs can be used to remove the cumulus cells Sur son’s Principles of Internal Medicine, 11th Ed., 1987). In rounding an egg prior to cryopreservation and other In Vitro addition, the estimates for appropriate dosages in humans fertilization techniques such an intracytoplasmic sperminjec 10 may be extrapolated from determinations of the level of enzy tion (ICSI). Hyaluronidase can be added to harvested oocytes matic activity of shASEGP in vitro and/or dosages effective between 10-200 U/ml in buffered salt solutions. Oocytes are in animal studies. For example, 70-300 TRU hyaluronidase is separated from the released cumulus cells through aspiration effective in reducing the tumor load in a scid mouse. Given and washed through several washes with media lacking this information, the corresponding dosages in the average 70 hyaluronidase. The eggs can then be processed for cryo 15 kg human would range from about 250,000-1,200,000 TRU preservation or IVF techniques. hyaluronidase. The amount of shASEGP administered to a sHASEGPs are also useful for the more effective penetra human patient is generally in the range of 1 TRU to 5,000,000 tion of chemotherapeutic agents into Solid tumors. SHA TRU of enzymatic activity, preferably between about 1,000 SEGPs can be injected intratumorally with anti-cancer agents TRU to 2,500,000 TRU, more preferably between about 100, or intravenously for disseminated cancers or hard to reach 000 TRU to 1,500,000 TRU, normally between about 250, tumors. The anticancer agent can be a chemotherapeutic, an 000 TRU and 1,200,000 TRU, with about 725,000 TRU rep antibody, a peptide, or a gene therapy vector, virus or DNA. resenting average prescribed doses. Additionally, shASEGPs can be used to recruit tumor cells In one embodiment, a shASEGP is formulated in a 0.15 M into the cycling pool for sensitization in previously chemore saline solution containing shASEGP at a concentration of fractory tumors that have acquired multicultural drug resis 25 about 150,000 TRU/cc. The formulation is then injected tance St Croix etal Cancer Lett 1998 Sep. 11; 131(1): 35-44). intravenously at 15,000 TRU/kg body weight of the patient. sHASEGPs are also useful to enhance delivery of biologics Alternatively, the enzyme formulation may also be injected Such as monoclonal antibodies, cytokines and other drugs to Subcutaneously to allow the hyaluronidase to perfuse around tumors that accumulate glycosaminoglycans. Many tumors the tumor site. In a preferred embodiment, shASEGP is delete genes involved with the catabolism of glycosami 30 injected peritumorally or into the tumor mass. In another noglycans such that localized accumulation can prevent anti preferred embodiment, shASEGP is formulated as a lipo neoplastic agents and the immune system from reaching the some and is delivered by injection either intravenously orator tumor maSS. near the site of cancerous cells associated with a defect in the sHASEGP can also be used to increase the sensitivity of Luca-1 (hpHAse) gene. Injection of shASEGP intrave tumors that are resistant to conventional chemotherapy. In 35 nously results in shASEGP in the tumor site. Moreover, one embodiment, shASEGP is administered to a patient hav Super Sialated shASEGP is preferably for parenteral admin ing a tumor associated with a LuCa-1 defect in an amount istration in that the terminal sialic acids on shASEGP prevent effective to increase diffusion around the tumor site (e.g., to the clearance of the enzyme from circulation by the reticu increase circulation of chemotherapeutic factors (e.g., to loendothelial system. Comparisons of Super sialated SHA facilitate circulation and/or concentrations of chemothera 40 SEGP to non-sialated bovine and ovine hyaluronidases reveal peutic agents in and around the tumor site), inhibit tumor cell that Substantially more favorable pharmacokinetics is motility (e.g., by HA degradation) and/or to lower the tumor achieved. cell(s) threshold of apoptosis (i.e., bring the tumor cell(s) to a Facilitation of Gene Therapy state of anoikis), a state that renders the tumor cell(s) more The efficacy of most gene delivery vehicles in vivo does not Susceptible to the action of chemotherapeutic agents or other 45 correspond to the efficacy found in vitro. Glycosaminogly agents that may facilitate cell death, preferably preferentially cans can hinder the transfer and diffusion of DNA and viral facilitate programmed cell death of cells in anoikis. Chemo vectors into many cell types. The levels such extracellular therapeutic agents as used herein is meant to encompass all matrix material can hinder the process considerably. Duben molecules, synthetic (e.g., cisplatin) as well as naturally sky et al., (Proc Natl AcadSci USA 1984 December; 81 (23): occurring (e.g., tumor necrosis factor IF)), that facilitate inhi 50 7529-33) demonstrated that hyaluronidase when combined bition of tumor cell growth, and preferably facilitate, more with collagenase could facilitate transduction of DNA in preferably preferentially facilitate tumor cell death. vivo. It has been demonstrated that adeno associated virus is Of particular interest is the use of shASEGP for the treat also amenable to hyaluronidase mediated gene therapy Favre ment of metastatic and non-metastatic cancers, particularly et al. (Gene Ther 2000 August: 7(16): 1417-20). metastatic cancers, having decreased to undetectable hyalu 55 We have determined herein that channels of defined size in ronidase activity relative to non-cancerous (normal) cells. the extracellular matrix are opened with shASEGP. These sHASEGP can be used as a chemotherapeutic agent (alone or pores generally do not enhance the diffusion of Substances in combination with other chemotherapeutics) in the treat greater than about 200-500 nm in diameter. However, smaller ment of any of a variety of cancers, particularly invasive molecules such as retroviruses, adenoviruses, adeno-associ tumors. For example, shASEGP can be used in the treatment 60 ated viruses and DNA complexes are amenable to shASEGP of small lung cell carcinoma, squamous lung cell carcinoma, mediated diffusion. The fact that shASEGPs generally act to as well as cancers of the breast, ovaries, head and neck, or any open channels that are Substantially large enough to permit other cancer associated with depressed levels of hyalu the movement of most therapeutic and other pharmacological ronidase or with a defective Luca-1 (hpHAse) gene (e.g., a agents of interest (including macromolecules Such as proteins LuCa-1 gene that does not provide for expression of adequate 65 and nucleic acids as well as macromolecular complexes such hpHAse levels or encodes a defective hpHAse that does not as gene therapy vectors) and yet are generally not so large as provide for an adequate level of hyaluronidase activity) or to promote the dislocation and movement of cells, provides US 7,871,607 B2 103 104 particularly advantageous applications in treating, preventing which is based on Tris-HCl (10 mM Tris-HCl, pH 7.5 to 8, 1 and/or diagnosing a variety of diseases for example. mM EDTA; 10 mM Tris-HCl, pH 7.5 to 8, 1 mM MgCl), a Alternatively, viruses can be armed with the shASEGP phosphate buffer (Krebs phosphate HO buffer), a sugar (glu gene to facilitate their replication and spread within a target cose, Sucrose, trehalose, etc.) Solution, or simply water. tissue for example. The target tissue can be a cancerous tissue 5 Hypodermoclysis whereby the virus is capable of selective replication within Hypodermoclysis, the Subcutaneous infusion of fluids, is a the tumor. The virus can also be a non-lytic virus wherein the useful and easy hydration technique suitable for mildly to virus selectively replicates under a tissue specific promoter. moderately dehydrated adult patients, especially the elderly. As the viruses replicate, the coexpression of shASEGP with The method is considered safe and does not pose any serious viral genes will facilitate the spread of the virus in vivo. 10 complications. The most frequent adverse effect is mild sub Alternatively the nucleic acid of interest and a shASEGP, cutaneous edema that can be treated by local massage or can be used simultaneously or consecutively or so as to be systemic diuretics. Approximately 3 L can be given in a staggered over time. Simultaneously refers to a coadminis 24-hour period at two separate sites. Common infusion sites tration. In this case, these two essential components can be are the chest, abdomen, thighs and upper arms. The preferred mixed to form a composition prior to being administered, or 15 Solution is normal saline, but other solutions, such as half can be administered at the same time to the cell or the host normal saline, glucose with Saline or 5 percent glucose, can organism. It is also possible to administer them consecutively, also be used. Potassium chloride can be added to the solution that is to say one after the other, irrespective of which com bag if needed. Additionally, other drugs can be delivered ponent of the combination product according to the invention through similar routes. Human shASEGP can be added to is administered first. Finally, it is possible to use a mode of enhance fluid absorption and increase the overall rate of administration which is staggered over time or is intermittent administration. Human shASEGP is preferable for repeated and which stops and restarts at intervals which may or may Hypodermoclysis over slaughter house-derived enzymes in not be regular. It is pointed out that the routes and sites of that it not likely to be immunogenic as the bovine enzyme is administration of the two components can be different. known to be. It may be administered at home by family According to one particularly preferred embodiment, the 25 members or a nurse; the technique should be familiar to every sHASEGP is administered before the nucleic acid, with the family physician. route of administration of the two components preferably In ambulatory patients, hypodermoclysis sites include the being similar. The time interval between the injections is not abdomen, upper chest, above the breast, over an intercostal critical and can be defined by the skilled person. It is possible space and the Scapular area. In bedridden patients, preferred to recommend an interval of from 10 minto 72 h, advanta 30 sites are the thighs, the abdomen and the outer aspect of the geously of from 30 minto 48 h, preferably of from 1 to 24h upper arm. After one to four days, the needle and tubing and, very preferably, of from 1 to 6 h. should be changed, although infusion sets have been left in In addition, the combination product according to the place for much longer periods without complications. invention can also be combined with one or more molecule(s) Administration of 500-mL boluses over one or two hours which is/are intended to improve the nucleic acid administra 35 tion. The molecules can be molecules which have a protective three times a day can also be given, with 150 U of shASEGP effect on the nucleic acid (protection with regard to degrada given at the Subcutaneous site before the first morning infu tion in the cell), which improve its penetration or its expres sion sion in the host cell (fusogenic peptide, nuclear localization Facilitation of Therapeutic Injections. signal, etc.), which enable one particular cell type to be tar 40 Many molecules injected percutaneously (e.g. by using a geted (ligand or antibody which recognizes a cell Surface needle or other device that penetrates the skin and is used to protein, etc.), or which prolong the therapeutic effect (immu deposit the molecules into a sub-dermal layer) reach circula nosuppressive agent, etc.). The combination product can also tion slowly or with very low efficiency. Several factors regu be combined with agents that facilitate transfection (proteins, late the pharmacokinetics and pharmacodynamics of mol etc.). 45 ecules injected subcutaneously (SC) or intramuscularly (IM). The combination product according to the invention can be Generally, larger molecules reach circulation more slowly prepared with a view to local or parenteral administration or and less efficiently without active transport into circulation. to administration by the digestive route. Routes which may in Subcutaneous bioavailability is determined by calculating the particular be mentioned are the intragastric, Subcutaneous, ratio of area under the curves for SC verses intravenous intracardiac, intravenous, intraperitoneal, intrasynovial, 50 administration (AUCs/AUC). A second factor is intratumor, intrapulmonary, intranasal and intratracheal charge and affinity for matrix molecules that may play a role routes, and, very particularly, the intramuscular route. The in sequestration of molecules Subcutaneously. If these mate administration can be effected by means of any technique of rials are degraded locally they may never reach their desired the art (injection, oral route, aerosol, instillation, etc.), as a targets and thus demonstrate a decreased overall systemic single dose or as a dose that is repeated once or several times 55 bioavailability to the target organs. after a particular time interval. The route of administration Large proteins are normally given intravenously so the can be adjusted to suit the gene of interest to be transferred medicament directly available in the blood stream. It would and the disease to be treated. The formulation can include however be advantageous if a medicament could be given pharmaceutically acceptable vehicles (excipients, adjuvants, Subcutaneously, intramuscularly or intradermally (ID) as etc.). The Substance leading to disorganization of the extra 60 these administration forms are much easier to handle for the cellular matrix and the nucleic acid of interest are preferably patient. Especially, if the medicament must be taken regularly dissolved in a buffer which is suitable for pharmaceutical use during the whole life and treatment is to start early, already and which can be hypertonic, hypotonic or isotonic. Various when the patient is a child. However, a medicament with a buffers can be envisaged. Those which may be mentioned by very large and labile molecule. Such as coagulation factor VIII way of illustration are a physiological saline solution (0.9% 65 of 170 to 300 kDa, have normally a very low bioavailability if NaCl), a nonphysiological saline solution (1.8% NaCl), a given Subcutaneously, intramuscularly or intradermally, Hepes-Ringer Solution, a Lactate-Ringer solution, a buffer since the uptake is not enough and degradation is severe. US 7,871,607 B2 105 106 In addition to the need to increase bioavailability of many the retina will occur as the vitreous body is removed. Subcutaneously administered biologics, more rapid pharma Examples of specific protease-free glycosaminoglycanase cokinetics is also critically important in instances of emer enzymes which may be used to bring about this vitreal disin gency medicine. The time required to reach intravenous sertion purportedly include: chondroitinase ABC, chondroiti access in many patients can prevent an otherwise rapid acting nase AC, chondroitinase B, chondroitin 4-sulfatase, chon drug when administered systemically from being utilized. In droitin 6-sulfatase, hyaluronidase and beta-glucuronidase. Some cases failure to reach intravenous access is then fol Although hyaluronidase enzyme has been known to be lowed by Subcutaneous injection, which leads to additional usable for various ophthalmic applications, including the Vit delay in reaching the target organs. Thus, the more rapid rectomy adjunct application described in U.S. Pat. No. 5.292, availability of subcutaneous drugs would be of benefit as a 10 509 (Hageman), published studies have indicated that the first line of treatment rather than to risk the time required to hyaluronidase enzyme may itself be toxic to the retina and/or achieve intravenous access. Examples of molecules that can other anatomical structures of the eye. See, The Safety of be delivered subcutaneously as well as intravenously include Intravitreal Hyaluronidase; Gottleib, J. L.; Antoszyk, A. N., epinephrine, atropine, narcan, lignocaine, and dextrose. Hatchell, D. L. and Soloupis, P., Invest Ophthalmol Vis Sci An additional benefit of the invention lies in the ability to 15 31: 11,2345-52 (1990). Moreover, the used of impure slaugh deliver equivalent or larger volumes of solutions ID, SC or IM terhouse preparations of hyaluronidase can cause uveitis or without the pain and morbidity associated with the pressure inflammation of the eye. The use of human shASEGP is thus and volume of the solution at the site of injection. preferable in both its increased potency, purity and lack of By way of illustration (and without limitation), there area animal origin that can give rise to immunogenic reactions and number of biologics that can be enhanced by combination antibody mediated neutralization following repeated admin with one or more shASEGPs (e.g. by co-formulation or co istration. In another embodiment, a pegylated form of a SHA administration of shASEGP before, coincident with or after SEGP can be injected into the eye. Such a pegylated shA administration of the biologic), including for example: SEGP is not cleared from the vitreous in such a rapid fashion Abciximabs (e.g. REOPROTM), Adalimumabs (e.g. and maintains its activity in the vitreous for a longer period of HUMIRATM), Agalsidase betas (e.g. FABRAZYMETM), 25 time. Aldesleukins (e.g. PROLEUKINTM), Alefacepts (e.g. The ophthalmic toxicity of Some hyaluronidase prepara AMEVIVETM), Alemtuzumabs (e.g. CAMPATHTM), tions has been confirmed by other investigators, who have Alteplases (e.g. ACTIVASETM). Alteplases (e.g. CATHFLO proposed that Such hyaluronidase preparations be used as a ACTIVASETM), Anakinras (e.g. KINERETTM), Arcitumom toxic irritant for causing experimentally induced neovascu abs (e.g. CEA-ScanTM), Asparaginases (e.g. ELSPARTM), 30 larization of the eye, in animal toxicity models, (see An Basiliximabs (e.g. SIMULECTTM), Becaplermins (e.g. Experimental Model of Preretinal Neovascularization in the REGRANEXTM), bevacizumabs (e.g. AVASTINTM), Botuli Rabbit; Antoszyk, A. N. Gottleib, J. L., Casey, R. C., Hatch numToxinTypeA’s (e.g. BOTOXTM), Botulinum ToxinType ell, D. L. and Machemer, R., Invest Ophthalmol Vis Sci32: 1. B’s (e.g. MYOBLOCTM), Capromab Pendetides (e.g. PROS 46-51 (1991). The use of a highly purified shASEGP devoid TASCINTTM), Cetuximabs (e.g. ERBITUXTM), Daclizumabs 35 of mercury-based and cattle or bacterially derived contami (e.g. ZENAPAXTM), Darbepoetin alfas (e.g. ARANESPTM), nants is preferable for intraocular procedures. Moreover, a Denileukin diftitoxs (e.g. ONTAKTM), Dornase alfas (e.g. recombinant human shASEGP is preferable over slaughter PULMOZYMETM), Drotrecogin alfas (e.g. XIGRISTM), house derived preparations in both purity lack of bovine Efalizumabs (e.g. RAPTIVATM), Epoetin alfas (e.g. EPO pathogens and reduced risk of immunogenicity. Most prefer GENTM), Etanercepts (e.g. ENBRELTM), Filgrastims (e.g. 40 ably a pegylated shASEGP is envisioned. NEUPOGENTM), Ibritumomabs/Tiuxetans (e.g. ZEVA LINTM), Imciromab Pentetates (e.g. MYOSCINTTM), Inflix An enzymatic method using a human shASEGP is thus imabs (e.g. REMICADETM), Interferon alfa-2as (e.g. ROF provided for treating ophthalmic disorders of the mammalian ERON-ATM), Interferon alfacon-1s (e.g. INFERGENTM), eye. In one embodiment of the invention, said shASEGP is Interferon alfa-n3s (e.g. ALFERON NTM), Interferon beta 45 PEGylated to prolong its residence within the vitreous and 1 as (e.g. AVONEXTM, BETASERONTM), Interferon beta-1as prevent localized uptake. Prevention of neovascularization, (e.g. REBIFTM), Interferon gamma-lbs (e.g. ACTIM and the increased rate of clearance from the vitreous of mate MUNETM), Laronidases (e.g. ALDURAZYMETM), Nofetu rials toxic to retina, are accomplished by administering an momabs (e.g. VERLUMATM), Omalizumabs (e.g. amount of hyaluronidase effective to liquefy the vitreous XOLAIRTM), Oprelvekins (e.g. NEUMEGATM), Palivizum 50 humor of the treated eye without causing toxic damage to the abs (e.g. SYNAGISTM), Pegfilgrastims (e.g. NEULASTATM), eye. Liquefaction of the vitreous humor increases the rate of Peg-interferon alfa-2as (e.g. PEGASYSTM), Peginterferon liquid exchange from the vitreal chamber. This increase in alfa-2bs (e.g. PEG-INTRONTM), Rasburicases (e.g. exchange removes those materials and conditions whose ELITEKTM), Reteplases (e.g. RETAVASETM), Rituximabs presence causes ophthalmologic and retinal damage. (e.g. RITUXANTM), Tenecteplases (e.g. TNKaseTM), Tositu 55 Cosmetic Uses of SHASEGP momab and Iodine 1-131s (e.g. BEXXARTM), Trastuzumabs It is known that hyaluronidase has the effect of depolymer (e.g. HERCEPTINTM), Urokinases (e.g. ABBOKINASETM). izing the long mucopolysaccharide chains of the fundamental Vitreous Hemorrhage substance, responsible for the retention of bound water and of In an effort to minimize the potential for causing further the slowing, by capillary compression, of the diffusion of detachment or tearing of the retina during performance of 60 organic liquids, which eliminate metabolic wastes. Such vitrectomy, it has previously been proposed in U.S. Pat. No. retention of water and wastes associated with fat overloading 5,292,509 (Hageman), to inject certain protease-free gly of the lipocytes, constitutes classical "pigskin' edema or cosaminoglycanase enzymes into the vitreous body, to cause "orange peel edema. This depolymerization will therefore the vitreous body to become uncoupled or “disinserted from cut the long chains of mucopolysaccharides into shorter the retina, prior to removal of the vitreous body. Such disin 65 chains, whence the elimination of the bound water, of wastes, sertion or uncoupling of the vitreous body is purported to restoration of the venous and lymphatic circulation and dis minimize the likelihood that further tearing or detachment of appearance of local edema. US 7,871,607 B2 107 108 Use of shASEGP by way of subcutaneous administration sHASEGP (preferably human versions thereof) can be is thus preferred for the removal of glycosaminoglycans administered intravenously to reach the brain tissue. involved in the accumulation of so-called cellulite and to Following ischemia and reperfusion of the brain as occurs promote lymphatic flow. Human shASEGP is preferred for in stroke, the hyaluronan content typically increases dramati the treatment of cellulite in that it is capable of removal of said 5 cally due to increased expression of HA synthases and glycosaminoglycans without the inflammatory components decreased catabolism. Failure of ion pumps and leakage of of slaughter house derived proteins and is of high purity and plasma into the interstitium results in fluid retention that if not is not likely to be immunogenic. The shASEGP can be properly cleared by the lymphatics, results in tissue necrosis. administered through repeated Subcutaneous injections, Some groups have attempted to prevent interstitial fluid accu through transdermal delivery in the form of ointments or 10 mulation following ischemia reperfusion by blocking vascu creams or through the use of injectable slow release formu lar permeability. However, once the fluid has extravasated, lations to promote the continual degradation of glycosami preventing vascular permeability can prevent resolution of noglycans and prevent their return. edema and exacerbate conditions. As noted above, human Organ Transplantation sHASEGP can be administered intrathecally (e.g. through a Hyaluronan has several biological effects, that are in part 15 shunt), or shASEGP, PEGylated shASEGP or Super related to its molecular size (West, D.C., Kumar, S. Exp. Cell. Sialated shASEGP (preferably human versions thereof) can Res. 183, 179-196, 1989). The content of hyaluronan in an be administered intravenously to reach the brain tissue. organ increases in different conditions of inflammation of that Human shASEGP can also be used in the treatment of organ. Thus, an increased concentration of hyaluronan has edema associated with brain tumors, particularly that associ been shown in tissue from different organs characterized by ated with glioblastoma multiform. The edema associated with inflammatory-immunological injury Such as alveolitis brain tumors results from the accumulation of hyaluronan in (Nettelbladt Oetal, Am Rev. Resp Dis 1989; 139: 759-762) the non-cancerous portions of the brain adjacent the tumor. and myocardial infarction (Waldenstrom et al., J. Clin Invest Administration of hyaluronidase to the sites of hyaluronan 1991; 8805): 1622-1628). Other examples are allograft rejec accumulation (e.g., by intravenous injection or via a shunt) tion after a renal (Hallgren et al, J Exp Med 1990a; 171: 25 can relieve the edema associated with Such malignancies by 2063-2076; Wells et al. Transplantation 1990: 50: 240-243), degrading the excess hyaluronan at these sites. Thus, hyalu small bowel (Wallander et al. Transplant Int 1993: 6: 133 ronidase is successful in the treatment of brain tumors not 137) or cardiac (Hällgren et al., J. Clin Invest 1990b: 85: only in the reduction of the tumor mass and inhibition of 668-673) transplantation; or a myocardial inflammation of tumor growth and/or metastasis, but it also is useful in reliev viral origin (Waldenstrdmetal, Eur J. Clin Invest 1993; 23: 30 ing edema associated with the malignancy. Human SHA 277-282). SEGP can be administered for treatment of edema in a man The occurrence of interstitial edemas in connection with ner similar to that for administration of bovine testicular the grafting of an organ constitutes a severe problem in the hyaluronidase to treat edema (see, e.g., Sa Earp Arq. Braz. field of transplantation Surgery. As much as 25% of the grafts, Med. 44:217-20). will swell to such a degree that the function will temporarily 35 Without being bound to a particular application or mecha be lost. Moreover, in 2-3% of the cases, the Swelling causes nism of action, shASEGPs can potentially ameliorate disruption of the kidney, resulting in a massive haemorrhage. ischemic or hemorrhagic stroke and/or other CNS damaging SHASEGP may be used to degrade accumulated gly conditions by one or more of the following: (i) increasing cosaminoglycans in an organ transplant. Removal of Such oxygenation of damaged tissue by reducing intracranial pres glycosaminoglycans promotes removal of water from the 40 Sure; (ii) facilitating the dissolution of clots and resorption of graft and thus organ function. Dose ranging from 500-10,000 toxic metabolites; (iii) Suppressing leukocyte extravasation Units/kg may be administered to reduce interstitial pressure into infarcted or hemorrhaged regions (e.g. by removal of as such. hyaluronan which is a receptor for CD44); (iv) promotion of Pathologic Accumulations of Glycosaminoglycans in the neural regeneration of damaged tissue; or (V) increasing cere Brain 45 bral vasculogenesis. Hyaluronan levels are elevated in a number of cerebrospi Treatment of Glycosaminoglycan Accumulation in Car nal pathologic conditions. Levels of cerebrospinal hyaluro diovascular Disease nan are normally less than 200 ug/L in adults (Laurent et al. It has been shown that the administration of hyaluronidase Acta Neurol Scand 1996 September; 94(3): 194-206). These in animal models following experimental myocardial infarct levels can elevate to over 8,000 ug/L in diseases such as 50 can reduce infarct size (Maclean, et. al Science 1976 Oct. 8: meningitis, spinal Stenosis, head injury and cerebral infarc 194(4261): 199-200). The proposed mechanism by which tion. Thus administration of shASEGP (for example by bovine hyaluronidase reduces infarct size in animals is by intrathecal delivery or systemic injection of a shASEGP, reducing hyaluronan accumulation that occurs following PEGylated shASEGP or super-sialated shASEGP) can be ischemia reperfusion. Reduction of infarct size is believed to utilized to degrade critically elevated levels of substrate. 55 occur from increased lymph drainage and increased tissue The lack of effective lymphatics in the brain can also lead oxygenation and reduction of myocardial water content. to life threatening edema following head trauma. Hyaluronan While reduced infarct size could be obtained in animal mod accumulation is a result of increased synthesis by HA syn els, the benefits were not realized in larger clinical studies in thases, and decreased degradation. Accumulation of hyaluro humans. Bovine testes hyaluronidase possesses a remarkably nan can initially serve the beneficial purpose of increasing 60 short serum half life of approximately 3 minutes in animals water content in the damaged tissue, to facilitate leukocyte and man Wolf, et. al., J Pharmacol Exp Ther 1982 August; extravasation, but continued accumulation can be lethal. 222(2): 331-7. This short half-life is due to the terminal man Administration of human shASEGP to a patient suffering nose residues that are readily recognized by the Scavenger from head trauma can thus removal tissue hyaluronan accu receptors of the reticuloendothelial system. While small ani mulation and the water associated with it. HumansPIASEGP 65 mals may benefit from hyaluronidase due to a smaller vascu can be administered intrathecally through a shunt or alterna lar bed, an enzyme with increased half-life is needed. Super tively, shASEGP, PEGylated shASEGP or Super-Sialated sialated shASEGP possesses more favorable pharmacokinet US 7,871,607 B2 109 110 ics due to Sialation for which there is no scavenger receptor. lian tissues in vivo. In that regard, shASEGP or a soluble Supersialated shASEGP in doses ranging from 100-200,000 human hyaluronidase domain thereof can be used to facilitate Units/kg may be utilized to facilitate resolution of excess the diffusion and therefore promote the delivery of small hyaluronan following ischemia reperfusion and to reduce molecule pharmacologic agents, such as anesthetics, anti infarct size. infectives and anti-inflammatories for example, as well as Supersialated shASEGP may also be used to limit coro larger molecule pharmacologic agents, such as proteins (in nary plaques from arteriosclerosis. Such plaques accumulate cluding, e.g., enzymes, monoclonal antibodies, and the like), glycosaminoglycans and mediate macrophage and foam cell nucleic acids (including deoxyribonucleic acids (e.g. genes adhesion Kolodgie etal, Arterioscler Thromb Vasc Biol. 2002 and antisense molecules) and ribonucleic acids (including Oct. 1; 22(10): 1642-8. Administration of Super Sialated 10 sHASEGP can be used to reduce plaque formation. As molecules for RNA interference (RNAi) and the like), and repeated administration of hyaluronidase is contemplated at macromolecular compositions which may comprise a com doses from 100-100,000 U/kg, the need to utilize a human bination of Such components (including combinations of recombinant protein with low risk of immunogenicity and nucleic acids, proteins, carbohydrates, lipids and lipid-based increased half-life will result in superior reduction of plaques. 15 molecules, and the like). By way of illustration and without Treatment of Peripheral Tissues Necrosis limitation, molecules and macromolecular complexes rang Tissue necrosis occurs in many diseases due to venous ing from less than about 10 nm to around 500 nm in diameter, insufficiency. The lack of Sufficient oxygenation is one of the particularly from about 20 to 200 nm, are likely to exhibit main obstacles for regrowth of the tissue. It has been demon dramatic improvements in delivery through interstitial spaces strated that intra-arterial hyaluronidase treatment signifi when the interstitial space has been previously, or is coinci cantly improves the clinical picture inpatients with peripheral dently, exposed to a shASEGP as described and illustrated arterial occlusive disease (Elder et. al. Lancet (1980) 648 herein. 649). SHASEGP can be injected intra-arterially 3-5 times a Without being restricted to a particular theory of mecha week at doses from 10-200,000 Units. nism of action, it is believed that shASEGP of the present Enhanced Delivery of Anesthetics and Other Pharmaco 25 invention functions principally by hydrolyzing glycosami logic Agents to Mammalian Tissues noglycans within the extracellular matrix Surrounding mam Slaughterhouse-derived hyaluronidase is commonly used malian cells to create pore-like passages or channels that for peribulbar block in local anesthesia prior ophthalmic sur facilitate diffusion or other movement of molecules within gery. The presence of the enzyme prevents the need for addi the exposed extracellular matrix; and, furthermore, that per tional blocks and speeds the time to the onset of akinesia (loss 30 meabilizing Such portions of the extracellular matrix has the of eye movement). Peribulbar and sub-Tenon's block are the greatest effect on the diffusion or “spreading of molecules most common applications of hyaluronidase for ophthalmic that are not so large as to be also hindered by other macro procedures. Since the discontinuation of WydaseTM, reports molecular components of the extracellular matrix, Such as an of increased diplopia and ptosis have been reported with overarching network of collagen. The extent of the spreading peribulbar block (Brown et al J Cataract Refract Surg 1999; 35 effect obtainable with a given shASEGP or a soluble human 25: 1245-9). hyaluronidase domain thereof in a particular tissue setting With Wyeth’s discontinuance of Wydase(R), bovine testes can be readily ascertained as illustrated herein by assessing derived hyaluronidase material is now Supplied by com the effect of the SHASEGP or domain thereof on the diffusion pounding pharmacies. However, there are several concerns of detectable test molecules of various sizes (such as fluores with using an extemporaneously compounded sterile product 40 ceinated or otherwise labeled beads) within the test tissue, (www. ashp.org/shortage? either with or without pretreatment or concomitant treatment hyaluronidase.cfm?cfid=11944667&CFToken—9426953 with shASEGP or domain thereof at a range of times and refiref). Compounded preparations are not FDA-approved concentrations. By way of illustration, in the case of recom products. As such, the FDA has no control over the quality or binant rhuPH20 injected subcutaneously into the lateral skin consistency of the manufacturing process. 45 of mice, fluoresceinated bead diffusion tests (as exemplified SHASEGP from 10-500 Units can be mixed directly with herein) indicated that the effects on diffusion were greatest 5 ml 2% lidocaine (Xylocaine), 5 ml 0.5% bupivacaine with particles of less than about 500 nm in diameter. (Marcaine) and optionally with epinephrine1: 200,000. shA It is further believed that larger macromolecules (exempli SEGP can be used to increase the onset of akinesia and to fied by beads of 500 nm or larger) may behindered in diffu remove the need for additional blocks. SHASEGP is also ideal 50 sion not only by their larger size but by their potential inter for akinesia for cosmetic Surgery in blepharoplasties and face action with other relatively more open networks within the lifts. SHASEGP can also be utilized following such surgical extracellular matrix, Such as a network formed from Strands procedures to diffuse antiinflammatories and to reduce tissue of interstitial collagen. In addition, and unlike described ani Swelling. mal-derived hyaluronidases, presently preferred shASEGPs SHASEGP may also be mixed with a buffering solution 55 of the present invention do not contain agents that promote Such as bicarbonate to prevent discomfort during the injection extravasation or contaminating agents such as collagenases procedure. SHASEGP can also be mixed with anesthesia for (that may open larger channels) or other contaminants that lacerations to both reduce the total volume of material may provoke immune reactions. In particular applications in required for injection and to reduce pain from Swelling of which larger pores or channels are desired, shASEGP or a tissue. 60 soluble human hyaluronidase domain thereof can be com Use of shASEGPs to Promote Dispersion of Pharmaco bined with preferably relativelty pure forms of other matrix logic and Other Agents degrading enzymes such as collagenases to facilitate further As described herein, the compositions and methods of the spreading of larger size macromolecular complexes or cells. present invention can be used to promote or enhance the For most applications, the use of an agent that promotes the delivery of anesthetics and other pharmacologic agents (such 65 permeability of macromolecules having a size of less than as members of the various classes of pharmaceutically effec about 500 nm, and more preferably less than about 200 nm, is tive agents referred to herein) to any of a variety of mamma very advantageous in that the agent does not substantially US 7,871,607 B2 111 112 promote the movement of cells within a treated tissue but of the body. For purposes of illustration (and without limita promotes the passage of Smaller complexes and agents. tion). Such agents include, for example: Anesthetics; Anti As described and illustrated herein, shASEGPs can be metbolites, anti-neoplastics and other anti-cancer agents; used to enhance the delivery of a variety of other pharmaco Anti-virals; Anti-infectives, including Anti-bacterials and logic and/or other agents to tissues within the human body 5 other antibiotics, Anti-fungals and other anti-infectives; (including tissues such as those at the back of the eye that are Immunomodulatory agents; Steroidal and non-steroidal anti typically relatively difficult to reach). Without wishing to be inflammtories: Beta blockers; Sympathomimetics: bound by theory, it is believed that shASEGPs can enhance Ducosanoids, prostaglandins and prostaglandin analogs; the pharmacokinetic and/or pharmacodynamic profiles of a Miotics, cholinergics and anti-cholinesterases; Anti-allergen number of Such agents by potentially enhancing their diffu 10 ics and Decongestants; Hormonal agents; Growth factors; sion or permeation within a tissue, and/or by enhancing their other synthetic and biologically derived agents; and combi convective transport by increasing bulk fluid flow within the nations thereof. Some illustrative examples of agents having interstitial space of a tissue. For purposes of illustration (and such properties that can be combined with shASEGPs in the without limitation), examples of pharmacologic agents that formulation of novel pharmacologics and/or reformulations can be combined with shASEGPs of the present invention 15 of existing pharmacologics include the various categories of (e.g. combined by co-formulation with one or more SHA agents described above and below (as well as other known SEGPs or by co-administration with one or more shASEGPs and novel agents that are regularly being identified for the (which may be administered prior to, coincident with or fol diagnosis, prevention or treatment of various disease): lowing administration of the agent) for treatment of various conditions affecting the eye include, for example, Angiogen Anesthetics, particularly non-inhalation local anesthetics esis inhibitors (such as angiostatins, anecortave acetates (e.g. (such as ambucaines; amoxecaines; amylocalnes; aptocaines; RETAANETM), endostatins, thrombospondins (e.g. thrombo articaines; benoximates; benzyl alcohols; benzocaines; betox spondin-1 and thrombospondin-2), anti-angiogenic antibod ycaines; biphenamines; bucricaines; bumecaines; bupiv ies and other anti-angiogenic agents or vascular targeting acaines; butacaines; butambens; butanilicaines; carbi agents (VTAs) (e.g. anti-VEGF antibodies such as bevaci 25 Zocaines; chloroprocaine; clibucaines; clodacaines; Zumabs (e.g. AVASTINTM), carboxyamido triazole (CAI), cocaines: dexivacaines; diamocaines; dibucaines; Interleukin-12 (IL-12), OLTIPRAZTM (5-2-pyrazinyl-4- dyclonines; elucaines; etidocaines; euprocins; fexicaines; methyl-1,2-3-thione), SU-5416, TNP-470, thalidomide), and fomocaines; heptacaines; hexylcaines; hydroxyprocaines; the like); Anti-cataract and anti-diabetic retinopathy Sub hydroxytetracaines; isobutambens; ketocaines; leucino stances (such as aldose reductase inhibitors (ARIs) for 30 caines; lidocaines; mepivacaines; meprylcaines; octocaines; example tolrestats (e.g. ALREDASETM) and Zopolrestats orthocaines; oxethacaines; oxybuprocaines; phenacaines; (e.g. ALONDTM), lisinoprils, enalaprils, statils, and thiol pinolcaines; piperocaines; piridocaines; polidocanols; cross-linking Substances and the like); Anti-inlammatory pramocaines; prilocalnes; procaines; propanocaines; propi agents (including steroidal and non-steroidal anti-inflamma pocaines; propoxycaines; proxymetacaines; pyrrocaines; tory agents as described herein and in the art); Anti-infectives 35 quatacaines; quinisocaines; risocaines; rodocaines; ropiv (including compounds as described below, as well as a num acaines; Salicylalcohols; Suicaines; tetracaines; trapencaines; ber of opthalmic preparations thereof such as BLEPHA trimecaines) and the like; as well as various other non-inha MIDETM, CILOXANTM, POLYTRIMTM, QUIXINTM, lation anesthetics (such as alfaxalones; amolanones: etoxa TOBRADEXTM, VIGAMOXTM, ZYMARTM); Beta-adrener drols; fentanyls, ketamines; levoxadrols; methiturals; metho gic blocking agents (such as betaxolols (e.g. BETOPTIC 40 hexitals; midazolams; minaxolones; propanidids; STM), timolols (e.g. BETIMOLTM and TIMOPTICTM) and propoXates; pramoxines; propofols; remifentanyls; Sufenta combinations thereof (e.g. timolols and a carbonic anhydrase nyls; tiletamines; Zolamine and the like); inhibitor such as a dorzolamide (e.g. COSOPTM); Carbonic Anti-metabolites (such as folic acid analogs (e.g., denop anhydrase inhibitors (such as acetazolamides, brinzolamides terins, edatrexates, methotrexates, piritrexims, pteropterins, (e.g. AZOPTM), dichlorophenamides, dorzolamides (e.g. 45 Tomudex, trimetrexates), purine analogs (e.g., cladribines, TRUSOPTTM, and in combinations with other agents as in fludarabines, 6-mercaptopurines, thiamiprines, thiagua e.g. COSOPTM), methazolamides and the like); Mydriatics nines), and pyrimidine analogs (e.g., ancitabines, azaciti (such as atropine Sulfates, cyclopentolates, homatropines, dines, 6-azauridines, carmofurs, cytarabines, doxifluridines, Scopolamines, tropicamides, eucatropines, and hydroxyam emitefurs, enocitabines, floXuridines, fluorouracils, gemcit phetamines and the like); Photodynamic therapy agents (such 50 abines, tegafurs) and the like; as verteporfin (e.g. VISUDYNETM); Prostaglandin analogs Anti-neoplastics (such as aclacinomycins, actinomycins, (such as bimatoprosts (e.g. LUMIGANTM), latanoprosts (e.g. adriamycins, ancitabilles, anthramycins, azacitidines, azaser XALATANTM), and travoprosts (e.g. TRAVATANTM); Sym ines, 6-azauridines, bisantrenes, bleomycins, cactinomycins, pathomimetics (such as brimonidines (e.g. ALPHAGANTM carmofurs, carmustines, carubicins, carZinophilins, chromo P); as well as combinations thereof (such as combinations of 55 mycins, cisplatins, cladribines, cytarabines, dactinomycins, a beta-adrenergic blocking agent and a carbonic anhydrase daunorubicins, denopterins, 6-diaZo-5-oxo-L-norleucines, inhibitor as in COSOPTTM); and numerous other classes of doxifluridines, doxorubicins, edatrexates, emitefurs, enocit agents such as anti-infectives and various other physiological abines, fepirubicins, fludarabines, fluorouracils, gemcitab modulators as described below or elsewhere herein, and/or as ines, idarubicins, loXuridines, menogarils, 6-mercaptopu known in the art or newly developed. 60 rines, methotrexates, mithramycins, mitomycins, A number of other pharmacologic agents that can be com mycophenolic acids, nogalamycins, olivomycines, peplomy bined with shASEGPs of the present invention (e.g. com cins, pirarubicins, piritrexims, plicamycins, porfironiycins, bined by co-formulation with one or more shASEGPs or by pteropterins, puromycins, retinoic acids, Streptonigrins, co-administration with one or more shASEGPs (which may streptozocins, tagafurs, tamoxifens, thiamiprines, thiogua be administered prior to, coincident with or following admin 65 nines, triamcinolones, trimetrexates, tubercidins, vinblas istration of the agent) can be used for treatment of various tines, Vincristines, Zinostatins, Zorubicins and the like) (see conditions affecting the eye or affecting various other tissues also the illustrative anticancer agents described herein); US 7,871,607 B2 113 114 Anti-virals (such as abacavirs (e.g. ZIAGENTM), acyclov TAZTM), cefterams, ceftezoles, ceftibutens, cefti irs (e.g. ZOVIRAXTM), amantadines and rimantadines (e.g. Zoximes, ceftriaxones (e.g. ROCEPHINTM), SYMMETRELTM), amprenavirs (e.g. AGENERASETM), cefuroximes (e.g. ZINACEFTM), cefuzonams, ceph cidofovirs (e.g. VISTIDETM), delavirdines (e.g. RESCRIP acetriles, cephalexins, cephaloglycins, cephaloridines, TORTM), didanosines (e.g. VIDEXTM), efavirenz (e.g. SUS cephalosporins, cephalothins, cephapirins, cephradines, TIVATM), famciclovirs (e.g. FAMVIRTM), fomivirsens (e.g. piveefalexins and the like): VITRAVENETM), foscarnets (e.g. FOSCAVIRTM), ganciclo Cephamycins (e.g., cefbuperaZones, cefinetazoles, cefini virs (e.g. CYTOVENETM) and valganciclovirs (e.g. VAL noxes, cefotetans, cefoxitins (e.g. MEFOXINTM), and CYTETM), idoxuridines (e.g. HERPLEXTM), indinavirs (e.g. the like): CRIXIVANTM), interferons (e.g. ALFERON NTM, 10 Clavams (e.g., clavulinic acid or clavulanates, 2- and INTRONTM A PEGASYSTM, PEG-INTRONTM, ROF 3-phenyl clavams, and combinations comprising Such ERON-ATM, and combinations of interferons and other anti agents such as amoxicillin/clavulanates (e.g. AUG Virals Such as ROBETRONTM and PEGASYS/COPE MENTINTM) and ticarcillin/clavulanates (e.g. TIMEN GUSTM), lamivudines (e.g. 3TCTM and EPIVIRTM) and TINTM), and the like): combinations of lamivudines and zidovudines (e.g. COM 15 Cyclic lipopeptides (e.g. daptomycins (e.g. CUBICINTM); BIVIRTM), monoclonal antibodies such as palivizumabs (e.g. Diaminopyrimidines such as 2,4-diaminopyrimidines SYNAGISTM), nelfinavirs (e.g. VIRACEPTTM), nevirapines (e.g., brodimoprims, tetroxoprims, trimethoprims and (e.g. VIRAMUNETM), oseltamivirs (TAMIFLUTM), penci the like): clovirs (e.g. DENAVIRTM), pleconarils, ribavirins (e.g. Ketolides (e.g. tellithromycins (e.g. KETEKTM), and COPEGUSTM and REBETOLTM), rimantadines (e.g. FLU bridged bicyclic ketolides (e.g. Enanta EP-013420 and MADINETM), ritonavirs (NORVIRTM), saquinavirs (e.g. 6-11 bycyclic ketolides as described in U.S. Published INVERASETM), stavudines (d4T, e.g., ZERITTM), vallacyclo patent application 20040157787), and the like): virs (e.g. VALTREXTM), vidarabines (e.g. VIRA-ATM), Zal Lincosamides (e.g., clindamycins, lincomycins and the citabines (e.g. HIVIDTM), Zanamivirs (RELENZATM), like): zidovudines (e.g. AZT, RETROVIRTM), and the like): 25 Macrollides (e.g., azithromycins (e.g. ZITHROMAXTM), Anti-bacterials and other antibiotics, including, for carbomycins, clarithromycins, dirithromycins, erythro example, Aminoglycosides; Amphenicols; Ansamycins; Car mycins, erythromycin acistrates, erythromycin esto bacephems; Carbapenems; Cephalosporins or Cephems; lates, erythromycin glucoheptonates, erythromycin lac Cephamycins; Clavams; Cyclic lipopeptides; Diaminopyri tobionates, erythromycin propionates, erythromycin midines; Ketolides; Lincosamides; Macrollides; Monobac 30 Stearates, josamycins, leucomycins, midecamycins, tams: Nitrofurans: Oxacephems; Oxazolidinones; Penems, miokamycins, oleandomycins, primycins, rokitamy thienamycins and miscellaneous beta-lactams; Penicillins: cins, rosaramicins, roXithromycins, spiramycins, trole Polypeptides antibiotics; Quinolones; Sulfonamides; Sul andomycins and the like); fones; Tetracyclines; and other antibiotics, illustrative mem Monobactams (e.g., aztreonams (e.g. AZACTAMTM), bers of which are provided below (and others known in the art 35 carumonams, tigemonams and the like); or newly developed): Nitrofurans (e.g., furaltadones, furazolium chlorides, Aminoglycosides (e.g., amikacins, apramycins, nifuradenes, nifuratels, nifurfolines, nifurpirinols, nifur arbekacins, bambermycins, butirosins, dibekacins, prazines, nifurtoinols, nitrofurantoins and the like); dihydrostreptomycins, fortimicins, gentamicins, isepa Oxacephems (e.g. flomoxefs, latamoxefs or moxalactams micins, kanamycins, micronomicins, neomycins, neo 40 (e.g. MOXAMTM), and the like): mycin undecylenates, netilmicins, paromomycins, Oxazolidinones (e.g. linezolids (e.g. ZYVOXTM), eper ribostamycins, Sisomicins, spectinomycins, streptomy eZolids, and the like): cins, tobramycins, trospectomycins and the like); Penems, thienamycins and miscellaneous beta-lactams Amphenicols (e.g., azidamfenicols, chloramphenicols, (e.g. ertapenems (e.g. INVANZTM), imipenems (e.g. florfenicols, thiamphenicols and the like): 45 with Cilastatin as in PRIMAXINTM), meropenems (e.g. Ansamycins (e.g., rifamides, rifampins or rifampicins (e.g. MERREMTM), and the like): RIFADINTM, RIFADIN IVTM, as well as combinations Penicillins (e.g., amdinocillins, amdinocillin pivoxils, comprising rifampins such as rifampin/isoniazid (e.g. amoxicillins, amplicillins (including combinations RIFAMATETM) and rifampin/isoniazid/pyrazinamide thereof (e.g. with sublactams as in UNASYNTM)), apal (e.g. RIFATERTM), rifamycins, rifapentines, rifaximins 50 cillins, aspoxicillins, azidocillins, azlocillins, bacampi (e.g. XIFAXAMTM), and the like): cillins, benzylpenicillinic acids, benzylpenicillin sodi Carbacephems (e.g. loracarbefs, 3-chloro-1-carbaceph ums, carbenicillins, carindacillins, clometocillins, ems, 3-thio Substituted carbacephems, carbacephems cloxacillins, coamoxiclavs, cyclacillins, dicloxacillins, described in U.S. Published Patent Application No. epicillins, fenbenicillins, floxacillins, hetacillins, lena 20050004095, and the like): 55 mpicillins, metampicillins, methicillin Sodiums, Carbapenems (e.g., biapenems, imipenems, meropenems, meZlocillins, nafcillins, oxacillins, penamecillins, panipenems and the like); penethamate hydriodides, penicillin G benethamines, Cephalosporins or cephems (e.g., cefaclors (e.g. penicillin G benzathines (e.g. BICILLINTML-A), peni CECLORTM), cefadroxils, cefamandoles, cefatrizines, cillin G benzhydrylamines, penicillin G calciums, peni cefazedones, cefazolins (e.g. ANCEFTM), cefcapene 60 cillin Ghydrabamines, penicillin G potassiums, penicil pivoxils, cefclidins, cefdinirs, cefditorens, cefepimes lin G procaines, penicillin N, penicillin 0, penicillin V. (e.g. MAXIPIMETM), cefetamets, cefiximes, penicillin V benzathines, penicillin V hydrabamines, cefinenoXimes, cefimetazoles, cefodizimes, cefonicids, penimepicyclines, phenethicillin potassiums, piperacil cefoperaZones, ceforanides, cefotaximes, cefotiams, lins (including combinations thereof (e.g. with beta cefotetans, cefoxitins, cefoZoprans, ce?pimizoles, cefpi 65 lactamase inhibitors such as taZobactam, as in ramides, cefpiromes, cefpodoxime proxetils, cefprozils, ZOSYNTM)), pivampicillins, propicillins, quinacillins, cefroxadines, cefsulodins, ceftazidimes (e.g. FOR Sulbenicillins, Sultamicillins, talampicillins, tazocillins, US 7,871,607 B2 115 116 temocillins, ticarcillins (e.g. with clavulanates as in griseofulvins, oligomycins, neomycin undecylenates, pyr TIMENTINTM), and the like): rolnitrins, siccanins, tubercidins, viridins); as well as Syn Polypeptides antibiotics (e.g., amphomycins, bacitracins, thetic anti-fungals such as allylamines (e.g., butenafines, naf capreomycins, colistimethates (e.g. COLI-MYCINTM), tifines, terbinafines); imidazoles (e.g., bifonazoles, colistins, enduracidins, enviomycins, fusafungines, butoconazoles, chlordantoins, chlormidazoles, cloconazoles, gramicidins, mikamycins, polymyxins, pristinamycins clotrimazoles, econazoles, enilconazoles, fenticonazoles, (and pristinamycin derivatives Such as quinupristins and flutrimaZoles, isoconazoles, itraconazole, ketoconazoles, dalfopristins (e.g. SYNERCIDTM)), ristocetins, teico lanoconazoles, miconazoles, omoconazoles, oxiconazole planins, thiostreptons, tuberactinomycins, tyrocidines, nitrates, Sertaconazoles, Sulconazoles, tioconazoles, Vori tyrothricins, Vancomycins, viomycins, Virginiamycins, 10 conazoles (e.g. VFENDTM)); thiocarbamates (e.g., tolci Zinc bacitracins and the like); clates, tolindates, tolnaftates); triazoles (e.g., fluconazoles Quinolones (incl. fluoroquinolones and analogs) (e.g., ala (e.g. DIFLUCANTM), itraconazoles (e.g. SPORANOXTM), trofloxacin mesylates, cinoxacins, ciprofloxacins (e.g. Saperconazoles, terconazoles); and other synthetics (e.g., CIPROTM), clinafloxacins, difloxacins, enoxacins, acrisorcins, amorolfines, biphenamines, bromosalicylchlo fleroxacins, flumequines, gatifloxacins (e.g. 15 ranilides, buclosamides, calcium propionates, chlorphenes TEQUINTM), grepafloxacins, levofloxacins (e.g. LEVA ins, ciclopiroxes, cloxyquins, coparaffinates, diamthazole QUINTM), lomefloxacins, miloxacins, moxyfloxacins dihydrochlorides, exalamides, flucytosines, halethazoles, (e.g. AVELOXTM), nadifloxacins, nalidixic acids, nor hexetidines, lipopeptides such as echinocandin (as in caspof floxacins, ofloxacins, oxolinic acids, paZufloxacins, ingin CANCIDASTM), loflucarbans, nifuratels, potassium pefloxacins, pipemidic acids, piromidic acids, roSOX iodides, propionic acids, pyrithiones, Salicylanilides, sodium acins, rufloxacins, sparfloxacins, temafloxacins, to Su propionates, Sulbentines, tenonitroZoles, triacetins, floxacins, trovafloxacin mesylates and the like); ujothions, undecylenic acids, Zinc propionate) and the like; Sulfonamides (e.g., acetyl sulfamethoxypyrazines, ben Amebicides and anti-protozoals (including, e.g., atova Zylsulfamides, chloramine-B, chloramine-T, dichloram quone, chloroquine hydrochloride, chloroquine phosphate, ine T, formylsulfisomidines, beta-glucosylsulfanil 25 metronidazole, metronidazole hydrochloride, pentamidine amides, mafenides, 4-(methylsulfamoyl) isethionate and the like); Anti-helmenthics and anti-parasitics Sulfanilanilides, noprylsulfamides, (including, e.g., mebendazole, pyrantel pamoate, thiabenda phthalylsulfacetamides, phthalylsulfathiazoles, Salazo Zole and the like); Anti-malarials (including, e.g., chloro Sulfadimidines. Succinylsulfathiazoles, Sulfabenza quine hydrochloride, chloroquine phosphate, doxycycline, mides, Sulfacetamides, sulfachlorpyridazines, Sulfach 30 hydroxychloroquine Sulfate, mefloquine hydrochloride, pri rysoidines, Sulfacytines, Sulfadiazines, Sulfadicramides, maquine phosphate, pyrimethamine, pyrimethamine with sulfadimethoxines, sulfadoxines, sulfaethidoles, Sulf sulfadoxine; Anti-tuberculotics and anti-leprotics (including, aguanidines, sulfaguanols, Sulfalenes, Sulfaloxic acids, e.g., clofazimine, cycloserine, dapsone, ethambutol hydro Sulfamerazines, Sulfameters, Sulfamethazines, Sulfame chloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifap thizoles, Sulfamethomidines, Sulfamethoxazoles, Sul 35 entine, Streptomycin Sulfate and the like, as well as combina famethoxypyridazines, sulfametroles, Sulfamidoch tions thereof such as rifampin/isoniazid (e.g. RIFAMATETM) rysoidines, Sulfamoxoles, Sulfanilamides, and rifampin/isoniazid/pyrazinamide (e.g. RIFATERTM). 4-sulfanilamidosalicylic acids, Sulfanilylsulfanil For purposes of further illustration (and without limita amides, Sulfanily lureas, n-sulfanillyl-3,4-Xylamides, tion), examples of immuno-modulatory agents that can be Sulfanitrans, Sulfaperines, sulfaphenazoles, Sulfaproxy 40 combined with shASEGPs of the present invention (e.g. lines, Sulfapyrazines, Sulfapyridines, Sulfasomizoles, combined by co-formulation with one or more shASEGPs or Sulfasymazines, Sulfathiazoles, Sulfathioureas, Sulfato by co-administration with one or more shASEGPs (which lamides, sulfisomidines, sulfisoxazoles and the like); may be administered prior to, coincident with or following Sulfones (e.g., acedapsones, acediasulfones, acetosulfone administration of the agent) include, for example, members Sodiums, dapsones, diathymosulfones, glucosulfone 45 of the following classes of agents: Sodiums, Solasulfones, Sublactams, succisulfones, Sul Immunosuppressants (including, e.g., azathioprine, basil fanilic acids, p-sulfanilylbenzylamines, Sulfoxone sodi iximab, cyclosporine, daclizumab, lymphocyte immune ums, thiazolsulfones and the like); globulin, muromonab-CD3, mycophenolate mofetil, Tetracyclines (e.g., apicyclines, chlortetracyclines, clo mycophenolate mofetil hydrochloride, sirolimus, tac mocyclines, demeclocyclines, doxycyclines, guamecy 50 rolimus and the like): clines, lymecyclines, meclocyclines, methacyclines, Vaccines and toxoids (including, e.g., BCG vaccine, chol minocyclines, oxytetracyclines, penimepicyclines, era vaccine, diphtheria and tetanus toxoids (adsorbed), pipacyclines, rollitetracyclines, Sancyclines, tetracy diphtheria and tetanus toxoids and acellular pertussis cline), and others (e.g., cycloserines, mupirocins, vaccine adsorbed, diphtheria and tetanus toxoids and tuberins and the like): 55 whole-cell pertussis vaccine, Haemophilus b conjugate and other antibiotics (such as clofoctols, fusidic acids, vaccines, hepatitis A vaccine (inactivated), hepatisis B hexedines, methenamines (including e.g., meth vaccine (recombinant), influenza virus vaccines (based enamine, methenamine anhydromethylene-citrates, on purified Surface antigens), influenza virus Vaccines methenamine hippurates, methenamine mandelates, (based on subvirions or purified subvirions), influenza methenamine Sulfosalicylates), nitrofurantoins (e.g. 60 virus vaccines (based on whole virions), Japanese FURADANTINTM), nitroxolines, ritipenems, tauro encephalitis virus vaccine (inactivated), Lyme disease lidines, Xibomols and the like): vaccine (recombinant OspA), measles and mumps and Anti-fungals, including polyenes such as amphotericin BS rubella virus Vaccine (live), measles and mumps and (e.g. ABELCETTM and AMBISOMETM), candicidins, dermo rubella virus vaccine (live attenuated), measles virus statins, filipins, fungichromins, hachimycins, hamycins, 65 vaccine (live attenuated), meningococcal polysaccha lucensomycins, mepartricins, natamycins (e.g. Natacyn), ride vaccine, mumps virus vaccine (live), plague vac nystatins, pecilocins, perimycins, and others (e.g., azaserines, cine, pneumococcal vaccine (polyvalent), poliovirus US 7,871,607 B2 117 118 vaccine (inactivated), poliovirus vaccine (live, oral, Ducosanoids, prostaglandins and prostaglandin analogs trivalent), rabies vaccine (adsorbed), rabies vaccine (hu (such as bimatoprosts, latanoprosts, travoprosts, uno man diploid cell), rubella and mumps virus vaccine prostones, and the like) as well as antiprostaglandins and (live), rubella virus vaccine (live, attenuated), tetanus prostaglandin precursors; toxoid (adsorbed), tetanus toxoid (fluid), typhoid vac Miotics, cholinergics and anti-cholinesterases (such as cine (oral), typhoid vaccine (parenteral), typhoid VI acetylcholine chlorides, carbachols, demecarium bro polysaccharide vaccine, varicella virus vaccine, yellow mides, diisopropyl fluorophosphates, eserines, physos fever vaccine and the like): tigmines, pilocarpines, phospholine iodines, salicylates, and the like): Altitoxins and antivenins (including, e.g., black widow and 10 Anti-allergenics (such as sodium antazolines, cetirizines, other spider antivenins, Crotalidae antivenom (polyva chlorpheniramines, cromoglycates, cromolyn Sodium lent), diphtheria antitoxin (equine), Micrurus fulvius (Crolom), emedastine difumarate, ketotifins (e.g. keto antivenin and the like); tifen fumarate), levocabastines, methapyrilines, Immune sera (including, e.g., cytomegalovirus immune nedocromil Sodium, olopatadines, pyrilamines, proph globulin (intraveneous), hepatitis B immune globulin 15 enpyridamines, and the like); (human), immune globulin intramuscular, immune Decongestants (such as phenylephrines, naphazolines, tet globulin intravenous, rabies immune globulin (human), rahydrozolines and the like): respiratory syncytial virus immune globulin intravenous Hormonal agents (such as estrogens, estradiols, progesta (human), immune globulins (human), tetanus immune tionals, progesterones, insulins, calcitonins, parathyroid globulin (human), varicella-Zoster immune globulin and hormone and peptide and vasopressin hypothalamus the like): releasing factor and the like); and other immunomodulators (including, e.g., aldesleukin, Growth factors (such as epidermal growth factor, fibroblast epoetin alfa, filgrastim, glatiramer acetate for injection, growth factor, platelet derived growth factor, transform interferon alfacon-1, interferon alfa-2a (recombinant), ing growth factor beta, Somatotropin and fibronectin and interferon alfa-2b (recombinant), interferon alfa-n3, 25 the like): interferon beta-1a, interferon beta-1b (recombinant), Analgesics (such as acetaminophen; alfentanil; aminoben interferon gamma-1b, levamisole hydrochloride, Zoate; aminobenzoate; anidoxime; anileridine; anilleri oprelvekin, Sargramostim and the like). dine; anilopam; anirolac, antipyrine; aspirin; benox Other agents that can be combined with shASEGPs of the aprofen; benzydamine; bici fadine hydrochloride: present invention (e.g. combined by co-formulation with one 30 brifentanil; brom adoline; bromfenac; buprenorphine; or more shASEGPs or by co-administration with one or more butacetn; butixirate; butorphanol; butorphanol; carbam sHASEGPs (which may be administered prior to, coincident azepine; carbaspirin calcium; carbiphene; carfentanil; with or following administration of the agent) include, e.g., ciprefadol Succinate; ciramadol; ciramadol; clonixeril; the following: clonixin; codeine; codeine phosphate; codeine Sulfate; Steroidal anti-inflammatories (such as alclomethasones, 35 conorphone; cyclazocine; deXOXadrol; dexpemedolac, algestones, beclomethasones, betamethasones, budes dezocine; diflunisal; dihydrocodeine; dimefadane; dipy onides, clobetasols, clobetaSones, clocortolones, clo rone, doxpicomine; drinidene; enadoline; epirizole; prednols, corticosterones, cortisones and hydrocorti ergo tamine tartrate; ethoxazene: etofenamate; eugenol; Sones (e.g. hydrocortisone acetate), cortivaZols, fenoprofen; fenoprofen calcium; fentanyl citrate; floc deflazacorts, desonides, desoximetasones, dexametha 40 tafenine; flufenisal; flunixin; flunixin meglumine; flu Sones (e.g. dexamethasone 21-phosphate), difluo pirtine; fluproduaZone; fluradoline; flurbiprofen; hydro roSones, diflucortolones, difluprednates, enoXolones, morphone; ibufenac; indoprofen, ketazocine; fluazacorts, flucloronides, flumethasones, flunisolides, ketorfanol, ketorolac; letimide; levomethadyl acetate; fluocinolones, fluocinonides, fluocortins, fluocortolo levomethadyl acetate hydrochloride; levonantradol; nes, fluorometholones, fluperolones, fluprednidenes, 45 levorphanol; lofemizole; lofentanil oxalate; lorcinadol; fluprednisolones, flurandrenolides, fluticasones, formo lomoxicarn; magnesium salicylate; mefenamic add: cortals, halcinonides, halobetasols, halometaSones, menabitan; meperidine; meptazinol; methadone; meth halopredones, hydrocortamates, hydrocortisones, adyl acetate; methopholine; methotrimeprazine; met loteprednol etabonate, maZipredones, medry Sones, kephamid acetate; mimbane; mirfentanil; molinaZone; meprednisones, methylprednisolones, mometasone 50 morphine Sulfate; moxazocine; nabitan; nalbuphine; furoate, paramethasones, prednicarbates, prednisolones nalmexone; namoxyrate; nantradol, naproxen; (e.g. prednisolone 25-diethylamino-acetate, predniso naproxen; naproXol; nefopam; nexeridine; noracymeth lone sodium phosphate), prednisones, prednivals, pred adol; ocfentanil; octaZamide; olvanil; OXetorone; oxyc nylidenes, rimexolones, tiXocortols, triamcinolones odone; oxycodone; oxycodone terephthalate; oxymor 55 phone; pemedolac, pentamorphone; pentazocine; (e.g. triamcinolone acetonide, triamcinolone bene pentazocine; phenazopyridine; phenyramidol; picena tonide, and triamcinolone hexacetonide) and the like); dol; pinadoline; pirfenidone; piroXicam olamine; prava Non-steroidal anti-inflammatories (such as cromoglicates, doline; prodilidine; profadol; propiram; propoxyphene; diclofenacs, flurbiprofens, indomethacins, ibuprofens, propoxyphene napsilate; proxazole; proXorphan; pyrro ketorolacs (e.g. ketorolac tromethamine), lodoxamides, 60 liphene; remifentanil; Salcolex; salethamide maleate; nedocromils, piroXicams, salicylates, and the like); Salicylamide; salicylate meglumine; Salsalate; salicy Beta blockers (such as betaxolols (e.g. betaxolol hydro late; spiradoline; Sufentanil; Sufentanil; Sucapsaicin; tal chloride), carteolols, esmolols, levobunolols, metacin; talniflumate; talosalate; tazadolene; metipranalols, timolols (e.g. timolol hemihydrate and tebufelone; tetrydamine; tifurac; tilidine, tiopinac; tona various forms of timolol maleate) and the like): 65 Zocine; tramadol; trefentanil; trolamine; Veradoline; Sympathomimetics (such as adrenaline, brimonidines, Verilopam; Volazocine; Xorphanol; Xylazine; Zenazocine dipivefrins, epinephrines and the like); mesilate; Zomepirac, and the like); US 7,871,607 B2 119 120 Other macromolecular pharmacologic agents such as other eye with a synthetic intraocular lens in place, pressure on the proteins, nucleic acids, and the like; as well as com corneal endothelium can cause significant damage to the cells plexes of macromolecules Such as various drug delivery, and Subsequent corneal Swelling and opacification can occur, protein delivery and/or gene delivery vehicles (includ which are associated with decreased vision. Typically, if a ing synthetically-prepared vehicles Such as liposomes patients intraocular pressure is significantly elevated at the and the like, as well as biologically-generated vehicles conclusion of the operative procedure, it is necessary to give Such as viral vectors and the like); and Such a patient large doses of carbonic anhydrase inhibitors, as Other agents used for a variety of conditions (and addi well as topical eye drops such as beta-blockers and alpha II tional information regarding usage, dosages, contrain agonists in order to decrease aqueous formation and/or to dications etc. of various agents including those identi 10 increase aqueous outflow. These agents all have significant fied herein), as known in the art, including for example side effects and, in some instances, are contraindicated in those additional agents identified in references such as patients with various types of medical conditions such as the Physicians' Desk Reference (PDR) (Thomson 2003, breathing problems, heart disease or high blood pressure. 2004, 2005); the American College of Physicians Com However, the use of shASEGP in these situations will elimi plete Home Medical Guide (DK Publishing 2003); and 15 nate the necessity of giving these patients large doses of Such additional agents indentified in patent references includ drugs. ing, for example, U.S. Published applications Furthermore, there is a significant amount of hyaluronic 20040224023, 20040224012 and 2005.0002865 (which acid in the trabecular meshwork. The shASEGP will break lists of agents are hereby incorporated by reference this down and therefore improve the outflow of the aqueous herein). through the trabecular meshwork. The patient’s intraocular Combinations of agents (e.g. two or more agents selected pressure will therefore decrease. The combination of shA from one or more of the preceding groups of agents and/or SEGP with other anterior chamber agents, such as a methyl from other groups or classes of pharmacologic and other cellulose (OcucoatTM for example, commercially available agents specified herein or known in the art). from StorZ Instrument Co.), used as spacers and/or protective Use of SHASEGPs for Reduction of Intraocular Pressure 25 agents in cataract Surgery, will also be efficacious in prevent and Other Opthalmic Indications ing significant pressure rises because it will in effect open the A common side effect occurring in postoperative cataract trabecular meshwork and allow more aqueous humor drain patients is a significant early, and occasionally prolonged, rise age by breaking down a significant amount of the hyaluronic in intraocular pressure. Such a condition is sometimes seri acid present in the trabecular meshwork. ous, especially in patients with glaucomatous optic disc 30 Removal of glycosaminoglycans from the trabecular changes. Although the pressure increase tends to be more meshwork is also useful for the reduction of intraocular pres severe when visco-elastic agents such as hyaluronic acid are sure in individuals suffering form open angle glaucoma. injected into the eye during Surgery, the intraocular pressure Human shASEGP can be administered by subconjunctuval can become elevated postoperatively even when such agents injection or injection directly in the anterior chamber. Since are not utilized. Furthermore, Such a pressure increase can 35 the shASEGP acts enzymatically, even relatively limited occur even when no additional medications are used during amounts of shASEGP can effectively function as a viscoelas the Surgical procedure. In some cases, it is advantageous to tic antidote or for other situations that are aided or improved leave a viscoelastic agent in the eye, which often necessitates by application of a glycosaminoglycanase. As with other giving patients large doses of carbonic anhydrase inhibitors. uses, the units of hyaluronidase activity provided by the shA These inhibitors lower the intraocular pressure by decreasing 40 SEGP for optimum application will depend in part on the the formation of aqueous humor, a fluid that is normally particular situation being treated—especially the amount of secreted in the eye, by the ciliary body. Current methods for glycosaminoglycan degradation that is desired—but can be relieving postoperative pressure increases in the eye include readily assessed using appropriate models and then optimized various types of eye drops such as beta-adrenergic blocking as appropriate for particular uses. By way of illustration, the agents, sympathomimetic agents, miotics, alpha II selective 45 units of shASEGP to be applied as an antidote will be influ agents, carbonic anhydrase inhibitors and prostaglandin enced, inter alia, by the particular procedure employed and agents. the amount of viscoelastic applied, and also on whether any of A preferred method of removing the Viscoelastic Such as the applied viscoelastic is to be removed using other proce hyaluronic acid is by injection of shASEGP during or imme dures such as irrigation and aspiration. Where shASEGP is diately following anterior segment or posterior segment Sur 50 used following irrigation and aspiration, generally amounts gical procedures, although other methods of administration providing in the range of 10 to 12,000 units, typically 100 to known in the art are possible as well. It is preferred if the 5,000 units, more typically 500 to 2,000 units may be applied, hyaluronic acid and the shASEGP are administered by injec although this will again depend on the particular application. tion into the anterior chamber during anterior segment ocular Where shASEGP is used to obviate the need for irrigation Surgical procedures to allow the hyaluronic acid to act as a 55 and aspiration, larger amounts will be applied in order to spacer during the start of the Surgical procedure. In some effectively digest a larger Volume of applied viscoelastic, cases of corneal transplantation, the hyaluronic acid and with amounts generally providing in the range of 50 to 20,000 sHASEGP combination may be placed on the surface of the units, typically 400 to 10,000, more typically 1,500 to 5,000 intraocular structures prior to Suturing the corneal transplant units may be applied, although this will again depend on the in place. This combination may also be used in posterior 60 particular application. segment Surgery, such as retina or vitreous Surgery. In the case of shASEGPs being used to reduce intraocular In some cases, it may be advisable to leave a visco-elastic pressure associated with glaucoma, application of SHA agent such as HealonTM, ViscoatTM, or other space-occupying SEGPs can also be used as a primary treatment to reduce IOP substances in the anterior chamber of the eye at the conclu associated with glaucoma and thereby potentially avoid the sion of surgery. This is especially true in positive pressure rise 65 need for Surgical procedures such as glaucomal filtration, when the intraocular contents tend to come forward and press glaucomal drainage implants (tube shunts), laser trabeculo against the posterior Surface of the cornea. If this occurs in an plasty, cyclophotocoagulation or iridotomy. Thus SHA US 7,871,607 B2 121 122 SEGPs of the present invention can be introduced, for administered intravenously (IV) and allowed to perfuse the example, by injection into the anterior chamber of the eye to CNVM, as well as the remainder of the body. An ophthal provide a minimally invasive means for reducing intraocular mologist then generally treats the CNVM with a red laser of pressure associated with glaucoma. Without wishing to be a specific wavelength (e.g. 689 nm) for about 90 seconds. The bound by theory, it is believed that the administration of 5 non-thermal laser light activates the dye (such as VISU sHASEGPs in such settings can enhance fluid outflow via the DYNETM) producing an active form of oxygen that both trabecular meshwork and thus lower IOP by reducing hydro coagulates and reduces the growth of abnormal blood vessels, static and/or oncotic pressures within the anterior chamber. which in turn inhibits the leakage of fluid from the CNVM. In the case of shASEGPs being used to used to dissolve sHASEGPs of the present invention can be used, for example, vitreous aggregates or “floaters', which are generally macro 10 to promote more localized administration of a dye (such as scopic opaque aggregates of vitreous fibers, the administra VISUDYNETM) directly to the eye; and/or to facilitate the tion of shASEGP to the vitreous body can be used to promote delivery of other agents that are usefully administered to the disaggregation. eye in connection with procedures such as PDT. Similarly, shASEGPs as described herein can be applied to Ganglion Cysts the vitreous to promote the resolution of vitreous hemor 15 The ganglion cyst (also known as a wrist cyst, Bible cyst, or rhages (i.e. extravasation of blood into the vitreous) which dorsal tendon cyst) is the most common soft tissue mass of the can occur in connection with conditions such as diabetic hand. It is a fluid filled sac that can be felt below the skin. It is retinopathy, retinal neovascularization, retinal vein occlu usually attached to a tendon sheath (lining which lubricates Sion, posterior vitreous detachment, retinal tears, ocular trau the tendon) in the hand or wrist or connected with an under mas and the like. The presence of vitreous hemorrhages, lying joint; however, some have no obvious connection to any which are typically slow to resolve, can delay, complicate or structures. These may also occur in the foot. It often occurs prevent procedures that require the retina to be visualized when there is a tear in the ligaments overlying the lining of through the vitreous for diagnosis and/or for treatment pro tendons or joints and the lining herniates out of the ligamen cedures such as laser photocoagulation and the like which are tous defect causing a bump under the skin. Because there is often primary treatments for conditions such as proliferative 25 often inflammation associated, the inflamed tissue produces a diabetic retinopathy. Use of shASEGPs to promote resolu jelly-like fluid that fills the protruding sac. They may be rock tion of Such vitreous hemorrhages can thus be used to facili hard due to a high pressure of the mucous like fluid contained tate diagnosis of retinal conditions and/or to obviate the need within the cyst, and are often mistaken for a bony promi for more radical procedures such as vitrectomy. CCC. In the case of shASEGPs being used to promote vitreo 30 sHASEGP can be used to ameliorate ganglion cysts. retinal detachment in treating conditions such as diabetic Intralesional injection of shASEGP from 5-1000 Units fol retinopathy, the administration of shASEGP into the vitreous lowed by fine needle aspiration will remove the cyst without can be used to promote detachment or uncoupling of the the need for Surgery. Corticosteroids may be optionally vitreous body from the retina. In this context, shASEGP may injected as well with the shASEGP. Additional injection may not only promote the detachment but help it to occur in a 35 be required for Some patients. manner that reduces the potential for retinal distortion or Myxedema tearing. Glycosaminoglycan (GAG) infiltration of the skin is a fea sHASEGPs of the present invention can also be used for ture of hyperthyroidism, hypothyroidism, pretibial myxe treating corneal opacities. In that regard, a variety of distor dema, Scleromyxedema, and Scleredema. Hyaluronic acid is tions or deformities can affect the normally organized struc 40 the main GAG in all the conditions and in normal skin. There ture of the cornea and lead to disruptions in the passage of is minimal histologic variability of GAG dermal distribution. light through the cornea and concomitant impairments in The acquired cutaneous mucinoses exhibit similar skin GAG vision. Thus, a number of conditions and causes lead to cor distribution and biochemical composition. The morphologic neal distortions that may appear as Scars, hazing or opacifi differences in fibroblastic activity Suggest that the mucinoses cation. In applying shASEGPs to the improvement or treat- 45 of scleredema and Scleromyxedema represent a local process, ment of such corneal conditions, the shASEGP (in a whereas the GAG infiltration of thyroid diseases may have a pharmaceutically acceptable solution which may comprise systemic origin. These disorders may be ameliorated with a one or more additional pharmacologic agents) can be intro sHASEGP from both a local and systemic route of adminis duced to the cornea by any of several routes of administration, tration. For chronic therapy, a PEGylated shASEGP may be depending in part on the extent and location of the corneal 50 envisioned. injury. By way of illustration, shASEGPs can be introduced Pulmonary Uses of shASEGP onto the outer Surface of the eye (using drops or other topical Levels of Hyaluronan in broncheoalveolar lavages (BAL) application for example), potentially encapsulated within car from normal individuals are generally below 15 ng/ml. How riers such as liposomes and the like that can be used to ever, BAL levels rise dramatically in conditions of respiratory facilitate transfer across the corneal epithelium, by injection 55 distress (Bjermer Br MedJ (Clin Res Ed) 1987 Oct. 3; 295 or microinjection onto or into the cornea itself, or by other (6602): 803-6). In ARDS for example, hyaluronan levels can approaches to the corneal tissue. In addition to biochemical or increase to 500 ng/ml whereas in farmers lung, BAL levels mechanical aids for promoting delivery, other means for pro can surpass 1000 ng/ml (Hallgren et al Am Rev. Respir Dis. moting permeation Such as iontophoresis can likewise be 1989 March: 139(3): 682-7), (Larrson etal Chest. 1992 Janu applied. 60 ary; 101(1): 109-14). The increased hyaluronan in the lung sHASEGPs of the present invention can also be used, for can prevent oxygen diffusion and gas exchange as well as example, to enhance the permeability of agents employed in activating neutrophil and macrophage responses. photodynamic therapy (PDT). PDT is a treatment for choroi Bovine preparations of hyaluronidase are no preferable for dal neovascular membranes (CNVM), which are generally the treatment of Such conditions for a number of reasons. leaky vascular structures under the retina in the “wet' form of 65 First, slaughterhouse testes-derived preparations of hyalu age-related macular degeneration (AMD). In PDT, a photo ronidase are known to be contaminated with serine proteases sensitive dye such as VISUDYNETM (verteporfin) is typically Such as acrosin. Secondly, the foreign nature of the bovine US 7,871,607 B2 123 124 enzymes increase the probability of an anaphylactic reaction, mMsaccharolactone for lysosomal Hyaluronidase; or 10 mM which could result in death of the patient. Thus a highly Hepes PH 7.4 with 1 mM CaCl and 1 mg/ml Human Serum purified preparation of recombinant human shASEGP can be Albumin (ICN) for neutral-active enzymes. A set of standards delivered by either pulmonary or intravenous delivery. for the calibration of enzyme activity against “relative Tur Human shASEGP can also be administered to patients suf- 5 bidity Reducing Units” (rTRU's) was generated by diluting fering from other pulmonary complications that are associ bovine testicular hyaluronidase (Sigma Type VI-S) in neutral ated with elevated glycosaminoglycans or to enhance the enzyme buffer from 1.0 to 1x10 rTRU/well and assaying delivery of other co delivered molecules to the lung. 100 ul/well in triplicate. Samples of acid-active Hyalu The invention will now be described in greater detail by ronidase were diluted in lysosomal assay buffer from 1:10 to reference to the following non-limiting examples 10 1: 130,000 were pipetted in triplicate at 100 ul/well. For most assays of tissue extracts and human plasma, a 30 min incu EXAMPLE1 bation at 37°C. was sufficient. Positive and negative control wells (no enzyme or no ABC (see below), respectively) were Microtiter Based Hyaluronidase Assays included in triplicate. The following example provides for a rapid assay for mea- 15 The reaction was terminated by the addition of 200 ul/well surement of the hyaluronidase activity of shASEGP. This of 6M Guanidine HC1 followed by three washes of 300 assay can be related to the TRU, the IU or NFU through use of ul/well with PBS, 2 MNaCl, 50 mMMgSO, 0.05%TWEEN a W.H.O. standard preparation of hyaluronidase. 20 detergent (Buffer B). An avidin biotin complex (ABC) kit Biotinylated Hyaluronan Microtiter Assay (Vector Labs: Burlingame Calif.) was prepared in 10 ml of The free carboxyl groups on glucuronic acid residues of 20 PBS containing 0.1%TWEEN 20 detergent, which was pre Hyaluronan are biotinylated in a one step reaction using incubated for 30 min at room temperature during the incuba biotin-hydrazide (Pierce), Sulfo NHS (Pierce) and 1-Ethyl tion. The ABC solution was added (100 ul/well) and incu dimethylaminopropyl-carbodiimide (Sigma). This biotiny bated for 30 min at room temperature. The plate was washed lated HA substrate is covalently coupled to a 96 well micro five times with Buffer B, then an o-phenylenediamine (OPD) titer plate in a second reaction. At the completion of the 25 substrate was added at 100 ul/well by dissolving one 10 mg enzyme reaction, residual Substrate is detected with an avi tablet of OPD in 10 ml of 0.1 M citrate-PO, buffer, pH 5.3 and din-peroxidase reaction that can be read in a standard ELISA adding 7.5ul of 30% HO. The plate was incubated in the plate reader. As the substrate is covalently bound to the micro dark for 10-15 min, then read using a 492 nm filter in an titer plate, artifacts such as pH-dependent displacement of the ELISA plate reader (Titertek Multiskan PLUS. ICN) moni biotinylated substrate does not occur. The sensitivity permits 30 tored by computer using the Delta Soft II plate reader soft rapid measurement of Hyaluronidase activity from cultured ware from Biometallics (Princeton N.J.). A standard curve cells and biological samples with an inter-assay variation of using the bovine testicular hyaluronidase was generated by a less than 10%. four parameter curve fit of the commercial hyaluronidase a. Protocol preparation and unknown samples were interpolated through Preparation of Biotinylated HA Substrate 35 their absorbance at 492 nm. One hundred mg of HA (Sigma Chemicals) was dissolved To analyze pH dependence of Hyaluronidases, purified in 0.1MMES, pH 5.0, to a final concentration of 1 mg/mland recombinant shASEGP and bovine testicular hyaluronidase allowed to dissolve for at least 24 hr at 4°C. prior to coupling are used. The pH dependence of enzyme activity is measured of biotin. Sulfo-NHS (Pierce; Rockford Ill.) was added to the by diluting purified shASEGP or partially purified bovine CSO MES solution to a final concentration of 0.184 mg/ml. 40 testicular hyaluronidase to 0.1 rTRU in the following buffers: Biotin hydrazide (Pierce) was dissolved in DMSO as a stock 50 mM formate, pH 3-4.5; 50 mM acetate, pH 5-6; 50 mM solution of 100 mM and added to the CSO solution to a final MES, pH 6-7: or 50 mM HEPES, pH 7-8. Samples are concentration of 1 mM. A stock solution of 1-ethyl-3-(3- assayed for 30 min at 37° C. and activity was expressed as a dimethylaminopropyl)carbidodiimide (EDAC) was prepared percent of maximal activity. NaCl was not used in buffers, as as a 100 mM stock solution in distilled water and added to the 45 it can alter the pH optima of testicular hyaluronidase prepa HA biotin solution at a final concentration of 30 mM. This rations (Gold, Biochem. J. 205: 69-74, 1982; Gacesa et al. solution was left stirring overnight at 4°C. Unlinked biotin Biochem. Soc. Trans. 7: 1287-1289, 1979); physiological salt and EDAC were removed by dialysis against water with 3 concentrations (0.15 M) decreased the apparent pH optimum, changes of 1000x volume of water. The dialyzed, biotinylated an effect that was more pronounced in purified preparations HA (bHA) was aliquoted and stored at -20° C. for up to 50 of the testicular enzyme than in the original crude sample. several months. b. Results Sulfo-NHS was diluted to 0.184 mg/ml in water with the Hyaluronan was biotinylated in a one step reaction using bHA at a concentration of 0.2 mg/ml and pipetted into 96 well biotin-hydrazide and EDAC. By limiting the EDAC, which COVALINK-NH plates (NUNC: Placerville N.J.) at 50 ul per couples the free carboxyl groups on HA with biotin well. EDAC was diluted to 0.123 mg/ml in water and pipetted 55 hydrazide, only a small fraction of the total glucuronic acid into the COVALINK-NH plates with the bHA solution result residues on HA were labeled. This amount of EDAC (3x10 ing in a final concentration of 10 ug?well bh A and 6.15 M) added to HA (2.8x10 M) results in a maximum of one ug?well EDAC. The plates were incubated overnight at 4°C. molecule of biotin hydrazide coupled per 93 disaccharide or for 2 hr at 23°C., which gave comparable results. After units of HA. covalent immobilization of b0S04 on the microtiter plates, 60 A four-parameter curve fit of bovine testicular hyalu the coupling Solution was removed by shaking and the plates ronidase standard reactions measured at pH 3.7, and diluted were washed 3 times in PBS containing 2MNaCl and 50 mM from 1.0 to 1x10'TRU/well, was prepared. Four parameter MgSO4 (Buffer A). The plates could be stored at 4°C. for up curve fits were established from the equation y=((A-D)/(1+ to one week. (conc/C)B))+D) where logy=ln(y'/1-y"), y'-(y-D)/(A-D), The COVALINK-NH plates with immobilized bHA were 65 B=-b/in 10 and C=EXP(a/B). The four parameters (A,B,C, equilibrated with 100 ul/well assay buffer—either 0.1 M for D) were calculated with a software program that utilized the mate, pH 3.7, 0.1 M NaCl, 1% TRITON X-100 detergent, 5 2+2 algorithm with linear regression (Rodbard et al., Clin. US 7,871,607 B2 125 126 Chem. 22:350, 1976). This curve fit incorporates the sigmoi truncated deletion mutants lacking predicted GPI anchor dal aspects the standard curve. Optimal accuracy for mea starting at position Y 482 and deleted progressively by one surement of a sample typically occurs from 0.001 to 0.01 amino acid. These primers were designed to have compatible TRU/well for a 30 min incubation. During a 60 min incuba Nhel (5') and BamHI (3') sites to clone the truncation mutants tion, /1000th of a TRU is detectable. A standard logarithmic in vector Irespuro2 either untagged with a stop codon in the 3' curve also can be utilized over a shorter range of values to primer, or as a C terminus His tagged protein for ease of establish a standard curve fit. Although the invention has been purification and detection. For example reverse primers SEQ described in connection with specific preferred embodi ID No. 8, SEQ ID No. 9, and SEQ ID No. 10 were used to ments, it should be understood that the invention as claimed generate deletion mutants ending at positionY482, F481 and should not be unduly limited to such specific embodiments. 10 I 480 without a 6 His tag. Other mutant primers were gener ated with the same base design with the appropriate modifi EXAMPLE 2 cations to include and exclude the particular amino acids. For generating His-tagged variants the same set of primers are Cloning of shASEGP cDNA used as for non tagged variants except that primers lack the Nucleic acid encoding Human shASEGP may be obtained 15 stop codon in the respective reverse primers, the forward by one skilled in the art through a number of procedures primer remaining the same (for His tagged construction refer including, but not limited to, artificial gene synthesis, RT to primers with SEQ ID No 19, 20, 21, 22, 23, 24 and 25 PCR, and cDNA library hybridization (for example see, which are the reverse primers without stop codon correspond Gmachl et al FEBS 336(3) 1993, Kimmel et al., Proc. Natl. ing to non tagged reverse primers for their respective con Acad. Sci. USA 90 1993 10071-10075). Alternatively, clones structs). Overlapping primers were used to construct a six encoding human shASEGP may be obtained from IMAGE, amino acid spacer followed by hexahistidine within BamHI or other Suppliers of human gene sequences (Invitrogen and Not sites in Irespuro2 vector Such that His-tagged Clone IDIOH10647). mutants were generated by ligation of the PCR amplified and The full length human PH20 cl NA was calculated to be restriction digested products within the NheI and BamHI sites 2009 nucleotides in length and contained an open reading 25 in the his tag containing Irespuro2 vector. frame of 1530 nucleotides. The 5' UTR is unusually large, To identify whether human shASEGP could be modified which can indicate a retained intron and can inhibit transla at its carboxy terminus to generate a secreted and neutral tion by preventing the ribosome from binding to the correct active enzyme, a series of truncations were made from the initiating methionine codon due to 9 non coding start codons GPI anchor attachment site to the predicted “catalytic in the 5' UTR. The protein (Genbank Accession number 30 domain based upon homology with the bee venom enzyme. NP 003.108) is predicted to comprise 509 amino acids SEQ DNA encoding the human shASEGP full length GPI ID No. 1 with a calculated molecular mass of 58 kDa. anchored clone in IRESPuro2 was used as a template to For sequencing of clones, PCR amplified bands were generate the various truncated deletion mutants. Software excised, and eluted with the Gel Extraction Kit (Qiagen) and modeling programs gave several predicted cleavage sites for cloned into the appropriate vectors with compatible ends after 35 the full length polypeptide. One of such predicted sites was at restriction digestion. All sequencing reactions were per amino acid position N483 (SEQID No. 1). PCR primers were formed on double stranded DNA with the Taq dye deoxy designed to successively truncate the protein from N483 to terminator cycle sequencing kit (Applied Biosystems) generate six deletion mutants starting at Y 482 (lacking N) according to the manufacturers instructions, and run on an and ending at E 477 (lacking P). ABI PrismTM automated sequencer (Applied Biosystems). 40 a. Protocol The human PH-20 open reading frame was obtained by amplifying a human testis clNA library (Clontech, Palo Alto Generating Truncation Mutant Lacking N483: Calif.) by Polymerase Chain Reaction using primers SEQID The full length GPI anchored shASEGP clone between NO 14 and SEQID NO.47. PCR products were digested with Nhe and BanhI site in plRESPuro2 was used as a template. Nheland BamHI and cloned into the Nheland BamHI sites of This template was amplified with 5' primer containing Nhel 45 site that starts at starting Methionine of the native signal the vector IRESpuro2 (Clontech). peptide at M1 (SEQID No. 14), and a 3' primer containing EXAMPLE-4 BamHI site that ends at Y 482 (SEQ ID No. 8). The PCR product was ran on a 1% agarose gel to resolve and confirm Isolation of SHASEGP from Human PH2O cDNA 50 the correct sized amplified band, gel purified, restriction A catalytically active secreted recombinant human SHA digested with Nhe and BamHI and cloned into vector plRE SEGP expression vector capable of effective glycosylation in SPuro2 (Clontech) between Nheland BamHI sites generating mammalian cells was generated as described below. Other an expression vector for expressing this truncation mutant of expression constructs with promoters and selection genes for SHASEGP ending at amino acid position N482 and lacking different species Such as yeast and insect cells that are also 55 the GPI anchor with amino acid sequence (SEQID No. 5 for capable of generating shASEGP are contemplated. Positive the sequence of the resulting polypeptide of shASEGP up to selection genes such as Glutamine Synthase or Dihydrofolate Y 482) and nucleotide sequence (SEQ ID No. 48—coding Reductase (DHFR) may also be used. The examples given nucleotides for polypeptide in SEQID No. 5) as indicated. below is not intended to restrict but is rather provided as an Generation of the other truncation mutants lacking Y 482, example of several plasmid expression systems that may be 60 F481, I 480, Q 479, and P478 respectively. used. The same strategy was used with the only difference being In order to construct secreted forms of shASEGP, trunca using the appropriate 3' primer for each mutant. The respec tion mutants that lack the hydrophobic C terminal end were tive 3' primers are as follows: constructed. Using a GPI cleavage prediction program the 3' primer for shASEGP mutant that lacks the Y 482-SEQ GPI anchor cleavage site was located around amino acid 65 ID No. 9 position N 483 in the full-length GPI-anchored protein. A set 3' primer for mutant that lacks the F 481-SEQID No. 10 of seven nested 3'primers were used to construct a set of seven 3' primer for mutant that lacks the 1480-SEQ ID No. 11 US 7,871,607 B2 127 128 3' primer for mutant that lacks the Q 479-SEQID No. 12 EXAMPLE-5 3' primer for mutant that lacks the P478-SEQID No. 13 Generating further deletion mutants to determine the mini Effects of Signal Peptide Modification on shASEGP mally active domain of shASEGP: Secretory Activity Further deletions, in blocks often to twenty amino acids were made from the 3' end of innermost neutral pH active Human shASEGP possesses an unusually long predicted truncation mutant of shASEGP, which is shASEGP up to E native leader peptide. Additionally, the existence of two adja 477. The Nhel forward primer SEQID No. 14) was used with cent cysteine residues in the leader peptide may lead to aggre an appropriately positioned 3' primer to PCR amplify a dele gation of polypeptide multimers within the endoplasmic tion mutant of shASEGP of the desired length from the 10 reticulum during high level expression and therefore prevent carboxy terminal end. For example PCR with primers high level expression of a shASEGP. A series of more effi described in SEQID No. 14 and SEQID No. 26 as the 5' and cient secretory leader peptides were therefore tested to exam 3' primers respectively was used to generate the polypeptide ine for their ability to enhance the targeting of shASEGP for in SEQID No. 49 when expressed from an expression con secretion. struct in IresPuro2 vector. Similarly, PCR with reverse 3' 15 a. Protocol primers described in SEQ ID No 27, 28, 29, 30, 31 and 32 The Kappa leader peptide was constructed by overlapping were used to generate deletion mutants ending at amino acid primer annealing and extension PCR with primers corre positions A 447, S 430, G 413, S 394, A 372, and S 347 sponding to sequences in SEQID No. 37, 38, 39 and 40. The respectively of the mature shASEGP. The PCR products in resulting PCR amplified kappa sequence was amplified with each case were digested with Nhel and BamHI enzymes and flanking primers containing Nhe site in the 5' end (as the digested product cloned into pIresPuro2 vector between described in SEQID No. 41) and EcoRI site at the 3' end (as Nhe and BamHI sites. A few independent clones in the final described in SEQID No. 42). This allowed cloning the Kappa expression construct from each group were tested for secreted leader peptide (the polypeptide sequence is as described in neutral active shASEGP activity by transient transfection in SEQID No. 43) in the Litmus 39 (NEB) vector between Nhel CHO cells in CD-CHO serum free media (Invitrogen, CA) 25 and EcoRI sites. SHASEGP has an internal EcoRI site; there and samples drawn at indicated time points for assay. Mini fore this kappa construct between Nhe site and EcoRI site prep DNA prepared from overnight cultures was transfected was further amplified with a 5' Spel primer (as described in with Genejuice (Novagen, CA) transfection reagent follow SEQID No. 44) and a 3' Mlul primer (as described in SEQID ing manufacturer recommended protocols. Hyaluronidase No. 45). shASEGP without GPI anchor ending at P 478 was 30 cut out from piresPuro2 with NheI and BamHI and cloned activity was measured by microtiter assay as described above. into a Litmus 39(NEB) vector within the Nhel and BamHI b. Results sites of the Litmus39 vector. This resulting shASEGP-con Hyaluronidase activity was measured in shASEGP trun taining Litmus vector was digested with Spel and Mlul cation mutants to identify the minimally active domain for restriction enzymes and the kappa leader construct amplified secreted neutral active hyaluronidase activity. 35 with Spel and Mlul was cloned into it. Site directed mutagen esis was performed on this Litmus 39 vector containing both Kappa and shASEGP sequences to generate the in frame fusion of Kappa leader sequence to the mature polypeptide of AMINO ACID 1 TO: UML 24 HRSPH 7.4 sHASEGP. Primer pairs corresponding to SEQID No.34 and 347 O.OOO 40 35 were used to generate the kappa leader with the native Asp 372 O.OOO 394 O.OOO as the terminal amino acid fused to the F38 of shASEGP (up 413 O.OOO to P478) (as described in SEQID No. 46 for the polypeptide 430 O.OOO sequence of the fusion protein). Other primer pair combina 447 O.OOO tions such as embodied by SEQID No. 33 with SEQID No. 467 O.O89 45 35 were used to generate Kappa leader ending at the terminal 477 0.567 478 O.692 Asp (D) fused to L36 of SHASEGP, SEQID No.33 with SEQ 479 0.750 ID No. 36 were used to generate Kappa leader ending at the 480 0.575 Gly (G) (before the terminal Asp (D)) fused to L 36 of SHA 481 O.740 482 O.329 SEGP, and SEQID No. 34 with SEQID No. 36 were used to 50 483 O.800 generate Kappa ending at the Gly (G) (before the terminal 509 O.O44 Asp (D)) fused to F 38 of SHASEGP. The Kappa shA SEGP fusions obtained by site directed mutagenesis were gel purified, digested with enzyme DpnI to digest any carryover The results showed that all six one amino acid deletion parental DNA, and then digested with Nhe and Bam HI and mutants ending at indicated amino acids from Y 482 to E 477 55 cloned in to the NheI/BamHI digested HisresPuro2 back gave higher secreted activity than GPI anchored shASEGP. bone which has the his tag (six amino acid spacer followed by The results also showed that deletions beyond A467 elimi six histidines) cloned in between BamHI and NotI sites in nated any secreted activity. Secreted neutral activity from the pIRESPuro2 vector. Therefore upon ligation we obtain a con A 467 clones decreased to approximately 10% of that found struct that is Nhe-kappa-SHASEGP-BamHI-His in pre P478 or N483 clones. It was therefore concluded that more of 60 sPuro2. Four sets of such construct were obtained that would the carboxy terminal domain of human shASEGP was correspond to the combinations of G or D at the Kappa leader required to create the neutral active hyaluronidase domain end and L36 or F38 at the beginning of mature shASEGP. A than previously assumed from the bee venom enzyme. The few independent clones from each type of construct were cysteines in the carboxy terminal domain are thus necessary transfected into CHO cells in CD-CHO medium (Invitrogen, for neutral activity. A very narrow range spanning approxi 65 CA) to test whether the presence of kappa secretion leader mately 10 amino acids before the GPI cleavage site at N 483 would promote increased levels of secreted protein as com thus defined the minimally active domain. pared to native secretion leader. Miniprep DNA prepared US 7,871,607 B2 129 130 from overnight cultures were transfected with Genejuice EXAMPLE-7 (Novagen, CA) transfection reagent following manufacturer recommended protocols and samples were drawn for testing Generation of a shASEGP Expressing Cell Line by microtiter assay at indicated time points. Hyaluronidase Non-transfected DG44 CHO cells growing in GIBCO activity was measured by microtiter assay as described above. 5 Modified CD-CHO media for DHFR(-) cells, supplemented with 4 mM Glutamine and 18 ml Plurionic F68/L (Gibco), Mouse IgG Kappa chain leader peptide shASEGP fusion were seeded at 0.5x10° cells/ml in a shaker flask in prepara constructs were tested to test for higher levels of secreted tion for transfection. Cells were grown at 37°C. in 5% CO neutral active shASEGP activity. humidified incubator with 120 rpm for shaking. Exponen b. Results 10 tially growing non-transfected DG44 CHO cells were tested for viability prior to transfection. 60,000,000 viable cells of the non-transfected DG44 CHO cell culture was pelleted and resuspended to a density of UML 24 HOURS 20,000,000 cells in 0.7 mL of 2x transfection buffer HUMAN SHASEGP GENE CONSTRUCT PH 7.4 15 (2xHeBS-40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mM IgG Kappa LeadershASEGPAA38–478 HIS6 3.0257 KCl, 1.4 mMNaHPO, 12 mM dextrose). To each aliquot of Native Leader SHASEGPAA 1-478 HIS6 O.4857 resuspended cells, 0.09 mL of the linear HZ24 plasmid (250 ug) was added, and the cell/DNA solutions were transferred into 0.4 cm gap BTX (Gentronics) electroporation cuvettes at The enzyme assay results indicated that the IgG Kappa room temperature. A negative control electroporation was leader was capable of enhancing secretion of shASEGP performed with no plasmid DNA mixed with the cells. The approximately 7 to 8 fold higher than the native secretion cell/plasmid mixes were electroporated with a capacitor dis leader when compared with clones P478, Y482 or N483 that charge of 330 V and 960 uF or at 350 V and 960 uF. lacked such a leader. Other kappa leader constructs with The cells were removed from the cuvettes after electropo variations of the leader fusion site from the Asp or the Gly of 25 ration and transferred into 5 mL of Modified CD-CHO media the Kappa leader to L36 or F38 of shASEGP yielded for DHFR(-) cells, supplemented with 4 mM Glutamine and increased levels of secreted neutral active hyaluronidase 18 ml Plurionic F68/L (Gibco), and allowed to grow in a well activity as well. These examples are intended to expand rather of a 6-well tissue culture plate without selection for 2 days at 37° C. in 5% CO, humidified incubator. than limit the scope of the invention, as other efficient secre 30 tory leader sequences may be utilized with the same technol Two days post electroporation, 0.5 mL of tissue culture media was removed from each well and tested for presence of Ogy. hyaluronidase activity. EXAMPLE 6 Initial Hyaluronidase Activity of HZ24 Transfected DG44 CHO Cells at 40 Hours Post Transfection 35 Generation of a Human shASEGP Expression Vector A shASEGP without an epitope tag was generated by Activity cloning into a bicistronic expression cassette, HZ24 (SEQID Dilution UnitSiml NO 51). The HZ24 plasmid vector for expression of shA 40 SEGP comprises a pCI vector backbone (Promega), DNA Transfection 1 1 to 10 O.25 330 V sequence encoding amino acids 1-482 of human PH20 hyalu Transfection 2 1 to 10 O.S2 ronidase, an internal ribosomal entry site (IRES) from the 350 V ECMV virus (Clontech), and the mouse dihydrofolate reduc Negative 1 to 10 O.O15 Control tase (DHFR) gene. The pCI vector backbone also includes 45 DNA encoding the Beta-lactamase resistance gene (Ampk), an fl origin of replication, a Cytomegalovirus immediate Cells from transfection 2 (350V), were collected from the early enhancer/promoter region (CMV), a chimeric intron, tissue culture well, counted and diluted to 10,000 to 20,000 and an SV40 late polyadenylation signal (SV40). The DNA viable cells per mL. A 0.1 mLaliquot of the cell suspension encoding the shASEGP construct contained a Kozak consen 50 was transferred to each well of five, 96 well round bottom Sus sequence in the Methionine of the native signal leader and tissue culture plates. 0.1 mL of CD-CHO media (GIBCO) a stop codon at Tyrosine 482. The resultant construct pCI containing 4 mMGlutamaX-1, and without hypoxanthine and PH20-IRES-DHFR-SV40pa (HZ-24) results in a single thymidine Supplements were added to the wells containing mRNA species driven by the CMV promoter that encodes cells (final volume 0.2 mL). amino acids 1-482 of PH20 and amino acids 1-187 of the 55 Ten clones were identified from the 5 plates grown without dihydrofolate reductase separated by the internal ribosomal methotrexate. entry site. The human PH20 open reading frame was amplified from an Invitrogen ORF clone (IOH10647. Invitrogen, Carlsbad 60 Relative Calif.) with a 5' Primer that introduced an Nhe site and Plate Well Hyaluronidase Kozack consensus sequence before the Methionine of PH20 ID Activity and a reverse primer that introduced a stop codon following 1C3 261 2C2 261 Tyrosine 482 and introduced a BamHI restriction site. The 3D3 261 resultant PCR product was ligated into the plasmid plRE 65 3E5 243 Spuro2 (Clontech, Palo Alto, Calif.) following digest of the 3C6 174 PH20 PCR fragment with Nhel and BamHI. US 7,871,607 B2 131 132 -continued -continued Relative Plate Well Hyaluronidase 3D3-5M Growth & SEIASEGP Production, 5L Bioreactor ID Activity 2G8 103 Run Viable % Units 1B9 304 2D9 273 Hours Cells x 10E5 Viable ml Vol (mL) Glucose Feed 4D10 3O2 1E11 242 10 144 60.2 100 423 4500 161 A1 (+) 333 control 168 55 100 478 4500 92 250 ml H12 (-) O Feed #1 control 192 66.6 98 512 4750 370 216 55.2 92 610 4750 573 250 ml 15 Six HZ24 clones were expanded in culture and transferred Feed #2 into shaker flasks as single cell Suspensions. Clones 3D3. 240 53 88 710 SOOO 573 3E5, 2G8, 2D9, 1E11, and 4D10 were plated into 96-well 264 49.8 84 852 SOOO 474 250 ml round bottom tissue culture plates using a two-dimensional Feed #2 infinite dilution strategy. Diluted clones were grown in a 288 40 70 985 5250 770 background of 500 non-transfected DG44 CHO cells per well, to provide necessary growth factors for the initial days 312 31 61 1467 S2SO 773 in culture. Ten plates were made per Subclone. 336 25.4 52 1676 5250 690 Clone 3D3 produced 24 visual subclones. Significant hyaluronidase activity was measured in the Supernatants from 25 8 of the 24 subclones (>50 Units/mL), and these 8 subclones EXAMPLE 9 were expanded into T-25 tissue culture flasks in the presence of 50 nM methotrexate. Clone 3D3 50 nM was further Purification of SHASEGP expanded in 500 nM methotrexate giving rise to clones pro 30 ducing in excess of 1,000 Units/ml in shaker flasks (clone Conditioned media from the 3D3 clone was clarified by 3D35M). depth filtration and tangential flow diafiltration into 10 mM Hepes pH 7.0. Soluble HASEGP was then purified by sequen EXAMPLE 8 tial chromatography on Q Sepharose (Pharmacia) ion 35 exchange, Phenyl Sepharose (Pharmacia) hydrophobic inter Production of SHASEGP action chromatography, phenyl boronate (Prometics) and A vial of 3D3 5M was thawed and expanded from T flasks Hydroxapatite Chromatography (Biorad, Richmond, Calif.). through 1 L spinner flasks in CHO CDM (Invitrogen, Carls SHASEGP bound to Q Sepharose and eluted at 400 mM bad Calif.) supplemented with 100 nM Methotrexate and 40 Glutamax (Invitrogen). Cells were transferred from spinner NaCl in the same buffer. The eluate was diluted with 2M flasks to a 5 L bioreactor (Braun) at an inoculation density of Ammonium sulfate to a final concentration of 500 mMASO4 4.0x1OE5 viable cells per ml. Parameters were temperature and passed through a Phenyl Sepharose (low Sub) column, setpoint, 3.7 C, pH 7.2 (starting Setpoint), with Dissolved followed by binding under the same conditions to a phenyl Oxygen Setpoint 25% and an air overlay of 0-100 cc/min. At 45 boronate resin. The shASEGP was eluted from the phenyl 168 hrs, 250 ml of Feed #1 Medium (CD CHO+50 g/L Glu sepharose resin in Hepes pH 6.9 after washing at pH 9.0 in 50 cose) was added. At 216 hours, 250 ml of Feed #2 Medium mM bicine without AS04. The eluate was loaded onto a (CD CHO+50 g/L Glucose--10 mM Sodium Butyrate) was added, and at 264 hours 250 ml of Feed #2 Medium was ceramic hydroxyapatite resin at pH 6.9 in 5 mM PO41 mM added. This process resulted in a final productivity of 1600 50 CaCl2 and eluted with 80 mM PO4 pH 7.4 with 0.1 mM Units per ml with a maximal cell density of 6 million cells/ml. CaCl. The addition of sodium butyrate was found to dramatically The resultant purified shASEGP possessed a specific enhance the production of shASEGP in the final stages of production. activity in excess of 65,000 USP Units/mg protein by way of 55 the microturbidity assay using the USP reference standard. Purified shASEGP eluted as a single peak from 24 to 26 minutes from a Pharmacia 5RPC styrene divinylbenzene col 3D3-5M Growth & SHASEGPProduction. SL Bioreactor umn with a gradient between 0.1%TFA/HO and 0.1%TFA/ Run Viable % Units 60 90% acetonitrile/10% HO and resolved as a single broad 61 Hours Cells x 10E5 Viable ml Vol (mL) Glucose Feed kDa band by SDS electrophoresis that reduced to a sharp 51 O 4.4 100 O 4500 547 kDa band upon treatment with PNG ASE-F. N-terminal 24 5.7 100 O 4500 536 48 10.1 100 37 4500 5O1 amino acid sequencing revealed that the leader peptide had 72 17.1 99 62 4500 421 been efficiently removed. 96 28.6 99 118 4500 325 65 120 28.8 99 240 4500 274 N-terminal Amino Acid Sequence Biochemically Purified SHASEGP. US 7,871,607 B2 Position 1 2 3 4 5 6 7 8 9 10 11 Theoretical Leu ASn Phe Arg Ala Pro Pro Val Ile Pro ASn Observed — ASn Phe Arg Ala Pro Pro Val Ile Pro ASn 10 EXAMPLE 10 ity of the undigested band could be attributed to N-linked sugar residues. PNGaseF partial digestion showed a series of Analysis of DG44 CHO-Derived shASEGP Glycosyla intermediates shifting from untreated and progressive shift tion with longer treatment. Although bands were somewhat dif Conflicting data exists as to whether shASEGPs from 15 fuse on a 7% Gel, at least 6 different intermediate isoforms different species require glycosylation for their catalytic could be visualized. activity. For example, it is reported that enzymatically active bee venom hyaluronidase can be synthesized in cells that lack Treatment of shASEGP with Neuramimidase revealed glycosylation machinery, i.e. Such as E. coli. Moreover, treat that CHO cells were in fact capable of synthesizing sialated ment of purified bovine testes hyaluronidase with PNGase human shASEGP. Upon treatment with neuramimidase and did not inactivate enzyme activity (Yamagata et al 1997). Western Blot analysis of shASEGP on 7% Gels, CHO Other studies report loss of activity following deglycosyla derived Human recombinantsHASEGPrevealed an approxi tion and that disulfide bonds are additionally required. mately 1-3 kDa shift in mobility compared to untreated shA As all Such previous tests were made using either crude or SEGP. This is thus the first report of the generation of a partially purified preparations however, it was not apparent 25 substantially sialated human shASEGP. This is very valuable whether the loss of activity was a result of exposure of deg for both stability and to enhance serum half-life of a human lycosylated enzyme to contaminating proteases in the crude sHASEGP as native sperm shASEGP from many species preparations or a direct functional relationship between gly lacks Sialation and does not react with Sialic acid specific cosylation and catalytic activity. lectins. a. Protocol 30 FACE Analysis of shASEGP To determine if functional N-linked glycosylation could be Analysis of active shASEGP oligosaccharides by FACE introduced into human shASEGP using a CHO based expres analysis permits rapid determination of profiles of catalyti sion system under protein free conditions, a cDNA encoding cally active shASEGPs. human shASEGP-HIS6 was expressed in CHO cells using an Protocol IRESpurobicistronic cassette in chemically defined media. 35 Purified Hyaluronidase from the 3D35M clone was evalu Cells were grown for 72 hours in CHO CDM (Invitrogen/ ated using FACETM N-Linked Oligosaccharide Profiling Gibco) followed by concentration and tangential flow diafil (Prozyme). Oligosaccharides were cleaved from 128.7 g of tration on a Pellicon TFF unit (Millipore) with 30 kDa cutoff glycoproteins by enzymatic digestion with N-Glycanase membranes. The concentrate was exchanged with 10 mM (a.k.a. PNGase), labeled using the fluorophore ANTS, and Hepes PH 7.450 mM. NaCl. The diafiltrate was then loaded 40 separated by electrophoresis. The relative positions of the on a DEAE streamline sepharose resin and eluted with a NaCl oligosaccharide bands were determined by running the gradient from 0-1 M NaCl on a Pharmacia FPLC resin. sample and dilutions of the sample alongside an oligosaccha Human SHASEGP eluted between 10-30% NaCl. Levels of ride standard ladder which designated the migration distance sHASEGP in column fractions determined that the majority in Degree of Polymerization (DP) units. of enzyme was recovered in the 10-30% NaCl gradient. The 45 enzyme from the 10-30% NaCl gradient was then further Results purified through affinity chromatography on an IMAC resin The N-Profile for the Hyaluronidase sample consists often charged with Ni. Human shASEGP was eluted from the bands of which six (running concomitant with the oligosac IMAC resin afterwashing with 10 mMImidizole with 50 mM charide standard bands G5-G12) had intensities greater than Acetate PH 5.0. The protein was concentrated and dialyzed 50 9%. Furthermore, the band running alongside the G9 standard against 10 mM Hepes PH 7.4. The highly purified enzyme was the most intense with intensities of 35%-46%. was determined to possess a specific activity of 97,000 Units/ sHASEGP Oligosaccharide Analysis mg protein in the presence of 1 mM Calcium and 1 mg/ml HSA in the ELISA-based biotinylated substrate microtiter assay. 55 To detect changes in protein relative molecular mass, puri SHASEGP Degree of Percent fied human SHASEGP was treated with PNG ASE or Oligosaccharide Polymerization of Total Neuramimidase overnight followed by gel electrophoresis, 1 1564 1.2 electotransfer and western blot analysis with an HRP linked 2 13.68 3.4 3 11.61 1O.O anti His6 monoclonal antibody (Qiagen) and ECL detection. 60 4 10.04 10.4 b. Results 5 8.37 35.4 Western blot analysis determined that the human shA 6 7.32 9.7 7 6.14 9.0 SEGP produced in CHO cells was sensitive to PNG ASE 8 5.57 12.4 treatment. The relative molecule mass of human sEASEGP 9 3.84 2.3 revealed that the protein was highly glycosylated. Upon com 65 10 3.26 O.S plete overnight digestion with PNGASE, human shASEGP reduced to a single species confirming that mild heterogene US 7,871,607 B2 135 136 EXAMPLE 11 noglycans ran as a Smear in the middle of the gel, the digested products showed the majority of alcian blue stain running at Dependence of shASEGP N-Linked Glycosylation for the dye front with a small amount of material running as an Enzyme Activity incremental ladder. a. Protocol 5 Samples of purified HIS6 shASEGP were mixed with EXAMPLE-13 buffer containing Neuraminidase and PNG ASE with and without 50 mm Octylglucoside overnight at 37 C. Oligosac Effects of Metal Ions on SHASEGP Activation charides were verified to have been removed by gel shift from Western Blot analysis. 10 In addition to the requirement of glycosylation for optimal b. Results enzyme activity, human shASEGP was found to be activated with cations for optimal enzyme activity. In the process of purification, shASEGP was found to have a low specific activity following Successive chromatography steps. The SAMPLE UML 15 HIS6tagged shASEGP was found to have a very low specific No Rx 22.01 activity when purified to homogeneity from DEAE followed Neuraminidase ON 50 mMOG 23.57 by successive Ni-IMAC purifications. As IMAC resins can PNGaseF W 50 mMOG O.O chelate metal ions, various metals were added back to shA PNGaseF without 50 mMOG on 10.74 SEGP to determine the relative enzyme activity. a. Protocol EXAMPLE-12 Purified shASEGP was tested following incubation with 0.1 mM nickel (Ni). Cobalt (Co) Zinc (Zn) Calcium (Ca) and Activity of shASEGP Towards Sulfated and Non-Sulfated Magnesium (Mg) for 2 hours at room temperature followed Glycosaminoglycans 25 by determination of hyaluronidase activity in microtiter In addition to the microtiter-based assay using HA, the based assay. substrate specificity of shASEGP towards other glycosami b. Results noglycans or proteoglycans can be tested using a gel shift assay with purified substrates to determine the activity of sHASEGP towards other glycosaminoglycans. Many Hyalu 30 ronidase assays have been based upon the measurement of the Metal Salt Additive Neutral Activity U/ml generation of new reducing N-acetylamino groups (Bonner NOADDITIVES 11.909 100 uMNi 6.0306 and Cantey, Clin. Chim. Acta 13: 746-752, 1966), or loss of 100 uM Co 8.972 viscosity (De Salegui et al., Arch. Biochem. Biophys. 121: 100 uMZn 3.7476 548-554, 1967) or turbidity (Dorfman and Ott, J. Biol. Chem. 35 100 uM Ca 101.9892 172: 367, 1948). With purified substrates all of these methods suffice for determination of the presence or absence of endo glucosamidic activity. A significant increase in hyaluronidase activity was found a. Protocol following incubation of shASEGP with 0.1 mM Calcium or GEL SHIFT ASSAY Purified Substrates are mixed with 40 0.1 mM Magnesium. No such activation was found following recombinant shASEGP to test for endoglucosidase activity incubation with other metals. The addition of Calcium to that give rise to increased mobility in substrate within the gel. sHASEGP increased the specific activity of the enzyme to Chondroitin Sulfate A, Aggrecan and D were from Calbio approximately 97,000 units per mg protein based upon A280 chem. Hyaluronan (Human Umbilical Cord) Chondroitin measurement. A dose response curve of Calcium and Mag Sulfate C, Dermatan sulfate, and Heparan-sulfate are 45 nesium metals was then tested to determine the optimal con obtained from Calbiochem. Human umbilical cord Hyaluro centration of metal ions to enzyme. nan was obtained from ICN. Each test substrate is diluted to 0.1 mg/ml. 10 ul samples of purified shASEGP or condi tioned media from sFHASEGP expressing cells as well as are mM Divalent Metal (Ca++) Mg++ mixed with 90 ul of test substrate in desired buffer and incu 50 bated for 3 hours at 37 C. Following incubation samples are 100 1 1.3 10 108 104 neutralized with sample buffer (Tris EDTA PH 8.0, Bro 1 169 164 mophenol Blue and glycerol) followed by electrophoresis on O.1 123 78 15% polyacrylamide gels. Glycosaminoglycans are detected O.O1 59 18 by staining the gels in 0.5% Alcian Blue in 3% Glacial Acetic 55 O.OO1 47 13 Acid overnight followed by destaining in 7% Glacial Acetic O.OOO1 39 13 Acid. Degradation is determined by comparison Substrate OOOOO1 55 15 mobility in the presence and absence of enzyme. b. Results Activation of shASEGP was found to occur in the micro 100 Units of shASEGPs in 10 ul was incubated with 90 60 molar range. Concentrations above 10 mM were inhibitory ul 10 mM Hepes Buffer with 50 ug/ml Human Serum Albu for both Calcium and Magnesium. To rule out nonspecific min for 2 hours at 37 C containing 10 ug of various gly activation of substrate rather than enzyme, Calcium Chloride cosaminoglycans and proteoglycans. Electrophoretic analy in 10 mM Hepes buffer was incubated with the immobilized sis followed by Alcian blue staining revealed increased biotinylated substrate on the microtiter plate followed by mobility shifts to a single species in Chondroitin Sulfate A, C 65 washing. No activation was found when the enzyme was and D. Aggrecan and Hyaluronan but not Heparan Sulfate nor added to the Calcium preincubated plate that had been Chondroitin Sulfate B. Whereas the undigested glycosami washed. The activation was also tested on phospholipase C US 7,871,607 B2 137 138 released native shASEGP which revealed a similar activation material was injected Subcutaneously with trypan blue dye. with Calcium ruling out an artifact of the carboxy terminus Areas were tested at various time points up to 15 minutes. HIS6 epitope tag. b. Results EXAMPLE 1.4 Effects of Albumin on the Activity of shASEGP 4 UNITS 4 UNITS HEAT INACTIVATED It was found that the dilution of recombinant rPUPH20 and minute post injection minute post injection other preparations of slaughterhouse testes-derived hyalu to = 52.38 to = 50.58 ronidases required albumin in addition to Calcium for opti 10 t = 116.51 t = 65.48 mal activity. tes = 181.93 Tss = 63.87 a. Protocol to = 216.96 To = 65.80 Human Serum Albumin (ICN) was diluted into 10 mM t = 279.99 T16 = 74.3 Hepes buffer with Calcium to determine the effects of albu min protein on enzyme activity. Enzyme assays with SHA 15 SEGP and commercial preparations were examined using EXAMPLE-17 both 1 mM CaCl and 1 mg/ml Human Serum Albumin. b. Results Activation of hyaluronidase activity was found at high Restoration of the Dermal Barrier Broken Down by dilutions in the presence of albumin. It was not clear whether SHASEGP this activation was a result of preventing denaturation or if the albumin affected the availability of the substrate. A preferable a. Protocol formulation of human shASEGP could therefore include To establish the regeneration time of the pores opened with Albumin and a metal salt consisting of either Calcium or sHASEGP following subcutaneous administration, 2 Units of Magnesium. 25 purified shASEGP or saline control was injected into two opposing lateral sites Subcutaneously in animals at t=0 fol EXAMPLE-15 lowed by injection with trypan blue at the same site at 30 min Spreading Activity of Purified shASEGP In Vivo 60 minand 24 hours. Area of the dye diffusion att=15 minutes 30 post injection was recorded for each time point compared to a. Protocol the control. Purified shASEGP in 10 mMHepes PH 7.4, 150 mMNaCl b. Results 0.1% Pluronic was diluted to 0.5 U?ul in pyrogen free water with 0.15M NaCl. A series of dilutions in 20 ul final of Saline were made to give a total of 0.01, 0.05, 0.1 Units per injection. 35 20 ul of Trypan Blue solution was added to a final volume of 2 UNITS SALINE CONTROL 40 uland injected Subcutaneously into the lateral skin on each Thour post injection shASEGP thour post injection shASEGP side ofbalbi' mice that had been previously anesthetized tos, = 183 tos, = 54 t1 = 167 t = 50 i.p. by ketaminefxylazine administration. Dye areas were t22 = 61 t2 = 48 measured in 2 dimensions with a microcaliper from t=0 to 40 t–45 min. Area was represented as mm. As a control recom binant Human HYAL1 that lacks neutral activity but is The results demonstrate that the dermal barrier reconsti secreted was included. tutes within 24 hours of administration of 2 Units of enzyme. b. Results 45 EXAMPLE-18 TESTARTICLE DYE AREA (a 45 MIN Determination of the Size of Channels Opened by SHASEGP A. Saline Control 51.5 mm B. SHASEGP 0.01 U 76.8 mm 50 C. SHASEGP O.OSU 98.22 mm It was shown that humanshASEGPopened channels in the D. SHASEGP O.1OU 180.4 mm interstitial space sufficient to permit the diffusion of a small E. HYAL1100 U 67.48 mm molecule, i.e. trypan blue dye. However, it was unknown what the upper limits were on the size of particles that could 55 diffuse in the presence of shASEGP. EXAMPLE-16 a. Protocol Kinetics of shASEGP Diffusion Activity Florescent molecules of varying sizes were used to deter mine the size of the channels opened by human shASEGP, a. Protocol 60 Fluoresceinated Dextrans of 4,400 and 2 million Da Average Recombinant purified shASEGP, was separated into 2 Molecular Weight (Sigma) as well as fluorescein labeled aliquots. One was heated to 95 C for 15 minutes in a ther beads of defined diameters from 20 nanometers to 500 mocycler with a heated lid. The other remained at room nanometers (Molecular Probes), were administered subcuta temperature. Thermal inactivation of enzyme activity was neously in a volume of 40 ul with following injection of verified in the microtiter based enzyme assay. For kinetic 65 sHASEGP or saline control in the same sites. Area of the dye analysis heat inactivated verses native material was tested. 4 front was then measured in two dimensions at 15 minutes post Units of purified shASEGP or equivalent heat inactivated injection. US 7,871,607 B2 139 140 b. Results injected i.v. into 2 animals. Measurement of the dye front area post 45 minute enzyme administration was calculated at 2.5, 5, 10 and 15 minutes for each dose or carrier control. b. Results Diffusion Test Results demonstrated that highly purified shASEGP was Diffusion Agent Particle Size Area at 15 min Stand Dev systemically available to distal tissues upon intravenous SHASEGP 4400 Da. 84.2 25.7 administration. The spreading activity of systemically admin Contro 4400 Da. 38.0 5.8 SHASEGP 2 x 1 OE6 Da 141.2 4.5 istered shASEGP was dose dependent, with a 10 unit injec Contro 2 x 1 OE6 Da 51.7 8.1 tion being indistinguishable from carrier control. SHASEGP 20 nm Diameter 92.3 2O6 10 Contro 20 nm Diameter S1.6 3.0 SHASEGP 100 mm 61.O 5.7 Diameter Dose Time Mean Contro 100 mm 40.O 7.0 Type IV Minutes Area (mm) SD Diameter SHASEGP 200 nm. 35.5 1.6 15 PH2O 1OOO 2.5 86.417 2.834193 Diameter PH2O 1OOO 5 102.17 2.221.146 Contro 200 nm. 27.9 8.2 PH2O 1OOO O 124.53 6.304.944 Diameter PH2O 1OOO 5 129.81 1434319 SHASEGP SOO mm 44.8 13.6 PH2O 3OO 2.5 59.137 7.218615 Diameter PH2O 3OO 5 73.638 7.5.1197 Contro SOO mm 41.2 9.8 PH2O 3OO O 87.092 8.686008 Diameter PH2O 3OO 5 92.337 10.66466 PH2O 1OO 2.5 56.308 7.741934 PH2O 1OO 5 63.156 1142052 The results demonstrated that molecules from approxi PH2O 1OO O 76.519 16.18449 mately 1 kDa (Trypan Blue) to 50 nm in diameter (Latex PH2O 1OO 5 77.432 17.32264 PH2O 30 2.5 S.O.S34 10.64287 25 Beads) showed enhanced diffusion following administration PH2O 30 5 59.493 S.163971 ofsHASEGP. While bovine serum albumin (66 kDA) showed PH2O 30 O 68.102 11.00071 similar kinetics of diffusion to trypan blue, the 50 nm latex PH2O 30 5 71.118 9.934.212 beads required significantly more time to diffuse. 500 nm PH2O 10 2.5 36.4 3.807072 PH2O 10 5 39.859 6.68.0932 beads showed no diffusion up to 480 minutes. PH2O 10 O 45.649 444936 30 PH2O 10 5 48.41 6.546835 EXAMPLE-19 Control O 2.5 34.652 5.935.037 Control O 5 36.279 3.614544 Serum Pharmacokinetics Profiles of Biotinylated Control O O 44.687 5.8.21216 Antibodies Following Subcutaneous Co-Injection of Control O 5 53.002 2.812439 Human SHASEGP 35 a. Protocol EXAMPLE 21 Female Balbic mice were anesthetized with a mixture of ketamine/xylazine. The mice were then injected Subcutane Illustrative Use of Pegylation to Generate Modified ously with 20 ul of 0.5 mg/ml solution of biotinylated mouse 40 sHASEGPS Exhibiting Improved Pharmacokinetics IgG mixed with 20 ul of either saline or 20 ul shASEGP containing 4Units of activity. As an illustrative example of the use of pegylation to gen b. Results erate modified forms of shASEGPs having improved phar macokinetics, such as increased serum half life, we attached 45 either linear or branched polyethylene glycol (PEG) moieties to an exemplary shASEGP, recombinant human PH20 TIME POST INJECTION CONTROL sHASEGP (4U) (rHuPH20) as described above. Serum IgG t = 0 hrs Ongfml Ongfml A number of different functionally activated groups can be Serum IgG t = 2hrs Ongfml 360 ng/ml used for attaching PEG moieties to shASEGPs such as Serum IgG t = 51 hrs 4152 ng/ml 4176 ng/ml 50 rHuPH20 (including, by way of illustration, aldehydes, suc cinimides, maleimides and others). A number of different The results demonstrate that shASEGP increases the pegylation reagents are commercially available from various kinetics of serum distribution of large molecules in circula Sources including Nektar Therapeutics (www.nektar.com) as tion. Where no biotinylated IgG could be detected in the well as a number of other suppliers. For purposes of illustra control group at 2 hours, 360 ng/ml was apparent by 2 hours 55 tion, rHuPH20 has been in the shASEGP group. In addition to varying the functional group, PEGs of a variety of different lengths and structures are available which EXAMPLE-20 can be incorporated using a particular functional group. For purposes of illustration, PEG lengths ranging from 5 to 40 Spreading Activity of Subcutaneously Injected 60 KDa have been conjugated to rHuPH20. Again a variety of Molecules Following Intravenous Injection of different PEGs having different lengths and structures are Human SHASEGP readily available from sources including Nektar, Sunbio, Celares and others. a. Protocol As an exemplary illustration of the pegylation of an illus Four sites for dye injection were utilized per dose of each 65 trative shASEGP, PEG aldehydes, succinimides and male Test Article and carrier control. Dye injection was 45 minutes imides have each been applied to conjugate PEG moieties to after i.v. injection. Each dose of test or control article was rHuPH20. Of these three chemistries, succinimidyl PEGs US 7,871,607 B2 141 142 have typically been the most convenient to use in the case of tissues of the posterior of the eye, particularly the choroid and rHuPH20. For example, rHuPH20 has been conjugated with retinal layers, which are important drug targets but are often exemplary succinimidyl monoPEG (MPEG) reagents includ difficult to efficiently reach with pharmacologic agents. ing mPEG-Succinimidyl Propionates (mPEG-SPA), mPEG New Zealand red male rabbits (approximately 2.1 to 3.2 Succinimidyl Butanoates (mPEG-SEBA), and mPEG2-N-Hy kg) were employed and were placed under general anesthesia droxylsuccinimide. These pegylated Succinimidyl esters using intramuscular injections of ketamine hydrochloride (21 contain different length carbon backbones between the PEG mg/kg) and Xylazine hydrochloride (5.25 mg/kg). Topical group and the activated cross-linker, and either a single or 0.5% proparacaine was used for anesthetizing the ocular Sur branched PEG group. These differences can be used, for face. Pupils were dilated with 1% tropicamide and 2.5% example, to provide for different reaction kinetics and to 10 phenylephrine. After opening the conjunctiva of the right eye potentially restrict sites available for PEG attachment to of each rabbit using Vannas scissors, the tip of a Medez rHuPH20 during the conjugation process. tri-port anesthetic cannula (Eagle Laboratories, Rancho Succinimidyl PEGs (as above) comprising either linear or Cucamonga, Calif.) was inserted into the episcleral space and branched PEGs have been conjugated to rhuPH20. Using 1.5 ml of either dex alone (American Pharmaceutical Part compositions and techniques as described in the art, Succin 15 ners, n=18) or dex plus shASEGP (3000 units of rHUPH20, imidyl PEGs have been used to generate rHuPH20s repro n=18) was injected adjacent to the posterior pole of the eye. A ducibly comprising a combination of molecules having cotton-tipped applicator was placed at the site of conjunctival between about three to six PEG molecules per shASEGP. entry to prevent egress of fluid. Such pegylated rHuPH20 compositions were readily purified At 1, 2, 3 or 6 hours after the injection, rabbits were anes to yield compositions having specific activities of approxi thetized as described above and sacrificed by intracardiac mately 25,000 Unit/mg protein hyaluronidase activity, and injection of pentobarbital Sodium (120 mg/kg). Following being substantially free of non-PEGylated rhuPH20 (less euthanasia, 1 ml of blood sample was taken and both eyes than 5% non-PEGylated). As is known in the art, and were enucleated from each animal. Immediately after the described for example in PEGylation references including enucleation, a 2-ml sample of aqueous humor was collected those cited above, a variety of reagents and techniques are 25 via anterior chamber paracentesis with tuberculin Syringe also available for achieving mono-disperse as opposed to from each eye. After removal of anterior segment from poly-disperse PEG products (see, e.g., the reviews and refer enucleated eyes, 1 ml of vitreous sample was withdrawn and ences cited above related to site-specific monoPEGylation placed into labeled Eppendorf tubes. The remainder of the eye and site-directed PEGylation). was dissected and 6-mm trephine (Fine Science Tools, Foster Improvements in both pharmacokinetics and pharmacody 30 City, Calif.) was used to punch the tissue sample inferior to namics have been observed in animal models following the optic nerve where drug was injected on the extrascleral administration of a PEGylated shASEGP as compared to the side. From the punched tissue the retina was scraped off with unmodified version of the shASEGP. For example, following a blunt blade and placed into labeled tube as was the choroid. intravenous administration to mice of either 1,000 Units of an Mass spectrometry was used to assess deX levels in tissues unmodified rhuPH20 or 1,000 Units of rhuPH20 conjugated 35 with the minimum quantifiable limits being on the order of 5 to PEGs having a molecular weight of approximately 40 kDa ng/ml. For the choroid and retinal samples, a buffer contain (rHuPH20-PEG40), it was observed that the serum half-life ing 50 mM Tris base, 1 mM MgCl, pH 9.0, was added to the of neutral active hyaluronidase activity (PH2O) in serum of tissue so the final concentration was 20 mg/mL. The tissue mice treated with rhuPH20-PEG40 was significantly longer was disrupted using an ultrasonic homogenizer (BioLogics, (16-20 fold) than that of mice treated with unmodified 40 Inc., model 150V/T) for 5-15 seconds. The samples of tissue rHuPH20. In addition, the effectiveness of rHuPH20-PEG40 homogenate, the vitreous fluid, aqueous fluid, and plasma has been compared to non-PEGylated rhuPH20 in a rat stroke were all analyzed by the following procedure. Samples con model. Initial results from these studies demonstrate greater taining dexamethasone and prednisolone as the internal stan effectiveness of rHuPH20-PEG4.0 (greater than 75% sur dard (I.S.) were extracted with methyl t-butyl ether by vortex vival, versus less than 50% in controls) in rat survival 7 days 45 mixing for five minutes. The organic and aqueous layers were post stroke-inducement. separated by centrifugation at 1,300 g for five minutes, the aqueous layer was frozen at -70° C. for fifteen minutes, and EXAMPLE 22 the organic layer was poured into new tubes and evaporated under a stream of nitrogen at 40°C. The residue was recon Illustrative Use of a SHASEGP to Promote 50 stituted with water: acetonitrile (50: 50 V/v). The processed Spreading of a Small Molecule Pharmacologic Agent samples were analyzed by high-performance liquid chroma In Vivo tography using a Phenomenex Synery Polar RP column maintained at 45°C. The mobile phase was nebulized using As an illustrative example of the use of shASEGP compo heated nitrogen in a Z-spray source/interface and the ionized sitions of the present invention to promote spreading or dif 55 compounds were detected using a tandem quadrupole mass fusion of pharmacologic agents into and within organs and spectrometer. Peak heights of dexamethasone and predniso tissues in a mammal, we assessed the use of rPUPH20 to lone were acquired and integrated using MassLynxTM soft promote penetration of dexamethasone (“dex’, an anti-in ware, Micromass, Beverly, Mass. The calibration curves were flammatory agent used as an exemplary Small molecule phar obtained by fitting the peak height ratios of the standards to macologic) into tissues of the mammalian eye in vivo 60 the internal standard a power equation in MassLynxTM. The For injection of agents such as anesthetics to the mamma equation of the calibration curve was then used to interpolate lian eye, commonly used periorbital routes for achieving the concentration of dexamethasone in the samples using anesthetic blocks include peribulbar, retrobulbar and sub their peak height ratios. Tenon's injections. For this illustrative example, we com The mass spec results revealed that the choroidal, retinal pared sub-Tenon's injections of dex, with or without con 65 and vitreous dexamethasone levels of rabbits treated with dex comitant administration of shASEGP, into the episcleral alone reached peak concentrations at the first time point (i.e. space, and then examined the concentrations of dex within 1 hour after injection); whereas the choroidal and retinal dex US 7,871,607 B2 143 144 concentrations of rabbits that received concomitant shA described above. In addition, we have observed that shA SEGP were higher and continued to increase to the second SEGPs can be used to increase hydraulic conductivity of time point (i.e. 2 hours after injection). Peak choroidal, retinal interstitial tissues over ten fold. and vitreous concentrations were approximately 10-fold, As confirmation of these principles in the context of tumor 5-fold and 8-fold higher, respectively, in tissues that had been cells, application of an exemplary shASEGP (rHuPH20, 50 treated with shASEGP, and the areas under the curve (AUC) Units) to tumor cells in vitro resulted in substantial removal of were also at least 50% higher. pericellular matrices, which are believed to be a major con These results indicate that the administration of shASEGP tributor to elevated tumor interstitial fluid pressure. can be used to substantially enhance the delivery of pharma In addition, administration of rhuPH20 in vivo, in a cologic agents such as dex, even to relatively difficult to reach 10 Xenograft human tumor model, resulted in a significant reduc tissues in vivo Such as segments at the posterior of the mam tion in tumor interstitial fluid pressure within one hour of malian eye. Furthermore, since the relatively thick scleral administration. layer forms a tissue barrier between the Sub-Tenon's injection As described in the detailed description of the invention space and the targeted choroid and retinal layers, the high above, shASEGPs of the present invention can be applied in levels of pharmacologic agent delivered to the latter layers 15 indicate that shASEGP can be used to effectively promote combination with any of a number of known or newly devel delivery of pharmacologics across tissue layers within the oped antitumor agents for use in the treatment of various body. CaCCS. In the case of promoting transcleral delivery, shASEGP can thus be applied in a minimally-invasive procedure to EXAMPLE 24 promote the delivery of agents used to treat conditions affect ing the back of the eye which are of particular interest in a Illustrative Use of a SHASEGP to Enhance the number of ocular disease situations. This can provide, for Pharmacokinetics of a Macromolecule Delivered by example, a less invasive delivery route for the treatment of Non-IV Parenteral Injection choroidal neovascularization (e.g. by administering steroids), 25 macular degeneration and other diseases affecting these tis As discussed above, shASEGPs can be used to modulate Sues which are typically treated via intravitreal injection. the pharmacokinetics of known or newly developed pharma There are a wide variety of agents currently used for these cologic and other agents by, example, facilitating their conditions, such as, but not limited to, dexamethasone, triam absorption and/or distribution with the body. Such improved cinolone, MacugenTM, and VEGFantagonists. 30 pharmacokinetic parameters can in turn be used to improve In addition to the enzymatic activities of shASEGP that the pharmacodynamics of a particular agent (e.g. reducing contribute to spreading in the context of delivery routes such local toxicity by lowereing the amount of an agent left near a as via sub-Tenon's injection, it is also believed that the intro site of injection and/or raising effectiveness by increasing the duction of the shASEGP in a volume of fluid that effectively amount of an agent delivered to (and/or through) a desired fills or distends the tissue space into which it is injected 35 target). Alternatively, since shASEGPs can be used to effec generates positive hydrostatic pressures that serve to further tively drive dispersion of a locally-administered pharmaco drive delivery offluids, and the agents comprised therein, into logic agent (e.g., through a site of injection and into the the Surrounding tissues (e.g. by promoting convective trans bloodstream), it is also possible to use the shASEGP to port). essentially improve the pharmacokinetics of absorption and 40 achieve similar pharmacodynamics with a reduced amount of EXAMPLE 23 applied agent. As an illustrative example of the use of shASEGPs to Illustrative Use of a SHASEGP to Reduce Tumor enhance the pharmacokinetics of a macromolecular drug that Interstitial Fluid Pressure Thereby Promoting the is normally administered by non-intravenous parenteral Susceptibility of a Tumor to Anti-Tumor 45 injection, we combined recombinant human PH20 Pharmacologic Agents (rHuPH2O) with a pegylated version of interferon alpha2b (PEG-INTRONTM available from Schering-Plough). PEG As described and illustrated herein, shASEGPs can be INTRON is a macromolecule of approximately 31 kD that is used to generate interstitial channels that can increase fluid typically administered by Subcutaneous injection for the flow and facilitate the delivery of pharmacologic and other 50 treatment of Hepatitis C virus (HCV) (generally in combina agents to desired tissue sites in vivo. Tumors present a par tion with the Small molecule drug ribavirin). Like many mac ticularly important target issue for a number of known and romolecules administered subcutaneously, PEG-INTRON is regularly developed pharmacologic agents, especially antine relatively slowly absorbed and a substantial portion of the oplastics, antimetabolites, antiangiogenics and various cyto agent never makes it to the bloodstream, with bioavailability toxic and cytostatic agents. The challenges associated with 55 on the order of 50-60%. In addition, a substantial fraction of targeting Such pharmacologics to tumor sites in vivo are exac patients (typically more than half) develop various injection erbated by the fact that many tumors exhibit elevated inter site reactions, presumably as a result of or exacerbated by the Stitial pressures that tend to impede infusion of administered retained local concentrations of the pharmacologic. As appre pharmacologics, particularly to sites deep within a tumor ciated by those of skill in the art, the consequences of Such mass. It is believed that shASEGPs can reduce this imped 60 pharmacokinetics are several-fold; and include not only the ance, and therefore faciliate delivery of any of a variety of “wasting of agent trapped near the injection site but also known and newly developed pharmacologic agents into reactions to the locally metabolized compound. tumors, thereby rendering them more sensitive to and more We administered Iodine-125 labelled PEG-INTRON by treatable by a given dose of a pharmacologic. In these regards, different routes of administration, with or without concomi sHASEGPs of the present invention have been shown to be 65 tant adminisration of rPuPH20, to examine the effects of capable of promoting the formation of interstitial channels rHuPH20 on the pharmacokinetic parameters of the PEG and to enhance the dispersion of markers of various sizes as INTRON. To determine the absolute bioavailability of US 7,871,607 B2 145 146 rHuPH20, we also measured the pharmacokinetic parameters the concentrations of agent in the bloodstream essentially of PEG-INTRON administered by intravenous administra reached those achieved by IV dosing, and thereafter appar tion. ently exceeded them. Briefly, eighteen Sprague-Dawley rats (each weighing EXAMPLE 25 approximately 225-250 grams) were divided into three groups of six animals each. A first group was administered Illustrative Use of a SHASEGP to Facilitate test article (i.e. I-125 labeled PEG-INTRON) by intravenous Intradermal Delivery of a Macromolecule Normally administration (1.5 micrograms/kg in a Volume of 100 micro Delivered by Intravenous Injection liters via tail vein injection). Specific activity of the I-125 10 labeled interferon was 55 uCi/microgram. Blood samples As discussed above, shASEGPs can be used to facilitate (100 microliters) were collected pre-dose and at 0.5, 1, 2, 4, 8, the delivery of known or newly developed pharmacologic and 24, 48, 72, 96 and 120 hours after dosing and processed for other agents by different routes of administration that may quantification of labeled drug by gamma counting (expressed provide for greater convenience, safety, reduced costs and/or 15 other benefits. As an illustrative example of such a reformu as Counts Per Minute (CPM) per gram plasma). A second lation, we used a shASEGP to facilitate intradermal delivery group of six animals was administered an equivalent dose and of a macromolecular drug that is normally administered by volume of test article by intradermal injection into a shaved intravenous injection. For this particular example, we com and prepared spot over the back of the neck, and blood bined recombinant human PH20 (rHuPH20) with a chimeric samples were processed as for the first group. A third group of human-murine anti-TNFalpha monoclonal antibody (Inflix six animals was administered an equivalent dose of testarticle imab, available as REMICADETM from Centocor). REMI mixed with rHuPH20 (100 I.U.) in a combined volume of 100 CADE is a macromolecule of approximately 150 kD that is microliters by intradermal injection as in the preceding group, typically administered by intravenous injection for the treat and blood samples were then processed as for groups one and mentofarthritis or Crohn's disease. Administration of REMI 25 CADE thus generally requires the involvement if intravenous two. The results of this study are shown below in Table 24-1. access and concomitant professional care and risks, and also requires on the order of two hours for each dosing. TABLE 24-1 REMICADE (20 mg/ml) was labeled with 'I to a specific Effects of SHASEGP on the pharmacokinetic profile of Interferon activity of 10 uCi/mg. Eighteen rats were divided into three 30 PEG groups of six animals each and were administered a mixture INTRON of radioactive and cold REMICADE (combined 10 mg/kg) in PEG- (1.5ugkg) + PEG a final volume of 100 ul (i) by intravenous administration, (ii) INTRON SHASEGP INTRON by intradermal administration or (iii) or by intradermal Time (1.5ug/kg) 1OOU (1.5ug/kg) administration in combination with 100 Unit shASEGP (Hours) Intradermal SD Intradermal SD Intravenous 35 (from a 100,000 U/ml stock); and then blood and tissues O.S 30,289 9,989 54,808 12,995 281,781 processed by gamma counting at various points after dosing, 1 43,137 9,434 76,672 17,570 185,630 essentially as described for the preceding example. The 2 54,655 11,128 102,159 17,626 155,591 results are summarized in Table: 3 55,261 11,055 115,329 24,910 128,656 6 61.214 15,528 118,364. 23,817 93,065 8 59.277 14,704 110,443 21,735 79,275 40 TABLE 25-1 12 46,696 12,752 85,710 28,078 58,457 24 16,233 6,595 26,047 9,006 16,966 Effects of SHASEGP on the Pharmacokinetic Parameters of REMICADE. 36 7,954. 1,777 12,498 1,762 9,277 48 3,862 499 5,517 1,030 5,847 CPM REMICADE AUC 1,222,952 2,212,583 1910,723 45 CPM (10 mg/kg) + CPM Cmax 61.214 118,364 REMICAD SHASEGP REMICADE Tmax 6 6 Time E (10 mg/kg) 1OOU (10 mg/kg) Absolute 64% 11.6% 100% (Hours) Intradermal SD Intradermal SD Intravenous Bioavail ability O.S 27.451 2,221 94,888 31,318 1,657,849 50 1 37,213 10,775 146,793 42,014 1,645,571 2 66,001 6,078 412,047 61,240 1,576,145 4 120,704 21,744 691,626 89,002 1,260,646 As can be seen in Table 24-1, the bioavailability of the 8 256,987 32,459 833,826 133,887 1,157,268 exemplary pharmacologic PEG-INTRON (as assessed by 24 493,004 75,694 853,423 65,777 790,357 AUC) was dramatically improved when the intradermal dos 48 515,439 53,431 728,302 74,848 684,599 72 454,717 54,388 640,303 81492 558,307 ing was combined with a dose of shASEGP. In fact, the 55 96 375,993 39,744 598.413 59,533 496,658 absolute bioavailability achieved with an intradermal dosing 120 336,067 66,964 518,861 53,643 434,008 in combination with shASEGP were statistically indistin AUC 53,350,555 118,424,609 82,718,550 Cmax 515,439 853,423 1,657,849 guishable from the direct intravenous administration, where Tmax 48 24 O all of the material is delivered directly into the bloodstreams. Ab- 62% 1.21% 100% 60 solute Thus, the use of shASEGP in combination with a pharmaco Bio logic that is introduced by non-IV parenteral administration avail can provide levels of delivery comparable to administration ability directly into the bloodstream. Although the Tmax is neces sarily longer following ID administration (since some 65 As can be seen in Table 5-1, the bioavailability of the amount of time is necessary for the agent to reach the blood exemplary pharmacologic REMICADE (as assessed by stream), within several hours after ID dosing with shASEGP, AUC) was dramatically improved when the intradermal dos US 7,871,607 B2 147 148 ing was combined with a dose of shASEGP. As was observed with enhanced delivery of PEG-INTRON, the overall levels TABLE 26-1-continued of REMICADE bioavailability achieved with an intradermal dosing in combination with shASEGP were statistically Effects of rhuPH20 on the pharmacokinetics of Factor VIII similar to those of the intravenous administration when all CPM material was delivered directly into the bloodstream. FACTOR Although the REMICADE Tmax is necessarily longer fol CPM VIII CPM FACTOR (50 IU/kg) + FACTOR lowing ID versus IV administration, within twenty four hours VIII SHASEGP VIII after ID dosing with shASEGP, the concentrations of agent in Time (50 IU/kg) 1OOU (50 IU/kg) the bloodstream exceeded that observed with IV dosing, and 10 (Hours) Intradermal SD Intradermal SD Intravenous thereafter approached or exceeded the IV concentrations. Cmax 56,087 84,066 Compared with ID dosing without shASEGP, the observed Tmax 4 4 T was reached about twice as quickly with shASEGP (24 Absolute 39.7% 51% 100% hours versus 48 hours); and the Cmax and bioavailability Bioavail (AUC) both substantially increased (by about 65% and 95%, 15 ability respectively). As shown in Table 26-1, co-adminsitration of Factor VIII EXAMPLE 26 with rHuPH20 significantly increased the systemic bioavail ability relative to Factor VIII alone (P<0.01). The use ofTCA Illustrative Use of a SHASEGP to Facilitate precipitation was necessary to eliminate the contribution of Intradermal Delivery of a Larger Macromolecular completely degraded I-125 labeled Factor VIII protein enter Complex Normally Delivered by Non-IV Parenteral Injection ing the blood stream. EXAMPLE 27 As another illustrative example of the use of shASEGPs to 25 enhance the pharmacokinetics of a macromolecular drug that Illustrative Use of a shASEGP to Improve Survival is normally administered by non-intravenous parenteral and/or Behavioral Outcomes Following Stroke injection, we combined recombinant human PH20 (rHuPH20) with recombinant human Factor VIII (RECOM In a first set of studies, shASEGPs were evaluated in a rat BINATETM available from Baxter). Factor VIII is a large 30 model of stroke. Briefly, stroke was surgically induced in glycoprotein complex comprising multiple polypeptides, and Sprague-Dawley rats by middle cerebral artery occlusion, has a combined molecular of approximately 280 kD. Factor followed at various times by intravenous administration of VIII is typically only administered by intravenous injection either a control Solution (saline) or a test Solution (Saline for the treatment of hemophilia. Like many macromolecules comprising a shASEGP). Animals were allowed to recover administered subcutaneously, Factor VIII is relatively slowly 35 and were followed over a period of time to assess rates of absorbed and a substantial portion of the agent never makes it Survival in control versus test groups. In addition, Survivors to the bloodstream, with bioavailability on the order of 0.5- were assessed over a range of timepoints using multiple 19/6. analyses of behavioral conditions typically associated with Eighteen rats were divided into three groups of six animals stroke. Only rats that performed normally in an initial quali each and were administered 125-I labeled Factor VIII (i) by 40 fying forelimb flexion test were included in the study, and intravenous administration, (ii) by intradermal administra were then randomly assigned to a control or test group of tion or (iii) by intradermal administration in combination animals. with shASEGP; and then blood and tissues processed at Aseptic Surgical procedures were performed by Zivic various points after dosing to measure counts per minute 45 Laboratories essentially as follows. Rats were anesthetized in (CPM) per gram plasma, essentially as described for the a chamber supplied with 2.5% halothane. A ventral midline preceding example. incision was made and the musculature was separated to expose the right external carotid artery, which was then iso TABLE 26-1 lated and double ligated. An untied but constrictive 5/O polya Effects of rhuPH20 on the pharmacokinetics of Factor VIII 50 mide ligature was then placed around the external carotid stump. A Small hole was cut into the lumen of the stump and CPM an 18-20 mm length of 470 microfilament suture material was FACTOR inserted into the lumen and advanced into the internal carotid CPM VIII CPM FACTOR (50 IU/kg) + FACTOR artery. When completely advanced, the internalized tip of the VIII SHASEGP VIII 55 monofilament should block flow into the middle cerebral Time (50 IU/kg) 1OOU (50 IU/kg) artery. The constrictive polyamide ligature was then tied to (Hours) Intradermal SD Intradermal SD Intravenous secure the monofilament. Retraction was removed and the O.S 29,748 12,002 46,376 5,503 670,743 skin incision closed with a wound clip. 1 43,767 9,415 70,249 14,614 379,558 Intravenous injection procedures were performed essen 2 49,507 14,592 72,736 14,985 247,397 3 56,087 17,621 76,631 14.454 200,602 60 tially as follows. Rats were anesthetized in a chamber sup 4 55.494 12,721 84,066 8,646 177,846 plied with 2.5% halothane. A ventral skin incision was made 5 55,277 14,181 79,735 9,363 165,914 to expose the jugular vein. A sterile 1 ml disposable Syringe 6 50,195 13,970 72,176 6,848 121,198 was fitted with a 30-gauge needle and loaded with injection 8 52.290 13,707 66.419 10,495 114,579 12 48,336 10,765 58,817 11,760 75,912 material. Using the clavicle for Support, the Syringe was 24 23,173 4,700 24,596 4.426 29,610 65 inserted into the jugular vein (using slight negative pressure AUC 1,014,521 1,303,078 2,554,607 on the syringe to draw bloodflow into the syringe and thereby confirm properplacement for IV injection) and then the injec US 7,871,607 B2 149 150 tion material was slowly injected into the jugular vein. After In addition to measuring rates of survival following stroke, injection, the needle was slowly withdrawn (and any bleeding survivors were also tested (at 1, 2, 3 and 4 weeks following controlled with a sterile cotton applicator), and then the skin Stoke) using other analyses designed to assess the incidence incision was closed with a wound clip. of certain behavioral problems associated with stroke. Behav Survival of animals in the various groups (approximately ioral conditions assessed included forelimb flexion, torso 40 animals in each group) was monitored following induction twist, hind limb, gait disturbances and wall walk, which were of stroke, and the number of survivors at each day were then used to generate a PNEGS behavorial score (higher scores plotted against days following stroke to generate a Kaplan reflecting an increase in behavioral problems associated with Meier stroke curve. Substantial differences between the the stroke). By the second week following induction of groups were apparent by the 3" day following stroke. By one 10 stroke, the shASEGP-treated animals exhibited significantly week following the induction of stroke, more than half of the higher behavioral scores (p=0.02), and the relative differ control animals had died, whereas nearly three quarters of the ences between the treated group and the control were even sHASEGP-treated animals had survived, which was a statis greater over subsequent weeks (p=0.007 and p=0.003 for tically significant difference (p=0.03). weeks three and four respectively). SEQUENCE LISTING <16 Os NUMBER OF SEO ID NOS: 53 <21 Oc SEO ID NO 1 <211 LENGTH: 509 <212> TYPE PRT <213> ORGANISM: Homo sapiens <22 Os FEATURE; <221 NAME/KEY: CARBOHYD LOCATION: 82, 166 235, 254, 368, 393, 490 <4 OOs SEQUENCE: 1 Met Gly Val Lieu Lys Phe His Ile Phe Phe Arg Ser Phe Wall Lys 1. 5 15 Ser Ser Gly Wall Ser Glin Ile Wall Phe Thir Phe Lieu. Luell le Pro 2O 25 3 O Lell Thir Luell Asn Phe Arg Ala Pro Pro Wall Ile Pro ASn Wall Pro 35 4 O 45 Phe Lell Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Luell Gly Phe SO 55 60 Asp Glu Pro Leu Asp Met Ser Luell Phe Ser Phe Ile Gly Ser Pro Arg 65 70 8O Ile Asn Ala Thr Gly Glin Gly Wall. Thir Ile Phe Wall Asp Arg Lieu. 85 90 95 Gly Tyr Pro Ile Asp Ser Ile Thir Gly Wall Thir Wall Asn Gly 105 110 Gly Ile Pro Glin Ile Ser Lieu. Glin Asp His Lieu. Asp Lys Ala 115 12O 125 Asp Ile Thir Phe Met Pro Wall Asp Asn Luell Gly Met Ala Wall 13 O 135 14 O Ile Asp Trp Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Pro 145 15 O 155 16 O Asp Wall Lys Asn Arg Ser Ile Glu Lieu Wall Glin Glin Glin Asn 1.65 17 O 17s Wall Glin Lell Ser Luell Thr Glu Ala Thr Glu Ala Glin Glu Phe 18O 185 190 Glu Ala Gly Asp Phe Leu Wall Glu. Thir Ile Lys Tell Gly 195 2 OO 2O5 Luell Lell Arg Pro Asn His Luell Trp Gly Luell Phe Pro Asp 210 215 22 O Tyr Asn His His Lys Pro Gly Asn Gly Ser Phe Asn 225 23 O 235 24 O US 7,871,607 B2 151 152 - Continued Wall Glu Ile Arg Asn Asp Asp Lieu. Ser Trp Lieu. Trp Asn Glu Ser 245 250 255 Thir Ala Luell Tyr Pro Ser Ile Tyr Lieu. Asn Thir Glin Glin Ser Pro Wall 26 O 265 27 O Ala Ala Thir Luell Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall 27s 28O 285 Ser Lys Ile Pro Asp Ala Lys Ser Pro Luell Pro Wall Phe Ala Thir 29 O 295 3 OO Arg Ile Wall Phe Thir Asp Glin Wall Lieu Phe Lell Ser Glin Asp Glu 3. OS 310 315 Lell Wall Thir Phe Gly Glu Thir Wall Ala Luell Gly Ala Ser Gly Ile 3.25 330 335 Wall Ile Trp Gly Thir Lell Ser Ile Met Arg Ser Met Lys Ser Luell 34 O 345 35. O Lell Luell Asp Asn Tyr Met Glu Thir Ile Luell ASn Pro Tyr Ile Ile Asn 355 360 365 Wall Thir Luell Ala Ala Met Cys Ser Glin Wall Lell Cys Glin Glu Glin 37 O 375 Gly Wall Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Luell His Luell 385 390 395 4 OO Asn Pro Asp Asn Phe Ala Ile Gln Lieu. Glu Gly Gly Phe Thir 4 OS 415 Wall Arg Gly Lys Pro Thir Lell Glu Asp Luell Glu Glin Phe Ser Glu Lys 425 43 O Phe Cys Ser Cys Ser Thir Lieu. Ser Glu Ala Asp 435 44 O 445 Wall Lys Asp Thir Asp Ala Wall Asp Wall Ile Ala Asp Gly Wall Cys 450 45.5 460 Ile Asp Ala Phe Lell Lys Pro Pro Met Glu Thir Glu Glu Pro Glin Ile 465 470 47s Phe Asn Ala Ser Pro Ser Thir Lieu. Ser Ala Thir Met Phe Ile Wall 485 490 495 Ser Ile Luell Phe Lell Ile Ile Ser Ser Wall Ala Ser Lell SOO 505 SEQ ID NO 2 LENGTH: 35 TYPE : PRT ORGANISM: Homo sapiens < 4 OOs SEQUENCE: 2 Met Gly Val Lieu Lys Phe Lys His Ile Phe Phe Arg Ser Phe Val Lys 1. 5 15 Ser Ser Gly Val Ser Glin Ile Val Phe Thr Phe Leu Lleu. Ile Pro Cys 25 Cys Lieu. Thr 35 <210s, SEQ ID NO 3 &211s LENGTH: 474 212. TYPE : PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 3 Lieu. Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp 1. 5 15 US 7,871,607 B2 153 154 - Continued Ala Trp Asn Ala Pro Ser Glu Phe Cys Luell Gly Lys Phe Asp Glu Pro 25 Lell Asp Met Ser Lell Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala 35 4 O 45 Thir Gly Glin Gly Wall Thir Ile Phe Wall Asp Arg Lell Gly Tyr SO 55 6 O Pro Ile Asp Ser Ile Thir Gly Wall Thir Wall Asn Gly Gly Ile Pro 65 70 Glin Ile Ser Lell Glin Asp His Luell Asp Ala Asp Ile 85 90 95 Thir Phe Met Pro Wall Asp Asn Luell Gly Met Ala Wall Ile Asp Trp 105 11 O Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Pro Asp Wall 115 12 O 125 Lys Asn Arg Ser Ile Glu Luell Wall Glin Glin Glin Asn Wall Glin Luell 13 O 135 14 O Ser Luell Thir Glu Ala Thir Glu Ala Glin Glu Phe Glu Ala 145 150 155 160 Gly Asp Phe Lell Wall Glu Thir Ile Lys Luell Gly Luell Luell Arg 1.65 17s Pro Asn His Luell Trp Gly Tyr Luell Phe Pro Asp Tyr Asn His 18O 185 19 O His Lys Pro Gly Tyr Asn Gly Ser Phe Asn Wall Glu Ile 195 Arg Asn Asp Asp Lell Ser Trp Luell Trp ASn Glu Ser Thir Ala Luell 21 O 215 Tyr Pro Ser Ile Tyr Lell Asn Thir Glin Glin Ser Pro Wall Ala Ala Thir 225 23 O 235 24 O Lell Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall Ser Lys Ile 245 250 255 Pro Asp Ala Lys Ser Pro Lell Pro Wall Phe Ala Thir Arg Ile Wall 26 O 265 27 O Phe Thir Asp Glin Wall Lell Phe Luell Ser Glin Asp Glu Luell Wall 28O 285 Thir Phe Gly Glu Thir Wall Ala Luell Gly Ala Ser Gly Ile Wall Ile Trp 29 O 295 3 OO Gly Thir Luell Ser Ile Met Arg Ser Met Lys Ser Lell Luell Luell Asp 3. OS 310 315 32O Asn Tyr Met Glu Thir Ile Lell Asn Pro Tyr Ile Ile Asn Wall Thir Luell 3.25 330 335 Ala Ala Met Cys Ser Glin Wall Luell Glin Glu Glin Gly Wall 34 O 345 35. O Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Luell His Lell Asn Pro Asp 355 360 365 Asn Phe Ala Ile Glin Lell Glu Gly Gly Lys Phe Thir Wall Arg Gly 37 O 375 Lys Pro Thir Luell Glu Asp Lell Glu Glin Phe Ser Glu Phe Cys 385 390 395 4 OO Ser Ser Thir Lell Ser Glu Lys Ala Wall Lys Asp 4 OS 415 Thir Asp Ala Wall Asp Wall Ile Ala Asp Gly Wall Ile Asp Ala 425 43 O Phe Luell Pro Pro Met Glu Thir Glu Glu Pro Glin Ile Phe Asn US 7,871,607 B2 155 156 - Continued 435 44 O 445 Ala Ser Pro Ser Thir Lieu. Ser Ala Thir Met Phe Ile Wal Ser Ile Lieu. 450 45.5 460 Phe Lieu. Ile Ile Ser Ser Wall Ala Ser Lieu. 465 470 SEQ ID NO 4 LENGTH: 4 48 TYPE : PRT ORGANISM: Homo sapiens < 4 OOs SEQUENCE: 4. Lieu. Asn. Phe Arg Ala Pro Pro Wall Ile Pro ASn Wall Pro Phe Luell Trp 1. 5 15 Ala Trp Asn Ala Pro Ser Glu Phe Cys Luell Gly Phe Asp Glu Pro 25 Lell Asp Met Ser Lell Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala 35 4 O 45 Thir Gly Glin Gly Wall Thir Ile Phe Tyr Wall Asp Arg Lell Gly Tyr SO 55 6 O Pro le Asp Ser Ile Thir Gly Wall Thir Wall Asn Gly Gly Ile Pro 65 70 8O Glin le Ser Lell Glin Asp His Luell Asp Ala Asp Ile 85 90 95 Thir Phe yr Met Pro Wall Asp Asn Luell Gly Met Ala Wall Ile Asp Trp 105 11 O Glu Glu Arg Pro Thir Trp Ala Arg Asn Trp Pro Asp Wall 15 12 O 125 Lys Asn Arg Ser Ile Glu Luell Wall Glin Glin Glin Asn Wall Glin Luell 13 O 135 14 O Ser Luell Glu Ala Thir Glu Ala Glin Glu Phe Glu Ala 145 150 155 160 Gly Asp Phe Lell Wall Glu Thir Ile Lys Luell Gly Luell Luell Arg 1.65 17s Pro Asn His Luell Trp Gly Tyr Luell Phe Pro Asp Tyr Asn His 18O 185 19 O His Lys Pro Gly Tyr Asn Gly Ser Phe Asn Wall Glu Ile 195 Arg Asn Asp Asp Lell Ser Trp Luell Trp ASn Glu Ser Thir Ala Luell 21 O 215 Tyr Pro Ser Ile Tyr Lell Asn Thir Glin Glin Ser Pro Wall Ala Ala Thir 225 23 O 235 24 O Lell Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall Ser Lys Ile 245 250 255 Pro Asp Ala Lys Ser Pro Lell Pro Wall Phe Ala Thir Arg Ile Wall 26 O 265 27 O Phe Thir Asp Glin Wall Lell Phe Luell Ser Glin Asp Glu Luell Wall 28O 285 Thir Phe Gly Glu Thir Wall Ala Luell Gly Ala Ser Gly Ile Wall Ile Trp 29 O 295 3 OO Gly Thir Luell Ser Ile Met Arg Ser Met Lys Ser Lell Luell Luell Asp 3. OS 310 315 32O Asn Met Glu Thir Ile Lell Asn Pro Tyr Ile Ile Asn Wall Thir Luell 3.25 330 335 US 7,871,607 B2 157 158 - Continued Ala Ala Met Cys Ser Glin Wall Luell Cys Glin Glu Glin Gly Wall 34 O 345 35. O Ile Arg Lys Asn Trp Asn Ser Ser Asp Luell His Lell Asn Pro Asp 355 360 365 Asn Phe Ala Ile Glin Lell Glu Gly Gly Phe Thir Wall Arg Gly 37 O 375 Lys Pro Thir Luell Glu Asp Lell Glu Glin Phe Ser Glu Phe Cys 385 390 395 4 OO Ser Ser Thir Lell Ser Glu Ala Wall Lys Asp 4 OS 415 Thir Asp Ala Wall Asp Wall Ile Ala Asp Gly Wall Ile Asp Ala 425 43 O Phe Luell Lys Pro Pro Met Glu Thir Glu Glu Pro Glin Ile Phe Asn 435 44 O 445 <210s, SEQ ID NO 5 &211s LENGTH: 482 212. TYPE : PRT &213s ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 5 Met Gly Val Lieu. Lys Phe His Ile Phe Phe Arg Ser Phe Wall 1. 5 15 Ser Ser Gly Wall Ser Glin Ile Wall Phe Thir Phe Lell Lell Ile Pro 25 Luell Thir Luell Asn Phe Arg Ala Pro Pro Wall Ile Pro Asn Wall Pro 35 4 O 45 Phe Luell Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Lell Gly Phe SO 55 6 O Asp Glu Pro Luell Asp Met Ser Luell Phe Ser Phe Ile Gly Ser Pro Arg 65 70 8O Ile Asn Ala Thir Gly Glin Gly Wall Thir Ile Phe Wall Asp Arg Luell 85 90 95 Gly Pro Tyr Ile Asp Ser Ile Thir Gly Wall Thir Wall Asn Gly 105 11 O Gly Ile Pro Glin Lys Ile Ser Luell Glin Asp His Lell Asp Ala 115 12 O 125 Asp Ile Thir Phe Met Pro Wall Asp ASn Lell Gly Met Ala Wall 13 O 135 14 O Ile Asp Trp Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Pro 145 150 155 160 Asp Wall Lys Asn Arg Ser Ile Glu Luell Wall Glin Glin Glin Asn 1.65 17O 17s Wall Glin Luell Ser Lell Thir Glu Ala Thir Glu Ala Glin Glu Phe 18O 185 19 O Glu Ala Gly Lys Asp Phe Luell Wall Glu Thir Ile Lys Luell Gly 195 Lell Luell Arg Pro Asn His Lell Trp Gly Lell Phe Pro Asp 21 O 215 Tyr Asn His His Tyr Lys Pro Gly ASn Gly Ser Phe Asn 225 23 O 235 24 O Wall Glu Ile Arg Asn Asp Asp Luell Ser Trp Lell Trp Asn Glu Ser 245 250 255 Thir Ala Luell Tyr Pro Ser Ile Luell Asn Thir Glin Glin Ser Pro Wall 26 O 265 27 O US 7,871,607 B2 159 160 - Continued Ala Ala Thir Luell Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall 27s 285 Ser Lys Ile Pro Asp Ala Lys Ser Pro Luell Pro Wall Phe Ala Thir 29 O 295 3 OO Arg Ile Wall Phe Thir Asp Glin Wall Luell Phe Lell Ser Glin Asp Glu 3. OS 310 315 Lell Wall Thir Phe Gly Glu Thir Wall Ala Luell Gly Ala Ser Gly Ile 3.25 330 335 Wall Ile Trp Gly Thir Lell Ser Ile Met Arg Ser Met Ser Luell 34 O 345 35. O Lell Luell Asp Asn Tyr Met Glu Thir Ile Luell ASn Pro Tyr Ile Ile Asn 355 360 365 Wall Thir Luell Ala Ala Met Ser Glin Wall Lell Glin Glu Glin 37 O 375 Gly Wall Ile Arg Lys Asn Trp Asn Ser Ser Asp Luell His Luell 385 390 395 4 OO Asn Pro Asp Asn Phe Ala Ile Glin Luell Glu Lys Gly Gly Phe Thir 4 OS 41O 415 Wall Arg Gly Lys Pro Thir Lell Glu Asp Luell Glu Glin Phe Ser Glu Lys 425 43 O Phe Cys Ser Cys Ser Thir Luell Ser Cys Glu Ala Asp 435 44 O 445 Wall Lys Asp Thir Asp Ala Wall Asp Wall Cys Ile Ala Asp Gly Wall Cys 450 45.5 460 Ile Asp Ala Phe Lell Lys Pro Pro Met Glu Thir Glu Glu Pro Glin Ile 465 470 47s 48O Phe Tyr SEQ ID NO 6 LENGTH: 153 O TYPE: DNA ORGANISM: Homo sapiens FEATURE: NAME/KEY: CDS LOCATION: (1) . . . (1530) OTHER INFORMATION: PH-2O GPI Anchored Hyaluronidase Glycoprotein <4 OOs, SEQUENCE: 6 atg gga gtg Cta a.a.a. tto aag CaC at C titt titc aga agc titt gtt a.a.a. 48 Met Gly Wall Luell Lys Phe Lys His Ile Phe Phe Arg Ser Phe Wall Lys 1. 5 1O 15 toa agt gga gta Cag ata gtt ttic acc titc citt Ctg att CC a tgt 96 Ser Ser Gly Wall Ser Glin Ile Wall Phe Thir Phe Lell Lell Ile Pro Cys 2O 25 3 O tgc ttg act Ctg aat tto aga gca cott cott gtt att C Ca aat gtg cott 144 Cys Luell Thir Luell Asn Phe Arg Ala Pro Pro Wall Ile Pro Asn Wall Pro 35 4 O 45 tto citc. tgg gcc tgg aat gcc CC a agt gala titt tgt citt gga a.a.a. titt 192 Phe Luell Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Lell Gly Phe SO 55 60 gat gag CC a Cta gat atg agc citc. ttic tot titc ata gga agc cc c cga 24 O Asp Glu Pro Luell Asp Met Ser Luell Phe Ser Phe Ile Gly Ser Pro Arg 65 70 7s 8O ata aac gcc acc 999 Cala ggit gtt aca at a titt tat gtt gat aga citt 288 Ile Asn Ala Thir Gly Glin Gly Wall Thir Ile Phe Wall Asp Arg Luell 85 90 95 ggc tac tat cott tac ata gat to a at C aca gga gta act aat gga 336 US 7,871,607 B2 161 162 - Continued Gly Pro Tyr Ile Asp Ser Ile Thir Gly Wall. Thir Wall Asn Gly 1OO 105 11 O gga at C cc c cag aag att t cc tta Cala gac Cat Ctg gac a.a.a. gct aag 384 Gly Ile Pro Glin Lys Ile Ser Luell Glin Asp His Lell Asp Lys Ala Lys 115 12 O 125 a.a.a. gac att aca titt tat atg CC a gta gac aat ttg gga atg gct gtt 432 Asp Ile Thir Phe Met Pro Wall Asp ASn Lell Gly Met Ala Wall 13 O 135 14 O att gac tgg gala gaa tgg aga cc c act tgg gca aga aac tgg a.a.a. cott Ile Asp Trp Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Pro 145 150 155 160 a.a.a. gat gtt tac aag aat agg tot att gala ttg gtt Cag Cala Cala aat 528 Asp Wall Lys Asn Arg Ser Ile Glu Luell Wall Glin Glin Glin Asn 1.65 17O 17s gta Cala citt agt citc. a Ca gag gcc act gag a.a.a. gca a.a.a. Cala gala titt 576 Wall Glin Luell Ser Lell Thir Glu Ala Thir Glu Ala Glin Glu Phe 18O 185 19 O gaa aag gca 999 aag gat tto Ctg gta gag act ata a.a.a. ttg gga a.a.a. 624 Glu Lys Ala Gly Lys Asp Phe Luell Wall Glu Thir Ile Lys Luell Gly 195 2OO 2O5 tta citt cgg CC a aat CaC ttg tgg ggt tat citt titt cc.g gat tgt 672 Lell Luell Arg Pro Asn His Lell Trp Gly Lell Phe Pro Asp Cys 21 O 215 22O tac aac Cat CaC tat aag a.a.a. cc c ggt aat gga agt tgc ttic aat 72 O Tyr Asn His His Tyr Lys Pro Gly ASn Gly Ser Cys Phe Asn 225 23 O 235 24 O gta gaa at a a.a.a. aga aat gat gat citc. agc tgg ttg tgg aat gaa agc 768 Wall Glu Ile Arg Asn Asp Asp Luell Ser Trp Lell Trp Asn Glu Ser 245 250 255 act gct citt tac C Ca t cc att tat ttg aac act Cag Cag tot cott gta 816 Thir Ala Luell Tyr Pro Ser Ile Luell Asn Thir Glin Glin Ser Pro Wall 26 O 265 27 O gct gct aca citc. tat gtg cgc aat cga gtt cgg gaa gcc at C aga gtt 864 Ala Ala Thir Luell Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall 27s 28O 285 t cc a.a.a. at a cott gat gca a.a.a. agt CC a citt cc.g gtt titt gca tat acc 912 Ser Lys Ile Pro Asp Ala Lys Ser Pro Luell Pro Wall Phe Ala Thir 29 O 295 3 OO cgc at a gtt titt act gat Cala gtt ttg a.a.a. titc citt tot Cala gat gala 96.O Arg Ile Wall Phe Thir Asp Glin Wall Luell Phe Lell Ser Glin Asp Glu 3. OS 310 315 32O citt gtg tat aca titt ggc gaa act gtt gct Ctg ggit gct tot gga att OO8 Lell Wall Thir Phe Gly Glu Thir Wall Ala Luell Gly Ala Ser Gly Ile 3.25 330 335 gta at a tgg gga a CC citc. agt at a atg cga agt atg a.a.a. tot tgc ttg Wall Ile Trp Gly Thir Lell Ser Ile Met Arg Ser Met Ser Cys Luell 34 O 345 35. O citc. Cta gac aat tac atg gag act at a Ctg aat cott tac at a at C aac 104 Lell Luell Asp Asn Tyr Met Glu Thir Ile Luell ASn Pro Tyr Ile Ile Asn 355 360 365 gtc aca Cta gca gcc a.a.a. atg agc Cala gtg citt cag gag Cala 152 Wall Thir Luell Ala Ala Met Cys Ser Glin Wall Lell Glin Glu Glin 37 O 375 38O gga gtg tgt at a agg a.a.a. aac tgg aat to a agt gac citt CaC citc. 2OO Gly Wall Cys Ile Arg Lys Asn Trp Asn Ser Ser Asp Luell His Luell 385 390 395 4 OO aac CC a gat aat titt gct att Cala citt gag a.a.a. ggit gga aag ttic aca 248 Asn Pro Asp Asn Phe Ala Ile Glin Luell Glu Lys Gly Gly Lys Phe Thir 4 OS 41O 415 US 7,871,607 B2 163 164 - Continued gta cgt gga a.a.a. cc.g a Ca citt gala gac Ctg gag caa titt tot gala a.a.a. 296 Wall Arg Gly Lys Pro Thir Lell Glu Asp Luell Glu Glin Phe Ser Glu Lys 42O 425 43 O titt tat tgc agc tgt tat agc acc ttg agt tgt aag gag a.a.a. gct gat 344 Phe Cys Ser Cys Ser Thir Luell Ser Cys Llys Glu Lys Ala Asp 435 44 O 445 gta a.a.a. gac act gat gct gtt gat gtg tgt att gct gat ggt gt C tgt 392 Wall Lys Asp Thir Asp Ala Wall Asp Wall Cys Ile Ala Asp Gly Wall Cys 450 45.5 460 ata gat gct titt Cta a.a.a. cott cc c atg gag aca gaa gaa cott Cala att 44 O Ile Asp Ala Phe Lell Lys Pro Pro Met Glu Thir Glu Glu Pro Glin Ile 465 470 47s 48O tto tac aat gct toa c cc t cc aca Cta tot gcc aca atg ttic att gtt 488 Phe Asn Ala Ser Pro Ser Thir Luell Ser Ala Thr Met Phe Ile Wall 485 490 495 agt att ttg titt citt atc. att tot tot gta gC9 agt ttg taa 53 O Ser Ile Luell Phe Lell Ile Ile Ser Ser Wall Ala Ser Lell SOO 505 SEO ID NO 7 LENGTH: 509 TYPE : PRT ORGANISM: Homo sapiens FEATURE: NAME/KEY: CARBOHYD LOCATION: 82, 166, 235, 254, 368, 393, 49 O <4 OOs, SEQUENCE: 7 Met Gly Val Lieu Lys Phe His Ile Phe Phe Arg Ser Phe Wall Lys 1. 5 15 Ser Ser Gly Wall Ser Glin Ile Wall Phe Thir Phe Lieu. Lell Ile Pro 25 Luell Thir Luell Asn Phe Arg Ala Pro Pro Wall Ile Pro Asn Wall Pro 35 4 O 45 Phe Luell Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Lell Gly Phe SO 55 6 O Asp Glu Pro Luell Asp Met Ser Luell Phe Ser Phe Ile Gly Ser Pro Arg 65 70 7s 8O Ile Asn Ala Thir Gly Glin Gly Wall Thir Ile Phe Tyr Wall Asp Arg Luell 85 90 95 Gly Tyr Pro Tyr Ile Asp Ser Ile Thir Gly Val Thir Wall Asn Gly 105 11 O Gly Ile Pro Glin Ile Ser Luell Glin Asp His Lieu. Asp Ala 115 12 O 125 Asp Ile Thir Phe Met Pro Wall Asp ASn Lell Gly Met Ala Wall 13 O 135 14 O Ile Asp Trp Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Pro 145 150 155 160 Asp Wall Lys Asn Arg Ser Ile Glu Lieu. Wall Glin Glin Glin Asn 1.65 17O 17s Wall Glin Luell Ser Lell Thir Glu Ala Thir Glu Lys Ala Glin Glu Phe 18O 185 19 O Glu Ala Gly Asp Phe Luell Wall Glu Thir Ile Lys Luell Gly 195 2O5 Lell Luell Arg Pro Asn His Lell Trp Gly Tyr Tyr Lieu. Phe Pro Asp 21 O 215 22O Tyr Asn His His Tyr Lys Pro Gly Tyr Asn Gly Ser Phe Asn 225 23 O 235 24 O US 7,871,607 B2 165 166 - Continued Wall Glu Ile Arg Asn Asp Asp Luell Ser Trp Lell Trp Asn Glu Ser 245 250 255 Thir Ala Luell Tyr Pro Ser Ile Luell Asn Thir Glin Glin Ser Pro Wall 26 O 265 27 O Ala Ala Thir Luell Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall 27s 285 Ser Lys Ile Pro Asp Ala Lys Ser Pro Luell Pro Wall Phe Ala Thir 29 O 295 3 OO Arg Ile Wall Phe Thir Asp Glin Wall Luell Phe Lell Ser Glin Asp Glu 3. OS 310 315 Lell Wall Thir Phe Gly Glu Thir Wall Ala Luell Gly Ala Ser Gly Ile 3.25 330 335 Wall Ile Trp Gly Thir Lell Ser Ile Met Arg Ser Met Ser Luell 34 O 345 35. O Lell Luell Asp Asn Tyr Met Glu Thir Ile Luell ASn Pro Tyr Ile Ile Asn 355 360 365 Wall Thir Luell Ala Ala Met Ser Glin Wall Lell Glin Glu Glin 37 O 375 Gly Wall Ile Arg Lys Asn Trp Asn Ser Ser Asp Luell His Luell 385 390 395 4 OO Asn Pro Asp Asn Phe Ala Ile Glin Luell Glu Gly Gly Phe Thir 4 OS 415 Wall Arg Gly Lys Pro Thir Lell Glu Asp Luell Glu Glin Phe Ser Glu Lys 425 43 O Phe Cys Ser Cys Ser Thir Luell Ser Glu Ala Asp 435 44 O 445 Wall Lys Asp Thir Asp Ala Wall Asp Wall Ile Ala Asp Gly Wall Cys 450 45.5 460 Ile Asp Ala Phe Lell Lys Pro Pro Met Glu Thir Glu Glu Pro Glin Ile 465 470 47s Phe Asn Ala Ser Pro Ser Thir Luell Ser Ala Thir Met Phe Ile Wall 485 490 495 Ser Ile Luell Phe Lell Ile Ile Ser Ser Wall Ala Ser Lell SOO 505 SEQ ID NO 8 LENGTH: 34 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Reverse Primer to generate GPI anchor lacking N483 and terminating at Y482 with BamHI site in the 5' end SEQUENCE: 8 aattggat.cc ticagtagaaa atttgaggitt citt c SEO ID NO 9 LENGTH: 33 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Reverse Primer to generate GPI anchor lacking Y482 and terminating at F481 with BamHI site in the 5' end SEQUENCE: 9 aattggat.cc ticagaaaatt tdaggttctt ctd US 7,871,607 B2 167 168 - Continued <210s, SEQ ID NO 10 &211s LENGTH: 34 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer to generate GPI anchor lacking F481 and terminating at I480 with BamHI site in the 5' end <4 OOs, SEQUENCE: 10 aattggat.cc ticaaatttga gottcttctg. tctic 34 <210s, SEQ ID NO 11 &211s LENGTH: 32 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer to generate GPI anchor lacking I480 and terminating at Q479 with BamHI site in the 5' end <4 OOs, SEQUENCE: 11 aattggat.cc ticattgaggit tottctgtct c c 32 <210s, SEQ ID NO 12 &211s LENGTH: 32 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer to generate GPI anchor lacking Q479 and terminating at P478 with BamHI site in the 5' end <4 OOs, SEQUENCE: 12 aattggat.cc ticaaggttct tctgtcticca td 32 <210s, SEQ ID NO 13 &211s LENGTH: 31 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Reverse Primer to generate GPI anchor lacking P478 and terminating at E477 with BamHI site in the 5' end <4 OOs, SEQUENCE: 13 aattggat.cc ticattcttct gttct coatgg g 31 <210s, SEQ ID NO 14 &211s LENGTH: 32 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Forward Primer with Nhe restriction site at 5' end <4 OOs, SEQUENCE: 14 aattgctago atgggagtgc taaaattcaa go 32 <210s, SEQ ID NO 15 &211s LENGTH: 1473 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . . (1473) <223> OTHER INFORMATION: sHASEGPup to P478 and His Tagged US 7,871,607 B2 169 170 - Continued <4 OOs, SEQUENCE: 15 atg gga gtg Cta a.a.a. tto aag CaC at C titt titc aga agc titt gtt a.a.a. 48 Met Gly Wall Luell Lys Phe Lys His Ile Phe Phe Arg Ser Phe Wall 1. 1O 15 toa agt gga gta t cc Cag ata gtt ttic acc titc citt Ctg att CC a tgt 96 Ser Ser Gly Wall Ser Glin Ile Wall Phe Thir Phe Lell Lell Ile Pro Cys 2O 25 3 O tgc ttg act Ctg aat tto aga gca cott cott gtt att C Ca aat gtg cott 144 Cys Luell Thir Luell Asn Phe Arg Ala Pro Pro Wall Ile Pro Asn Wall Pro 35 4 O 45 tto citc. tgg gcc tgg aat gcc CC a agt gala titt tgt citt gga a.a.a. titt 192 Phe Luell Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Lell Gly Phe SO 55 60 gat gag CC a Cta gat atg agc citc. ttic tot titc ata gga agc cc c cga 24 O Asp Glu Pro Luell Asp Met Ser Luell Phe Ser Phe Ile Gly Ser Pro Arg 65 70 7s 8O ata aac gcc acc 999 Cala ggit gtt aca at a titt tat gtt gat aga citt 288 Ile Asn Ala Thir Gly Glin Gly Wall Thir Ile Phe Wall Asp Arg Luell 85 90 95 ggc tac tat cott tac ata gat to a at C aca gga gta act gtg aat gga 336 Gly Pro Tyr Ile Asp Ser Ile Thir Gly Wall Thir Wall Asn Gly 1OO 105 11 O gga at C cc c cag aag att t cc tta Cala gac Cat Ctg gac a.a.a. gct aag 384 Gly Ile Pro Glin Lys Ile Ser Luell Glin Asp His Lell Asp Ala Lys 115 12 O 125 a.a.a. gac att aca titt tat atg CC a gta gac aat ttg gga atg gct gtt 432 Asp Ile Thir Phe Met Pro Wall Asp ASn Lell Gly Met Ala Wall 13 O 135 14 O att gac tgg gala gaa tgg aga cc c act tgg gca aga aac tgg a.a.a. cott Ile Asp Trp Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Pro 145 150 155 160 a.a.a. gat gtt tac aag aat agg tot att gala gtt Cag Cala Cala aat 528 Asp Wall Lys Asn Arg Ser Ile Glu Luell Wall Glin Glin Glin Asn 1.65 17O 17s gta Cala citt agt citc. a Ca gag gcc act gag a.a.a. gca a.a.a. Cala gala titt 576 Wall Glin Luell Ser Lell Thir Glu Ala Thir Glu Lys Ala Glin Glu Phe 18O 185 19 O gaa aag gca 999 aag gat tto Ctg gta gag act ata a.a.a. ttg gga a.a.a. 624 Glu Lys Ala Gly Lys Asp Phe Luell Wall Glu Thir Ile Lys Luell Gly 195 2OO 2O5 tta citt cgg CC a aat CaC ttg tgg ggt tat citt titt cc.g gat tgt 672 Lell Luell Arg Pro Asn His Lell Trp Gly Lell Phe Pro Asp Cys 21 O 215 22O tac aac Cat CaC tat aag a.a.a. cc c ggt aat gga agt tgc ttic aat 72 O Tyr Asn His His Tyr Lys Pro Gly ASn Gly Ser Cys Phe Asn 225 23 O 235 24 O gta gala at a a.a.a. aga aat gat gat citc. agc tgg ttg tgg aat gala agc 768 Wall Glu Ile Arg Asn Asp Asp Luell Ser Trp Lell Trp Asn Glu Ser 245 250 255 act gct citt tac C Ca t cc att tat ttg aac act Cag Cag tot cott gta 816 Thir Ala Luell Tyr Pro Ser Ile Luell Asn Thir Glin Glin Ser Pro Wall 26 O 265 27 O gct gct aca citc. tat gtg cgc aat cga gtt cgg gaa gcc at C aga gtt 864 Ala Ala Thir Luell Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall 27s 28O 285 t cc a.a.a. at a cott gat gca a.a.a. agt CC a citt cc.g gtt titt gca tat acc 912 Ser Lys Ile Pro Asp Ala Lys Ser Pro Luell Pro Wall Phe Ala Thir 29 O 295 3 OO US 7,871,607 B2 171 172 - Continued cgc at a gtt titt act gat caa gtt ttg a.a.a. titc citt tot Cala gat gala 96.O Arg Ile Wall Phe Thr Asp Glin Val Luell Lys Phe Lell Ser Glin Asp Glu 3. OS 310 315 32O citt gtg tat aca titt ggc gala act gtt gct Ctg ggit gct tot gga att OO8 Lell Wall Thir Phe Gly Glu Thr Wall Ala Luell Gly Ala Ser Gly Ile 3.25 330 335 gta at a tgg gga acc ctic agt at a atg cga agt atg a.a.a. tot ttg Wall Ile Trp Gly Thir Lieu. Ser Ile Met Arg Ser Met Ser Cys Luell 34 O 345 35. O citc. Cta gac aat tac atg gag act at a Ctg aat cott tac at a at C aac 104 Lell Luell Asp Asn Tyr Met Glu Thr Ile Luell ASn Pro Tyr Ile Ile Asn 355 360 365 gtc aca Cta gca gcc aaa atg tdt agc Cala gtg citt cag gag Cala 152 Wall Thir Luell Ala Ala Lys Met Cys Ser Glin Wall Lell Cys Glin Glu Glin 37 O 375 38O gga gtg tgt at a agg aaa aac tig aat to a agt gac tat citt CaC citc. 2OO Gly Wall Cys Ile Arg Lys Asn Trp Asn Ser Ser Asp Luell His Luell 385 390 395 4 OO aac CC a gat aat titt gct att caa citt gag a.a.a. ggit gga aag ttic aca 248 Asn Pro Asp Asn Phe Ala Ile Glin Luell Glu Lys Gly Gly Lys Phe Thir 4 OS 41O 415 gta cgt gga a.a.a. ccg aca citt gala gac Ctg gag Cala titt tot gala a.a.a. 296 Wall Arg Gly Lys Pro Thir Lieu. Glu Asp Luell Glu Glin Phe Ser Glu 42O 425 43 O titt tat tgc agc tgt tat agc acc ttg agt tgt aag gag a.a.a. gct gat 344 Phe Cys Ser Cys Tyr Ser Thr Luell Ser Cys Lys Glu Lys Ala Asp 435 44 O 445 gta a.a.a. gac act gat gct gtt gat gtg tgt att gct gat ggt gt C tgt 392 Wall Lys Asp Thir Asp Ala Val Asp Wall Cys Ile Ala Asp Gly Wall Cys 450 45.5 460 ata gat gct titt Cta aaa Cct CCC atg gag aca gaa gaa cott gga to c 44 O Ile Asp Ala Phe Lieu Lys Pro Pro Met Glu Thir Glu Glu Pro Gly Ser 465 470 47s 48O ggit tot ggt gct CaC cat Cac Cat CaC Cat taa 473 Gly Ser Gly Ala His His His His His His 485 490 <210s, SEQ ID NO 16 &211s LENGTH: 490 212. TYPE : PRT &213s ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 16 Met Gly Val Lieu. Llys Phe Llys His Ile Phe Phe Arg Ser Phe Wall 1. 5 15 Ser Ser Gly Wall Ser Glin Ile Wall Phe Thir Phe Lell Lell Ile Pro 2O 25 Luell Thir Luell Asn Phe Arg Ala Pro Pro Wall Ile Pro Asn Wall Pro 35 4 O 45 Phe Luell Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Lell Gly Phe SO 55 6 O Asp Glu Pro Luell Asp Met Ser Lieu. Phe Ser Phe Ile Gly Ser Pro Arg 65 70 Ile Asn Ala Thir Gly Glin Gly Val Thir Ile Phe Wall Asp Arg Luell 85 90 95 Gly Pro Tyr Ile Asp Ser Ile Thir Gly Wall Thir Wall Asn Gly 105 11 O Gly Ile Pro Glin Lys Ile Ser Lieu. Glin Asp His Lell Asp Ala 115 12 O 125 US 7,871,607 B2 173 174 - Continued Asp Ile Thir Phe Met Pro Wall Asp ASn Lell Gly Met Ala Wall 13 O 135 14 O Ile Asp Trp Glu Glu Trp Arg Pro Thir Trp Ala Arg Asn Trp Lys Pro 145 150 155 160 Asp Wall Lys Asn Arg Ser Ile Glu Luell Wall Glin Glin Glin Asn 1.65 17O 17s Wall Glin Luell Ser Lell Thir Glu Ala Thir Glu Lys Ala Glin Glu Phe 18O 185 19 O Glu Ala Gly Asp Phe Luell Wall Glu Thir Ile Lys Luell Gly 195 Lell Luell Arg Pro Asn His Lell Trp Gly Tyr Lell Phe Pro Asp 21 O 215 Tyr Asn His His Tyr Lys Pro Gly Tyr ASn Gly Ser Phe Asn 225 23 O 235 24 O Wall Glu Ile Arg Asn Asp Asp Luell Ser Trp Lell Trp Asn Glu Ser 245 250 255 Thir Ala Luell Tyr Pro Ser Ile Luell Asn Thir Glin Glin Ser Pro Wall 26 O 265 27 O Ala Ala Thir Luell Tyr Wall Arg Asn Arg Wall Arg Glu Ala Ile Arg Wall 27s 285 Ser Lys Ile Pro Asp Ala Lys Ser Pro Luell Pro Wall Phe Ala Thir 29 O 295 3 OO Arg Ile Wall Phe Thir Asp Glin Wall Luell Phe Lell Ser Glin Asp Glu 3. OS 310 315 Lell Wall Thir Phe Gly Glu Thir Wall Ala Luell Gly Ala Ser Gly Ile 3.25 330 335 Wall Ile Trp Gly Thir Lell Ser Ile Met Arg Ser Met Ser Luell 34 O 345 35. O Lell Luell Asp Asn Tyr Met Glu Thir Ile Luell ASn Pro Tyr Ile Ile Asn 355 360 365 Wall Thir Luell Ala Ala Met Ser Glin Wall Lell Glin Glu Glin 37 O 375 Gly Wall Ile Arg Lys Asn Trp Asn Ser Ser Asp Luell His Luell 385 390 395 4 OO Asn Pro Asp Asn Phe Ala Ile Glin Luell Glu Lys Gly Gly Phe Thir 4 OS 415 Wall Arg Gly Lys Pro Thir Lell Glu Asp Luell Glu Glin Phe Ser Glu 425 43 O Phe Cys Ser Ser Thir Luell Ser Glu Ala Asp 435 44 O 445 Wall Lys Asp Thir Asp Ala Wall Asp Wall Cys Ile Ala Asp Gly Wall Cys 450 45.5 460 Ile Asp Ala Phe Lell Lys Pro Pro Met Glu Thir Glu Glu Pro Gly Ser 465 470 47s 48O Gly Ser Gly Ala His His His His His His 485 490 SEO ID NO 17 LENGTH: 39 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: SpacerHis For Primer SEQUENCE: 17