CA2264484C - Human plasma hyaluronidase - Google Patents

Abstract

The invention is based on the discovery of methods for purification of an acid active hyaluronidase found in human plasma (hpHAse), including both biochemical and immunoaffinity purification methods. The method of immunoaffinity purification of the invention is based on the discovery of a method for identifying antibodies that specifically bind native hpHAse (anti-native hpHAse antibodies), and anti-native hpHAse antibodies identified by this screening method. The invention also features an assay for sensitive detection of HAse activity using biotinylated hyaluronic acid (bHA). Purification and characterization of hpHAse lead to the inventors' additional discovery that hpHAse is encoded by the LuCa-1 gene, which gene is present in the human chromosome at 3p21.3, a region associated with tumor suppression. The invention additionally features methods of treating tumor-bearing patients by administration of hpHAse and/or transformation of cells with hpHAse-encoding DNA.

Description

CA 02264484 1999-03-03W0 98ll6655 PCT/US97/180891HUMAN PLASMA HYALURONIDASECROSS—REFERENCE TO RELATED APPLICATIONSThis application is a continuation-in-part of U.S. application serial no.5 08/733,360, filed October 17, 1996, which application is hereby incorporated by1015202530reference.STATEMENT REGARDING FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENTThis invention was made with government support under grant nos.CA44768, CA58207, and GM46765, awarded by the National Institutes of Health.The government may have certain rights in this invention.FIELD OF THE INVENTIONThis invention relates generally to the field of B-1,4—endoglycosidases,particularly hyaluronidases.BACKGROUND OF THE INVENTIONHyaluronidases (HAses; E.C. 3.1.25) are a group of neutral- andacid-active enzymes found throughout the animal kingdom in organisms as diverseas microbes (e.g, Streptococcus pyogenes, T reponema palladium, and nematodes),bees, wasps, hornet, spiders, scorpions, fish, snakes, lizards, and mammals.Hyaluronidases degrade hyaluronan (HA; also known as hyaluronic acid) and, toa lesser extent, chondroitin sulfates (for a review, see Kreil et al. 1995 Protein Sci.4: 1666-9). Vertebrate hyaluronidases are separated into two general classes: 1) theneutral hyaluronidases, such as the predominantly sperrn—associated protein PH2O(Liu et al. 1996 Proc. Natl. Acad. Sci. USA £27832-7; Primakoff et al. 1985 J.Cell Biol. _1_(_)l:2239—44; Lin et al. 1993 Proc. Natl. Acad. Sci. USA 29210071-5);and 2) the acid-active hyaluronidases, which have a distinct pH optimum betweenpH 3.5 to 4.0 and have been described in extracts of liver (Fiszer-Szafarz et al.1995 Acta Biochim Pol. @2316), kidney (Komender et al. 1973 Bull. Acad. Pol.WO 98/166551015202530CA 02264484 1999-03-03PCT/U S97/ 180892Sci. [Biol.] ;_1_:637-41), lung (Thet et al. 1983 Biochem. Biophys. Res. Commun._1fl:71-7), brain (Margolis et al. 1972 J. Neutroc/zem. _l_2:2325-32), skin (Cashmanet al. 1969 Arch. Biochem. Biophys. §§:387-95), placenta, macrophages,fibroblasts (Lien et al. 1990 Biochim Biophys. Acta @1318-25; Ruggiero et al.1987 J. Dent. Res. ®:1283—7), urine (Fiszer-Szafarz et al. supra) and humanplasma (De Salegui et al. 1967 Arch. Biochem. Biophys. @260-67). Acid-activehyaluronidase activity has also been described in the sera of mammals, thoughsome species exhibit no detectable activity at all (Fiszer-Szafarz et al. 1990 Biol.Cell 68:95-100; De Salegui et al. 1967 supra).Hyaluronan, the main substrate for hyaluronidase, is a repeatingdisaccharide of [GlcNAcfl1-4GlcUA61—3],, that exists in vivo as a high molecularweight linear polysaccharide. Degradation of hyaluronan by hyaluronidase isaccomplished by either cleavage at B~N-acetyl-hexosamine—[1—>4]—glycosidic bondsor cleavage at B-gluconorate-[1->3]-N-acetylglucosamine bonds.Hyaluronan is found in mammals predominantly in connective tissues, skin,cartilage, and in synovial fluid. Hyaluronan is also the main constituent of thevitreous of the eye. In connective tissue, the water of hydration associated withhyaluronan creates spaces between tissues, thus creating an environment conduciveto cell movement and proliferation. Hyaluronan plays a key role in biologicalphenomena associated with cell motility including rapid development, regeneration,repair, embryogenesis, embryological development, wound healing, angiogenesis,and tumorigenesis (Toole 1991 Cell Biol. Extracell. Matrix, Hay (ed), PlenumPress, New York, 1384-1386; Bertrand et al. 1992 Int. J. Cancer _5;:1—6;Knudson et al, 1993 FASEB J. 111233-1241). In addition, hyaluronan levelscorrelate with tumor aggressiveness (Ozello et al. 1960 Cancer Res. @600-604;Takeuchi et al. 1976, Cancer Res. $22133-2139; Kimata et al. 1983 Cancer Res.fl:1347-1354).Hyaluronidase is useful as a therapeutic in the treatment of diseasesassociated with excess hyaluronan and to enhance circulation of physiological fluidsand/or therapeutic agents at the site of administration. For example, hyaluronidasehas been used to reduce intraocular pressure in the eyes of glaucoma patientsW0 98l166551015202530CA 02264484 1999-03-03PCT/US97I180893through degradation of hyaluronan within the vitreous humor (USPN 4,820,516,issued April 11, 1989). Hyaluronidase has also been used in cancer therapy as a"spreading agent" to enhance the activity of chemotherapeutics and/or theaccessibility of tumors to chemotherapeutics (Schiiller et al., 1991, Proc. Amer.Assoc. Cancer Res. 3_2:173, abstract no. 1034; Czejka et al., 1990, Phamzazie4_§:H.9) and has been used in combination with other chemotherapeutic agents inthe treatment of a variety of cancers including urinary bladder cancer (Horn et al.,1985, J. Surg. Oncol., E2304-307), squamous cell carcinoma (Kohno et al., 94,J. Cancer Res. Oncol. , Q9293-297), breast cancer (Beckenlehner et al., 1992, J.Cancer Res. Oncol. L8:591—596), and gastrointestinal cancer (Scheithauer et al.,1988, Anticancer Res. §:391-396). Administration of hyaluronidase also inducesresponsiveness of previously chemotherapy—resistant tumors of the pancreas,stomach, colon, ovaries, and breast (Baumgartner et al., 1988, Reg. Cancer Treat.1:55-58; Ziinker et al., 1986, Proc. Amer. Assoc. Cancer Res. 212390). Serumhyaluronidase prevents growth of tumors transplanted into mice (De Maeyer et al.,1992, Int. J. Cancer _5_1:657-660), while injection of hyaluronidase inhibits tumorformation caused by exposure to carcinogens (Pawlowski et al., 1979, Int. J.Cancer Q2105-109; Habennan et al., 1981, Proceedings of the 17th AnnualMeeting of the American Society of Clinical Oncology, Washington, D.C. , ;g:105,abstract no. 415). Intravenous or intramuscular injection of hyaluronidase iseffective in the treatment of brain cancer (gliomas) (PCT published application no.W088/02261, published April 7, 1988).Hyaluronidase expression, and levels of hyaluron, have been associated withtumor development and progression. Levels of a secreted neutral hyaluronidaseactivity in carcinomas derived from ovary (Miura et al. 1995 Anal. Biochem.2_fi_:333-40), prostate (Lokeshwar et a1. 1996 Cancer Res §_6:65l—7), brain,melanocyte, and colon (Liu et al. 1996 Proc. Natl. Acad. Sci. USA 9?;:7832—7837)are higher than in normal tissue. This secreted neutral hyaluronidase activityappears similar or identical to the neutral hyaluronidase activity of the spermhyaluronidase PH20.hyaluronidase activity is significantly decreased in metastatic carcinomas of theIn contrast to neutral activity, the acid active serumWO 981166551015202530CA 02264484 1999-03-03PCT/U S97! 180894lung, breast, and colon (Northrup et al. 1973 Clin. Biochem. _6_:220-8; Kolarovaet al. 1970 Neoplasma fl:641-8). Further, mice having an allele of the hyal-1locus that is associated with lower levels of serum hyaluronidase activity exhibitfaster rates of growth of transplanted tumors than mice having an hyal-1 allele thatis associated with 3-fold higher hyaluronidase activity levels (Fiszer-Szafarz et al.1989 Somat. Cell. Mol. Genet. §:79—83; De Maeyer et al. supra).At present, the only hyaluronidase activity available for clinical use is ahyaluronidase isolated from a testicular extract from cattle (WYDASE°,Wyeth—Ayerst). The bovine extract is not optimum not only because of its non-human source, but also because the extract contains multiple types ofhyaluronidases and other as yet undefined components. While the human serumacid-active hyaluronidase activity would be preferred for administration, thishyaluronidase has not been previously isolated or purified. Although previousstudies were able to determine that the serum acid-active hyaluronidase activity isnot a component of platelets since hyaluronidase activity levels in plasma arecomparable to those found in serum (De Salegui et al. 1967 supra), attempts toisolate this acid active hyaluronidase activity from human serum have met withlimited success due in part to the stability of the purified activity and the inabilityto obtain an adequately high specific activity. Immunopurification attempts havebeen hindered by the inability to identify and isolate antibodies that specificallybind the activity in its native form in plasma. Monoclonal antibodies identified byconventional ELISA techniques bind denatured human plasma hyaluronidase in theELISA screening assay do not bind the native, folded protein (Harrison et al. 1988J Reprod Fertil 8_2:777-85).Conventional methods for hyaluronidase activity include ELISA-like assays(Delpech et al. 1987 J. Immunol. Methods _1Q4:223—9; Stern et al. 1992 Matrix_1__2_:397-403; Afify et al. 1993 Arch. Biochem. Biophys. @434-41; Reissig et al. ,1955, J. Biol. Chem. _2_1_7_:956-966) in which a sample containing hyaluronidase isapplied to the well of a microtiter dish having hyaluronan or hyaluronectin non-covalently bound to its surface. HAse present in the sample degrades the HAsubstrate. The plates are then washed, and HAse activity is detected by examiningW0 98l166551015202530CA 02264484 1999-03-03PCT/US97/18089the plates for remaining HA substrate.Hyaluronidase activity can also be detected by substrate gel zymography(Guentenhoner et al. , 1992, Matrix 12.5388-396). In this assay a sample is appliedto a SDS-PAGE gel containing hyaluronan and the proteins in the sample separatedby electrophoresis. The gel is then incubated in an enzyme assay buffer andsubsequently stained to detect the hyaluronan in the gel. Hyaluronidase activity isvisualized as a cleared zone in the substrate gel.These conventional methods for detecting hyaluronidase activity arehampered by both the difficulties in producing a detectably-labeled hyaluronic acidsubstrate and the technical difficulties in achieving rapid, sensitive, andreproducible detection of hyaluronidase activity. For example, biotin labeling ofhyaluronic acid for use in ELISA-like assays has proved reticent to biotinylationsince HA contains no free amine groups, the moiety with which activated biotincovalently binds. Prior attempts to solve this problem have focused on use of abiotinylated-HA binding aggrecan peptide from bovine nasal cartilage (Levvy et al.1966 Method Enzymol. §:571-584), which requires tedious, time-consuming steps.Furthermore, conventional hyaluronidase assays use assay plates having HAsubstrate non-covalently bound to the plate surface, which can lead to both falsepositive and false negative results. Because the HA substrate is non-covalentlybound to the plate surface, the washing step following exposure of the plates to theHAse—containing sample often results in non-specific removal of non-degraded HAsubstrate on the plate. Thus, the sensitivity of the conventional HAse assay iscompromised. HAse activities using gel zymography avoid the problem associatedwith ELISA-like assays, but are time-consuming (e. g. , the test sample and the HA-containing gel are normally incubated for about 18 hr to 24 hr) and can result inartifacts if the gel is improperly loaded with excess protein sample. Moreover,analyses of crude preparations is impossible by gel zymography.Thus, despite the presence of a desirable acid active plasma hyaluronidaseactivity, and human blood product companies’ economic motivation to obtain anyand all useful components from a resource as precious and scarce as human blood,W0 98/ 166551015202530CA 02264484 1999-03-03PCT/U S97! 180896the human plasma fractions containing this acid active hyaluronidase activity arediscarded for want of an acceptable method for its isolation and purification.Given the value of hyaluronidases in chemotherapy, there is a need in thefield for a method of identifying and isolating the polypeptide associated with theacid active hyaluronidase activity in human serum.SUMMARY OF THE INVENTIONThe invention is based on the discovery of methods for purification of anacid active hyaluronidase found in human plasma (hpHAse), which include bothThe method ofimmunoaffinity purification of the invention is based on the discovery of a methodbiochemical and immunoaffinity purification methods.for identifying antibodies that specifically bind native hpHAse (anti—native hpHAseantibodies), and anti-native hpHAse antibodies identified by this screening method.The invention also features an assay for sensitive detection of HAse activity usingbiotinylated hyaluronic acid (bHA). Purification and characterization of hpHAselead to the inventors’ additional discovery that hpHAse is encoded by the LuCa-1gene, which is located in the human chromosome at 3p21.3, a region associatedwith tumor suppression.Thus, in one aspect the invention features an assay device for detection ofhyaluronidase activity comprising an insoluble support and biotinylated hyaluronicacid (bHA) covalently bound to the support. Preferably, the bHA is prepared ina one-step reaction of hyaluronic acid, 1~ethy1-dimethylaminopropyl carbodiamide(EDC), and biotin hydrazide. In preferred embodiments, the bHA comprises atleast one biotin moiety per every 100 disaccharide units in the hyaluronic acidmoiety, and the bHA is covalently bound to the support by at least one covalentbond per every 50 disaccharide units in the hyaluronic acid moiety.In another aspect the invention features a method of purifying a native acidactive hyaluronidase (aaHAse) from a sample, the method comprising the steps of:(a) dissolving a sample suspected of containing aaHAse in a solution at atemperature substantially less than room temperature, the solution comprising anon-ionic detergent; (b) raising the temperature of the solution to a temperatureW0 98/166551015202530CA 02264484 1999-03-03PCT/US97ll80897substantially greater than room temperature, said raising resulting in the formationof a detergent—rich phase comprising aaHAse and a detergent—poor phase; and (c)isolating aaHAse from the detergent-rich phase. Preferably the aaHAse is hpHAseand steps (a)—(c) are repeated twice.In yet another aspect of the invention, the invention features a method forscreening candidate antibodies for binding to a native aaHAse comprising the stepsof: (a) incubating a candidate antibody with a sample comprising native aaHAse,said incubating being for a time sufficient for formation of antibody-aaHAsecomplexes; (b) contacting the sample with an insoluble support having anti-antibody and detectably—labeled hyaluronic acid bound thereto for a time sufficientfor formation of anti—antibody-candidate antibody—hpHAse complexes; and (c)exposing the sample in contact with the support to an acidic pH of about 3.4 to3.7, thereby allowing hpHAse in the antibody-aaHAse complex to degrade thedetectably labeled hyaluronic acid, wherein samples associated with hyaluronic aciddegradation comprise an anti—aaHAse antibody. In related aspects, the inventionfeatures anti-native hpHAse antibodies, hybridoma cell lines secreting suchantibodies, and assay devices for immunopurification and/or detection of hpHAsecomprising anti-hpHAse antibodies bound thereto.In still another aspect, the invention features a method of purifying hpHAsefrom a sample, the method comprising contacting a sample comprising hpHAsewith an anti-hpHAse antibody. In preferred embodiments, the sample is humanblood, serum, plasma, or urine or the hpHAse is a recombinant hpHAse.In another aspect, the invention features substantially purified nativehpHAse characterized by a fatty acid moiety that is resistant to cleavage byphospholipase C, phospholipase D, and N-glycosidase—F. In related aspects theinvention features formulations comprising native hpHAse, particularly liposomeformulations.In yet another aspect the invention features methods for recombinantlyexpressing hpHAse. The recombinant expression system of the invention provideshigh levels of secreted hpHAse suitable for commercial production of hpHAse andfor production of hpHAse for use in therapeutic applications. In related aspects,_......._.........t.a..........__....__..»..............H.t.,.... . . .. .., ,..,,...................l..............t.a..,.,....,W0 98/ 166551015202530CA 02264484 1999-03-03PCT/US97/180898the invention features recombinant hpHAse and methods of making recombinanthpHAse.In additional related aspects, the invention features methods of treatingpatients having or susceptible to a condition for which administration ofhyaluronidase is desirable. In specific embodiments, the invention featuresadministration of human plasma hyaluronidase to a patient having" or susceptibleto cancer associated with defective hpHAse expression comprising administeringhpHAse polypeptide to the patient in an amount effective to suppress tumorgrowth. In further embodiments, the invention features methods of treating apatient after suffering a myocardial infarction or a patient suffering from alysosomal storage disease.Another aspect of the invention features a method of treating a patienthaving or susceptible to cancer associated with a defective LuCa-1 gene, themethod comprising introducing into a cell of the patient a construct comprising anucleotide sequence encoding a human plasma hyaluronidase polypeptide and aeukaryotic promoting sequence operably linked thereto, resulting in the genetictransformation of the cell so that the nucleotide sequence expresses hpHAase.The invention also features a method for identifying a patient having orsusceptible to a condition associated with defective human plasma hyaluronidaseactivity comprising the steps of contacting a plasma sample from the patient withan anti-native human plasma hyaluronidase antibody and detecting complexes ofthe enzyme and the antibody.A primary object of the invention is to provide a purified hyaluronidase,hpHAse, which can be used in a variety of clinical therapies including cancertherapy, particularly cancers associated with a defect in the tumor suppressor geneLuCa—1.An advantage of the present invention is that purified hpHAse is moreappropriate for therapeutic uses than the presently available commercialformulations of hyaluronidase which are from a non-human source, which containtwo hyaluronidases (rather than one), and which, as determined by SDS—PAGEanalysis, is a very crude mixture that contains various proteins, including severalW0 981166551015202530CA 02264484 1999-03-03PCT/US97/ 180899unidentified proteins and proteins having various biological activities includingPurifiedhpHAse provides a ''clean'' source of hyaluronidase, is less likely to induce someanticoagulant activities (Doctor et al., Thrombosis Res. 30:565-571).of the side effects associated with the presently available commercial formulation,and allows better control of the level of activity associated with specific dosages.Another advantage of the invention is that hpHAse can be purified from alipid fraction of plasma that, according to present commercial practices, isotherwise discarded.An advantage of the HAse assay of the invention is that it provides for anassay for HAse activity that is at least 1,000—fold more sensitive than conventionalELlSA—like assays. In addition, the invention provides a biotinylated hyaluronicacid substrate that can be easily and efficiently prepared.An advantage of the anti-native aaHAse assay of the invention is that theassay provides a rapid screen for antibodies that bind native aaHAse.Another advantage of the invention is that specific detection of hpHAseallows a specific means of measuring LuCa—l expression and correlation of levelsof hpHAse in serum or urine of a patient with susceptibility to or presence of adisease associated with a LuCa—l defect (e.g., cancer, e.g., small cell lung cellcarcinoma).These and other objects, advantages and features of the present inventionwill become apparent to those persons skilled in the art upon reading the details ofthe invention as more fully set forth below.BRIEF DESCRIPTION OF THE DRAWINGSFig. 1 is a schematic showing the chemical and physical configuration ofa surface for covalent attachment of biotinylated HA.Fig. 2 is a schematic showing the chemical structure of biotinylated HA.Fig. 3 is a schematic showing the steps in the anti-native acid active HAseantibody assay of the invention.Fig. 4 is a graph showing hyaluronidase activity associated withsupernatantsof human plasma after immunoprecipitation with varyingW0 98/1665510152025CA 02264484 1999-03-03PCT/U S97] 1808910concentrations of anti—native hpHAse l7E9 antibody (open circles) or bound controlantibody (open squares).Fig. 5 is a schematic showing the alignment of the predicted amino acidsequence of LuCa-1 (SEQ ID NO:3) with the amino acid sequence of the neutralHAse PH20 (SEQ ID NO:11). Identical amino acid residues are shaded.Fig. 6 is a hydropathy plot of the LuCa-1/hpHAse.Fig. 7A is a graph showing the acid active HAse activity of detergentextracts of LuCa-1~expressing cos-7 cells and conditioned medium (shadedcolumns) and of mock-transfected cells and conditioned medium (unshadedcolumns).Fig. 7B is a graph showing the acid active Hase activity of detergentextracts of LuCa-1 expressing Hek 293 cells and conditioned medium (shadedcolumns) and of mock-transfected cells and conditioned medium (unshadedcolumns).Fig. 8 is a graph showing the pH optima of the HAse activity of hpHAsepurified from human plasma (open squares and solid line), recombinant LuCa-1expressed in cos-7 cells (closed squares with dashed line), and Type VI—S testicularhyaluronidase (closed circles with dashed line).Fig. 9 is a graph showing hpHAse expression in the cell layer andconditioned media of normal human keratinocytes and several carcinoma cell lines.Fig. 10 is a graph showing the effects of hpHAse peritumoral injection upongrowth of HSC—3 head and neck squamous cell carcinoma cells in an animalmodel.Fig. 11 is a graph showing the effects of hpHAse expression in transformedHSC-3 cells upon tumor growth in an animal model.Fig. 12 is a graph showing the effects of hpHAse expression in transformedHSC-3 cells upon cachexia in an animal model.W0 98ll66551015202530CA 02264484 1999-03-03PCT/U S97/ 180891 1DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTSBefore the present purified hyaluronidase and DNA encoding same aredescribed, it is to be understood that this invention is not limited to the particularmethodology, protocols, cell lines, vectors and reagents described as such may, ofcourse, vary. It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by the appended claims.It must be noted that as used herein and in the appended claims, thesingular forms "a", "and", and "the" include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to "a transformed cellcontaining DNA encoding a hyaluronidase" includes a plurality of such cells andreference to "the transformation vector" includes reference to one or moretransformation vectors and equivalents thereof known to those skilled in the art,and so forth.Unless defined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood to one of ordinary skill in the art towhich this invention belongs. Although any methods, devices and materials similaror equivalent to those described herein can be used in the practice or testing of theinvention, the preferred methods, devices and materials are now described.All publications mentioned herein are incorporated herein by reference forthe purpose of describing and disclosing the cell lines, vectors, and methodologieswhich are described in the publications which might be used in connection with thepresently described invention.DefinitionsBy “acid active hyaluronidase" or "aaHAse" is meant a hyaluronidasehaving B-l,4—endoglycosidase activity in the cleavage of hyaluronan and a pHoptimum of HAse activity at about pH 3.7. aaHAse as used herein encompasseshuman plasma hyaluronidase.By "human plasma hyaluronidase," "human plasma acid activehyaluronidase, " and "hpHAse" is meant a hyaluronidase naturally found in humanW0 98/ 16655202530CA 02264484 1999-03-03PCT/US97ll808912plasma and having the following characteristics: 1) B—1,4-endoglycosidase activityin the cleavage of hyaluron; 2) a pH optimum of HAse activity at about pH 3.7;3) a molecular weight of about 57 kDa as determined by 12.5% SDS-PAGEnon~reducing gel electrophoresis; 4) a specific enzymatic activity of about 2 x 105to 8 x 105 turbidity reducing units (TRU)/mg protein following purification; 5) anisoelectric point, as determined by elution in chromatofocusing on Mono-P f .p.1.c. ,of pH 6.5; 6) partitioning into the Triton X-114 detergent-rich phase upontemperature-induced detergent phase extraction; 7) a fatty acid post-translationalmodification (e.g. , a lipid anchor) that is resistant to cleavage by phospholipase C,phospholipase D, and N-glycosidase-F; 8) at least two N —1inked glycosylation sites;and 9) has the amino acid sequence:MAGHLLPICALFLTLLDMAQGFRGPLLPNRPFTTVWNANTQWCLERHGVDVDVSVFDVVANPGQTFRGPDMTIFYSSQLGTYPYYTPTGEPVFGGLPQNASLIAHLARTFQDILAAIPAPDFSGLAVIDWEAWRPRWAFNWDTKDIYRQRSRALVQAQHPDWPAPQVEAVAQDQFQGAARAWMAGTLQLGGALRPRGLWGFYGFPDCYNYDFLSPNYTGQCPSGIRAQNDQLGWLWGQSRALYPSIYMPAVLEGTGKSQMYVQHRVAEAFRVAVAAGDPNLPVLPYVQIFYDTTNHFLPLDELEHSLGESAAQGAAGVVLWVSWENTRTKESCQAIKEYMDTTLGPFILNVTSGALLCSQALCSGHGRCVRRTSHPKALLLLNPASFSIQLTPGGGPLSLRGALSLEDQAQMAVEFKCRCYPGWQAPWCERKSMW (SEQ ID N021)where MAGHLLPICALFLTLLDMAQG (SEQ ID N022) is a signal sequencecleaved during post-translational modification. "hpHAse" as used herein is meantto encompass hpHAse polypeptides having the amino acid sequence of naturally-occurring hpHAse, as well as hpHAse polypeptides that are modified relative tothe naturally—occurring amino acid sequence due to amino acid substitution,Preferably,"hpHAse" encompasses hpHAse polypeptides that are biologically active (e.g. , candeletion, and/or addition (e.g., fusion proteins) and the like.bind anti—hpHAse antibodies and/or exhibit hyaluronidase activity).By "urine form of hpHAse" or "urine hpHAse" is meant a form of hpHAseis found in human urine and characterized by: 1) B—1,4—endoglycosidase activity inthe cleavage of hyaluron; 2) a pH optimum of HAse activity at about pH 3.7; 3)WO 98/166551015202530CA 02264484 1999-03-03PCT/US97/1808913a molecular weight of about 57 kDa as determined by gel zymography using 12.5 %SDS—PAGE non-reducing gel electrophoresis; 4) immunoprecipitation with anti-native hpHAse monoclonal antibodies specific to LuCa—1; 5) an isoelectric point,as determined by elution in chromatofocusing on Mono-P f.p.l.c., or pH 6.5; and6) partitioning into the Triton X-114 detergent—rich phase upon temperature-induceddetergent phase extraction.By "native hpHAse" is meant hpHAse that is folded in its naturally-occurring configuration (i.e. , hpHAse is not denatured). Where the native hpHAsedoes not comprise the entire amino acid sequence of natural1y—occurring hpHAse(SEQ ID NO:1), native hpHAse polypeptides are those polypeptides that, whenfolded, mimic a three-dimensional epitope of native, fu1l—length hpHAse such thatantibodies that bind native hpHAse bind to the hpHAse polypeptide. "NativehpHAse" encompasses both hpHAse that is naturally found in human plasma,blood, serum, and urine, as well as hpHAse that is recombinantly produced (e.g.,by expression in a mammalian cell).By "polypeptide" is meant any chain of amino acids, regardless of lengthor post-translational modification (e.g., glycosylation. phosphorylation. or fattyacid chain modification).By a "substantially pure polypeptide" a polypeptide that has been separatedfrom components which naturally accompany it (e.g., a substantially pure hpHAsepolypeptide purified from human plasma is substantially free of componentsnormally associated with human plasma). Typically, the polypeptide issubstantially pure when it is at least 60%, by weight, free from the proteins andnaturally-occurring organic molecules with which it is naturally associated.Preferably, the preparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight, hpHAse polypeptide. A substantially purehpHAse polypeptide can be obtained, for example, by extraction from a naturalsource (e.g., mammalian plasma, preferably human plasma); by expression of arecombinant nucleic acid encoding hpHAse polypeptide; or by chemicallysynthesizing the protein. Purity can be measured by any appropriate method, e. g. ,W0 98/166551015202530CA 02264484 1999-03-03PCT/U S97! 1808914chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.A protein is substantially free of naturally associated components when itis separated from those contaminants which accompany it in its natural state.Thus, a protein which is chemically synthesized or produced in a cellular systemdifferent from the cell from which it naturally originates will be substantially freefrom its naturally associated components. Accordingly, substantially purepolypeptides include those derived from eukaryotic organisms but synthesized inE. coli or other prokaryotes.By "antibody" is meant an immunoglobulin polypeptide that is capable ofbinding an antigen. Antibody as used herein is meant to include the entireantibody as well as any antibody fragments (e. g. F(ab’)2, Fab’, Fab, Fv) capableof binding the epitope, antigen or antigenic fragment of interest. Antibodies of theinvention are immunoreactive or irnmunospecific for and therefore specifically andselectively bind to native hpHAse polypeptide. Anti-hpHAse antibodies arepreferably immunospecific (i.e., not substantially cross-reactive with relatedmaterials). Antibodies may be polyclonal or monoclonal, preferably monoclonal.By "binds specifically" is meant high avidity and/or high affinity bindingof an antibody to a specific polypeptide i.e. , epitope of hpHAse. Antibody bindingto its epitope on this specific polypeptide is preferably stronger than binding of thesame antibody to any other epitope, particularly those which may be present inmolecules in association with, or in the same sample, as the specific polypeptideof interest e. g. , binds more strongly to hpHAse than to other components in humanplasma. Antibodies that bind specifically to a polypeptide of interest may becapable of binding other polypeptides at a weak, yet detectable, level (e.g., 10%or less of the binding shown to the polypeptide of interest). Such weak binding,or background binding, is readily discernible from the specific antibody binding tothe compound or polypeptide of interest, e. g. by use of appropriate controls. Ingeneral, antibodies of the invention which bind to native hpHAse with a bindingaffinity of 107 liters/mole or more, preferably 108 l/mole or more, even morepreferably 109 l/mole or more, are said to bind specifically to hpHAse. In general,an antibody with a binding affinity of 10‘ l/mole or less is not useful in that it willW0 98/ 166551015202530CA 02264484 1999-03-03PCT/U S97/ 1808915not bind an antigen at a detectable level using conventional methodology currentlyused.By "anti-native hpHAse antibody" or "anti-hpHAse antibody" is meant anantibody that specifically binds native (i.e., non-denatured) hpHAse. Preferably,such antibodies can be used to immunopurify (e.g., by immunoprecipitation orimmunoaffinity column chromatography) natura1ly—occurring hpHAse from humanplasma and/or recombinant hpHAse expressed by, for example, mammalian cells.By "operably linked" is meant that a DNA of interest (e.g., DNA encodingan hpHAse polypeptide) and a regulatory sequence(s) are connected in such a wayas to permit gene expression of the DNA of interest when the appropriatemolecules (e.g., transcriptional activator proteins) are bound to the regulatorysequence(s), thus facilitating production of, e.g., an hpHAse polypeptide, arecombinant protein, or an RNA molecule.By "transformation" is meant a permanent genetic change induced in a cellfollowing incorporation of new DNA (i.e. , DNA exogenous to the cell). Where thecell is a mammalian cell, the permanent genetic change is generally achieved byintroduction of the DNA into the genome of the cell.By "vector" is meant any compound, biological or chemical, whichfacilitates transformation of a host cell with DNA encoding an hpHAse polypeptideof the invention.By "substantially identical" is meant a polypeptide or nucleic acid exhibitingat last 50%, preferably 85%, more preferably 90%, and most preferably 95%homology to a reference amino acid or nucleic acid sequence. For polypeptides,the length of comparison sequences will generally be at least 16 amino acids,preferably at least 20 amino acids, more preferably at least 25 amino acids, andmost preferably 35 amino acids. For nucleic acids, the length of comparisonsequences will generally be at least 50 nucleotides, preferably at least 60nucleotides, and most preferably 110 nucleotides.Sequence identity is typically measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison,W0 98/166551O15202530CA 02264484 1999-03-03PCT/US97/1808916WI 53705). Such software matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, substitutions, and other modifications.Conservative substitutions typically include substitutions within the followinggroups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine.The terms "treatment", "treating" and the like are used herein to generallymean obtaining a desired pharmacologic and/ or physiologic effect. The effect maybe prophylactic in terms of completely or partially preventing a disease or symptomthereof and/or may be therapeutic in terms of a partial or complete cure for adisease and/or adverse effect attributable to the disease. "Treatment" as usedherein covers any treatment of a disease in a mammal, particularly a human, andincludes: (a) preventing the disease (e.g. , cancer) from occurring in a subject whomay be predisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; or (c) relieving thedisease, i.e., causing regression of the disease (e. g, reduction in tumor volume,slowing of cachexia). The invention is directed toward treatment of patients havingor susceptible to cancer associated with a defect in the LuCa-1 gene, the gene thatencodes hpHAse.By "therapeutically effective amount of a substantially pure hpHAsepolypeptide" is meant an amount of a substantially pure hpHAse polypeptideeffective to facilitate a desired therapeutic effect. The precise desired therapeuticeffect will vary according to the condition to be treated. For example, the desireddegradation of hyaluronan is the desired therapeutic effect where hpHAse isadministered to the subject in the treatment of a condition associated with excesshyaluron, undesirable cell motility (e.g., tumor cell metastasis), and/ or to enhancecirculation of physiological fluids at the site of administration and/or inhibit tumorgrowth or progression. Where hpHAse is administered to treat a patient havingor susceptible to cancer associated with a LuCa-1 defect, one desired therapeuticeffects include, but are not necessarily limited to, an inhibition of tumor cellgrowth and a decrease in the tumor cell’s threshold to apoptosis (i.e. , increase theW0 98/1665510152025CA 02264484 1999-03-03PCT/U S97/ 1808917cell’s sensitivity to triggers to programmed cell death). Therapeutic effects ofhpHAse may be associated with hpHAse’s hyaluronidase activity, chondroitanaseactivity, or both.By "LuCa-1 defect" or "hpHAse defect" is meant a genetic defect in a cellat chromosome position 3p.21.3 associated with a decreased level of hpHAseactivity relative to hpHAse activity levels in normal cells and/or a decreased levelof hpHAse activity in the serum or plasma of the affected patient (e.g., due todecreased expression of hpHAse or expression of a defective hpHAse). “LuCa-1defect” or “hpHAse defect’ are also meant to encompass cellular defects associatedwith a decreased level of hpHAse activity in the serum or plasma of the affectedpatient (e. g. , a defect in transport of hpHAse into the bloodstream). For example,plasma from patients have a LuCa—l defect-associated lung cancer exhibits about50% less hpHAse activity than plasma from normal patients (i.e. , patients who donot have a LuCa—l defect-associated cancer). Normal human plasma exhibits about15 rTRU/mg hpHAse activity as determined using the HAse assay of the invention.The hpHAse activity of plasma from LuCa—l defect-associated lung cancer patientsis about 7.5 rTRU/mg.By "having or susceptible to a condition associated with a LuCa—l defect"is meant to describe a patient having a heterozygous, homozygous, or epigeneticdefect at the LuCa—l locus and/or some other genetic defect outside the LuCa—llocus associated with decreased levels of hpHAse (e.g., serum hpHAse or urinehpHAse, preferably serum hpHAse) relative to hpHAse levels associated withnormal patients (i.e., patients having no LuCa—l defect that results in alteredhpHAse levels). Exemplary patients having or susceptible to a condition associatedwith a LuCa—l defect are patients bearing tumor or pre-tumor cells that do noexpress normal levels of hpHAse (e.g., a metatstatic carcinoma).The invention will now be described in further detail.W0 98/ 166551015202530CA 02264484 1999-03-03PCT/US97/ 1808918Hyaluronidase Activity AssayThe HAse assay of the invention involves labeling of HA by biotinylationof free carboxyl groups through a simple, one step reaction with 1—ethyl-dimethylaminopropyl carbodiamide (EDC, CICHZCHZCI, Sigma) and biotinhydrazide (Pierce). Briefly, a solution composed of dissolved HA and dissolvedbiotin hydrazide is combined with EDC. Preferably, biotin is present in excessrelative to EDC and HA. The molar amount of EDC present is varied accordingto the desired number of covalent bonds to be found between the carboxyl groupsof the disaccharide units of HA and the NH groups of the biotin moieties (i.e, thedesired number of biotin moieties relative to the number of HA disaccharide units).Preferably, the molar ratio of HA disaccharide units to EDC is 85:1, while themolar ratio of biotin hydrazide to EDC is 38: 1. An exemplary chemical structureof the resulting HA—bHA compound is shown in Fig. 1.After the HA-EDC-biotin hydrazide reaction is complete, uncoupled biotincan be removed through dialysis, preferably dialysis against distilled water. Thebiotinylated HA substrate (bHA) that results comprises an EDC—biotin moietycovalently bound to the HA molecule at a ratio of 1 EDC—biotin moiety for every200 disaccharide moieties, preferably every 150 disaccharide moieties, morepreferably every 85 to 100 disaccharide units in the HA molecule. The ratio ofEDC—biotin to HA molecule can range from as high as 1:1 (e.g. , one EDC—biotinmoiety bound to a disaccharide unit) to 5:1 (e.g., one EDC—biotin moiety boundto an HA molecule containing 100 disaccharide units). The bHA reagent can bestored, preferably at -20°C, until use.Assay devices for detection of HAse activity are prepared by coupling bHAto, for example, the surface of a microtiter well having a covalently attachedmoiety NH-CH3 (e.g., Covalink—NH microtiter plates, NUNC, Placerville, NJ).An exemplary chemical and physical configuration of surface for covalentattachment is shown in Fig. 2. Methods for preparation of such substrates and theuse of such substrates to covalently immobilize compounds are well known in theart (see, e.g., U.S. Patent No. 5,427,779, issued June 27, 1996, and PCTpublished application number WO 8905329, published June 15, 1989). CouplingW0 98/ 166551015202530CA 02264484 1999-03-03PCT/US97l1808919of bHA to the assay device is accomplished by, for example, incubation of bHAin the Covalink-NH well with EDC according to the manufacturer’s specifications.Unbound bHA is removed by washing the wells with buffer. bHA that remainsTheresulting assay device comprises bHA covalently bound to the surface to bein the well of the plate is covalently bound to the surface of the well.exposed to the test sample such that the bHA is bound to the surface by at least onecovalent bond, preferably at least one covalent bond per every 200 disaccharideunits in the bHA molecule, more preferably at least one covalent bond per 100disaccharide units, most preferably at least one covalent bond per 50 disaccharideunits in the bHA molecule. The plates are preferably used within about 1 week to10 days after preparation.The HAse activity assay is performed by placing a test sample in the wellof the assay device having covalently-bound bHA, and incubating the sample fora period sufficient to allow for hpHAse in the test sample to degrade bHA in thewell. The test sample can be any sample suspected of having HAse activity (e.g.,a biological sample (e.g., blood, serum, plasma, urine, or a sample derived froma recombinant source) or a sample from a step during biochemical purification).Preferably, a sample having a known amount of hpHAse activity is subjected to theassay as a control.The pH of the incubation buffer can be adjusted according to the desired pHoptima of the HAse activity in the sample (e. g. , neutral HAse activity (pH of about7.0 to 7.5) or acid active HAse activity (pH below about 4.5, preferably at aboutpH 3.0 to 3.7)) . Following incubation, the wells are washed to remove degradedbHA and remaining, undegraded bHA is detected, e.g. , by reaction the remainingbHA with avidin peroxidase and detection with a microplate reader. Covalentbinding of bHA to the assay device prevents the undegraded bHA from beingwashed off the plate, thus increasing the sensitivity and accuracy of the assay.Moreover, the assay requires only 60 minutes or less to complete and is 1,000times more sensitive than conventional colorimetric assays (Afify et al. supra) andabout 1,000 to 5,000 times more sensitive than SDS-based gel zymography. TheHAse assay of the invention can be used in a variety of applications where oneW0 98/16651015202530CA 02264484 1999-03-03PCT /U S97/ 1808920desires to determine (qualitatively or quantitatively) the presence of acid active orneutral HAse activity in a sample. In one embodiment, the HAse activity assay ofthe invention is useful in the identification of patients having a defect in HAseactivity (e.g., a LuCa—1/hpHAse defect associated with reduced hpHAse plasmaactivity). determine , and/or ._ Specific activity of hyaluronidase is expressed in turbidity reducing units(TRU). One TRU is defined as the amount of hyaluronidase activity required toreduce the turbidity of an acidified solution of hyaluronan and is equivalent to theU.S.P./National Formulary (NF XIII) units (NFU). The results using the assaysdescribed above are related to the TRU, the NFU, and U.S.P. units through astandard curve of a sample of hyaluronidase (e.g., WYDASE°,Wyeth-Ayerst)standardized through the U.S.P. For example, a standard curve can be generatedthrough co-incubation of serial dilutions of bovine testicular hyaluronidase(WYDASE°) and activity of unknown samples interpolated through a fourparameter curve fit to yield values in relative TRU (rTRU)/ml (Dorfman et al.,1948, J. Biol. Chem. 1_72:367).Biochemical hpHAse Purification MethodAcid active hyaluronidase activity can be significantly enriched and/orpurified using temperature-induced detergent phase extraction with a non-ionicdetergent (e.g., Triton X-114). In general, a sample comprising or suspected ofcomprising an acid active HAse (aaHAse) such as hpHAse is dissolved in asolution comprising a non-ionic detergent at low temperature (e.g., substantiallybelow room temperature, preferably less than about 15°C, more preferably about4°C).plasma without platelet degranulation (e.g., through addition of citrate), a lipidThe sample can be, for example, raw human plasma, outdated humanfraction of human plasma, human blood, human serum, human urine, orconditioned medium or lysates from cells expressing recombinant aaHAse (e.g.,mammalian, insect, bacterial, or yeast cells, preferably mammalian cells).Preferably, the sample is human plasma or human urine, which is a particularlyrich source of hpHAse. Purification from plasma is advantageous over purificationWO 98/1665510152025CA 02264484 1999-03-03PCT/US97ll808921from whole blood or serum since the plasma fraction contains less total proteinthan either serum or whole blood.After the sample is dissolved, the temperature of the solution is raised toat least room temperature or above (preferably above about 25°C, more preferablyabout 37°C), thereby resulting in formation of detergent-rich and detergent-poorphases. The aaHAse partitions into the detergent-rich phase. The detergent-richphase can be further enriched for aaHAse by removal of the detergent-rich phaseand repetition of temperature-induced detergent phase extraction. Repeating thistemperature-induced detergent phase extraction three times results in at least about10-fold, preferably at least about 20-fold, more preferably at least about 60-foldenrichment of aaHAse activity relative to aaHAse activity in the starting material.The aaHAse activity of the detergent-rich phase can be further enriched andpurified by, for example, cation exchange chromatography and/or hydroxylapatiteresin.Generation and Identification of Anti—Native aaHAse AntibodiesAlthough there are known procedures for producing antibodies from anygiven antigen, previous attempts to produce anti—native aaHAse antibodies (e. g.,Although antibodies that binddenatured anti-aaHAse have been generated, these antibodies, generated usinganti-native hpHAse antibodies) have failed.conventional methods and a conventional ELISA assay, did not bind native (i.e.,non-denatured) aaHAse (Harrison et al. 1988 J Reprod Fertil _8_2:777-85). Byfollowing the procedures described herein, antibodies that bind native aaHAseenzymes (e.g., native hpHAse) have been obtained. Likewise, the ordinarilyskilled artisan can follow the procedures outlined herein to generate other anti-native aaHAse antibodies, including other anti-native hpHAse antibodies. Ingeneral, the invention overcomes the problems associated with production ofaaHAse antibodies by providing a screening assay that detects antibodies that bindnative aaHAse.... ,,.,..m~-w4 m~.-. . , ...m..n...........,.....................................».............,:.,....~...,...w~ W0 98/166551015202530CA 02264484 1999-03-03PCTIU S97! 1808922Generation of anti—hpHAse antibodiesaaHAse can be used as an antigen in the immunization of a mammal (e.g. ,mouse, rat, rabbit, goat) and production of hybridoma cell lines according tomethods well known and routine in the art (see, for example, Harlow and Lane,1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY; Schrier et al., 1980, Hybridoma Technigues, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY). The aaHAse used inthe antigenic preparation can be purified from the source in which it naturallyoccurs (e.g., hpHAse purified from human plasma), recombinant aaHAse,biologically active (e.g., antigenic and/or enzymatically active) aaHAsepolypeptides, native aaHAse, and/or denatured aaHAse polypeptides. Where theaaHAse is a recombinant aaHAse, the recombinant aaHAse can comprise the aminoacid sequence of a naturally-occurring aaHAse, or can be modified relative tonative aaHAse (e.g., by amino acid substitution, deletion, or addition (e.g. , fusionprotein)). Preferably, the antigenic preparation is native aaHAse (e.g., aaHAsepurified from the source in which it naturally occurs or recombinant, full-lengthaaHAse). aaHAse can be any acid active hyaluronidase; preferably the aaHAse ishpHAse.Assay for anti—aaHAse antibodiesThe antibodies secreted by the hybridoma cell lines are screened in the anti-native aaHAse antibody assay of the invention. In general, the assay involves aninsoluble support (e.g., the surface of a well of a microtiter plate) to which isbound: 1) an anti—antibody; and 2) detectably labeled hyaluronic acid (HA). Theanti—antibody is capable of binding antibodies produced by the aaHAse-immunizedmammalian host regardless of antigen specificity. For example, where theimmunized host is a mouse, the anti—antibody is a goat anti-mouse antibody (i.e.,an antibody from a goat immunized with mouse antibodies). Preferably, the anti-antibody binds an Fc portion of antibodies produced by the aaHAse-immunizedmammalian host, and may specifically bind immunoglobulin classes or subclasses(e.g. , specifically bind IgG or an IgG subclass such as IgG, or IgG2). Preferably,the anti-antibody is covalently bound to the surface of the insoluble support. TheW0 98/11565510152025CA 02264484 1999-03-03PCT/US97l1808923detectably labeled HA is preferably biotinylated HA (bHA) in the above-describedHAse assay, and is preferably covalently bound to the surface of the insoluble plateas described above.A schematic of the anti-aaHAse assay of the invention is shown in Fig. 3.The anti-aaHAse antibody assay of the invention takes advantage of the inventors’observation that aaHAses do not bind their HA substrates under non—acidicconditions (i.e., under conditions in which the aaHAse is not enzymatically active).In general, the assay is performed by contacting the candidate antibody with asample comprising native aaHAse (e. g. , native hpHAse) to allow for formation ofnative aaHAse/antibody complexes. Preferably, this contacting step is performedat a non-acidic, preferably neutral, pH. The sample is then contacted under non-acidic (preferably neutral) conditions with the insoluble support having bound anti-antibody and detectably labeled HA to allow for formation of nativeaaHAse/antibody/anti-antibody complexes by binding of the anti-antibody to thecandidate antibody. Preferably, excess or unbound material is washed away witha non—acidic (preferably neutral) solution.The wash buffer is replaced with an acidic solution having a pH that allowsfor enzymatic activity of the aaHAse. Preferably the acidic solution has a pH thatapproximates the optimum pH for HAse activity of the aaHAse. For example,where the aaHAse is hpHAse, the acidic solution preferably has a pH of 3.7. Thesample is incubated with the insoluble support for a time sufficient for degradationof the detectably labeled HA by the immunoprecipitated aaHAse bound in theThesamples are then washed to remove degraded HA and undegraded HA is detectednative aaHAse/antibody/anti-antibody complex, preferably about 60 min.by virtue of its label. For example, where the detectable label is biotin,undegraded bHA is detected as described in the above-described HAse assay.Degradation of HA is correlated with the presence of aaHAse in the sample whichin turn is correlated with the presence of an anti-native aaHAse antibody. Thegeneral characteristics of antibodies of the invention are described below.WO 981166551015202530CA 02264484 1999-03-03PCT/US97Il808924Antibody/antigen binding forcesThe forces that hold an antigen and antibody together can be classified intofour general areas: (1) electrostatic; (2) hydrogen bonding; (3) hydrophobic; and(4) Van der Waals. Electrostatic forces are due to the attraction betweenoppositely charged ionic groups on two protein side-chains. The force of attraction(F) is inversely proportional to the square of the distance (d) between the charges.Hydrogen bonding forces are due to formation of reversible hydrogen bridgesbetween hydrophilic groups such as -OH, —NH2 and -COOH. These forces arelargely dependent upon close positioning of two molecules carrying these groups.Hydrophobic forces operate in the same way that oil droplets in water merge toform a single large drop. Accordingly, non—polar, hydrophobic groups such as theside—chains on valine, leucine and phenylalanine tend to associate in an aqueousenvironment. Lastly, Van der Waals are forces created between molecules byinteraction between the external electron clouds. Further information about thedifferent types of forces is known in the art (see, e. g. , Essential Immunology, I .M.Roitt, ed., 6th Ed. Blackwell Scientific Publications, 1988.Useful antibodies of the present invention exhibit all of these forces. Byobtaining an accumulation of these forces in greater amounts, it is possible toobtain an antibody that has a high degree of affinity or binding strength to nativeaaHAse, and in particular an antibody that has a high degree of binding strengthto aaHAse in the material in which it naturally occurs (e.g., human plasma).Measuring antibody/antigen binding strengthThe binding affinity between an antibody and an antigen ia an accumulative -measurement of all of the forces described above. Standard procedures forcarrying out such measurements are known in the art and can be directly appliedto measure the affinity of anti-native aaHAse antibodies of the invention.One standard method for measuring antibody/antigen binding affinity usesa dialysis sac, composed of a material permeable to the antigen but impermeableto the antibody. Antigens that bind completely or partially to antibodies are placedwithin the dialysis sac in a solvent (e.g., water). The sac is then placed within alarger container which does not contain antibodies or antigen but contains only theW0 98/1615551O15202530CA 02264484 1999-03-03PCT/US97/1808925solvent. Since only the antigen can diffuse through the dialysis membrane theconcentration of the antigen within the dialysis sac and the concentration of theantigen within the outer larger container will attempt to reach an equilibrium. Theamount of antigen that remains bound to antibody in the dialysis sac and theamount that disassociated from the antibody are calculated by determining theantigen concentrations within the dialysis sac and within the solvent outside thedialysis sac. By constantly renewing the solvent (e.g., the water) within thesurrounding container so as to remove any diffused antigen, it is possible to totallydisassociate the antibody from antigen within the dialysis sac. If the surroundingsolvent is not renewed, the system will reach an equilibrium, and the equilibriumconstant (K) of the reaction, i.e., the association and disassociation between theantibody and antigen, can be calculated. The equilibrium constant (K) is calculatedas an amount equal to the concentration of antibody bound to antigen within thedialysis sac divided by the concentration of free antibody combining sites times theconcentration of free antigen. The equilibrium constant or "K" value is generallymeasured in terms of liters per mole. The K value is a measure of the differencein free energy (AG) between the antigen and antibody in the free state as comparedwith the complexed form of the antigen and antibody. Anti-native aaHAseantibodies having an affinity or K value of 107 1/mole to 10° l/mole or more arepreferred.Antibody avidityAs indicated above the term "affinity" describes the binding of an antibodyto a single antigen determinate. The term "avidity" is used to express theinteraction of an antibody with a multivalent antigen. The factors that contributeto avidity are complex and include both the heterogeneity of the antibodies in agiven serum that are directed against each determinate on the antigen and theheterogeneity of the determinants themselves. The multivalence of most antigensleads to an interesting "bonus" effect in which the binding of two antigenmolecules by an antibody is always greater, usually many fold greater, than thearithmetic sum of the individual antibody links. Thus, it can be understood thatthe measured avidity between an antiserum and a multivalent antigen will beW0 98/166551015202530CA 02264484 1999-03-03PCT/US97/1808926somewhat greater than the affinity between an antibody and a single antigendeterminate.Uses of Anti—Native aaHAse Antibodies Anti—native aaHAse antibodies are useful in various immunotechniques,including immunopurification and immunodetection techniques. Anti-nativeaaHAse antibodies useful in such immunotechniques may be either polyclonal ormonoclonal antibodies, preferably monoclonal antibodies.Preferably, anti-native aaHAse antibodies useful in immunotechniquesexhibit an equilibrium or affinity constant (Kd) of at least 107 l/mole to 109 l/moleor greater. The binding affinity of 107 l/mole or more may be due to (1) a singlemonoclonal antibody (i.e., large numbers of one kind of antibody) (2) a pluralityof different monoclonal antibodies (e.g., large numbers of each of five differentmonoclonal antibodies) or (3) large numbers of polyclonal antibodies. It is alsopossible to use combinations or (1)—(3).Preferred antibodies bind 50% or more of native aaHAse in a sample.However, this may be accomplished by using several different antibodies as per(l)—(3) above.effective than a single antibody in binding a larger percentage of antigen in aAn increased number of different antibodies is generally moresample. Thus, a synergistic effect can be obtained by combining combinations oftwo or more antibodies which bind native aaHAse, i.e. , by combining two or moreantibodies that have a binding affinity K, for native aaHAse of 107 l/mole or more.Immunopurification using anti-native aaHAse antibodiesAnti-native aaHAse antibodies can be used in, for example,immunopurification of aaHAse from its naturally occurring source (e.g., hpHAsefrom human blood, plasma, serum, or urine) or from a source of recombinantaaHAse production (e.g., from supematants or cell lysates of transformed cellsexpressing hpHAse). Immunopurification techniques useful with the anti-nativeinclude,aaHAse antibodies of the inventionbut are not limited to,imrnunoprecipitation, immunoaffinity isolation on beads, irnmunoaffinity columnWO 981166551015202530CA 02264484 1999-03-03PCT/U S97] 1808927chromatography, and other methods well known in the art. Anti-native aaHAseantibodies useful in immunopurification techniques The immunopurificationmethods using the antibodies of the invention can use a single anti—native aaHAseantibody (e. g, a monoclonal or polyclonal antibody, preferably a monoclonalantibody) or multiple anti-native aaHAse antibodies.In addition, the anti-native aaHAse antibodies of the invention can be usedto prepare a device for immunopurification of native aaHAse, preferably nativehpHAse. In general, such devices are prepared by covalently binding an anti-native aaHAse antibody to an insoluble support (e.g., bead, affinity columncomponent (e.g., resin), or other insoluble support used in immunoaffinitypurification). Alternatively, the antibody may be bound to a metal particle whichallows separation of anti-native aaHAse-aaHAse complexes from a solution by useof a magnetized column. The anti-aaHAse antibody may be a monoclonal orpolyclonal antibody, preferably a monoclonal antibody. The antibodies bound tothe insoluble support (or otherwise employed in purification) may also comprisea mixture of anti-native aaHAse antibodies to provide a device that can bind to atleast 50% of the native aaHAse in the sample. Such immunopurification devicescan be used to isolate aaHAse from a source in which it naturally occurs (e.g.,hpHAse from raw serum, raw plasma, or urine) or from a source ofrecombinantly-produced aaHAse.Qualitative and guantitative immunodetection using anti-native aaHAseantibodiesAnti-native aaHAse antibodies can be used in‘ immunodetection assay todetect and, where desirable, quantitate aaHAse in a sample. Immunodetectionassays using anti-native aaHAse antibodies can be designed in a variety of ways.For example, anti-native aaHAse antibodies can be used to produce an assay devicecomprising anti-native aaHAse antibodies bound to a soluble support (e.g., animmunoassay column, beads, or wells of a microtiter plate). Methods for covalentor non—covalent attachment of an antibody to a soluble support are well known inthe art. A sample suspected of containing an aaHAse is then contacted with theassay device to allow formation of anti-native aaHAse antibody-aaHAse complexes.WO 98/166551015202530CA 02264484 1999-03-03PCT/US97/ 1808928The anti-native aaHAse-aaHAse complexes can then be detected by virtue of anaaHAse activity associated with the complex as described in the anti-native aaHAseassay described above, or by contacting the complex with a second detectably-labeled anti-native aaHAse antibody.By "detectably labeled antibody", "detectably labeled anti-aaHAse" or"detectably labeled anti-aaHAse fragment" is meant an antibody (or antibodyfragment that retains antigen binding specificity), having an attached detectablelabel. The detectable label is normally attached by chemical conjugation; wherethe label is a polypeptide, the label can be attached by genetic engineeringtechniques. Detectable labels may be selected from a variety of such labels knownin the art, but normally are radioisotopes, fluorophores, paramagnetic labels,enzymes (e.g. , horseradish peroxidase), or other moieties or compounds that eitheremit a detectable signal (e. g., radioactivity, fluorescence, color) or emit adetectable signal after exposure of the label to its substrate. Various detectablelabel/substrate pairs (e.g., horseradish peroxidase/diaminobenzidine,avidin/streptavidin, luciferase/luciferin)), methods for labelling antibodies, andmethods for using labeled antibodies are well known in the art (see, for example,Harlow and Lane, eds. (Antibodies: A Laboratory Manual (1988) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY)).Alternatively, detectably labeled anti-native aaHAse antibodies can bedirectly used to detect and/or quantify aaHAse in a sample. For example,detectably labeled anti-native aaHAse antibodies can be contacted with a tissuesample suspected of having a LuCa—1/hpHAse defect (e.g. , a tissue sample derived‘from breast, ovaries, or lung) for a time sufficient to allow for formation ofcomplexes between the anti-native hpHAse antibody and hpHAse in the tissuesample (e.g., hpHAse in the plasma membrane of cells of the tissue sample).Binding of the anti-native hpHAse antibody can then be detected and/or quantifiedby virtue of a detectable label bound to the anti-native hpHAse antibody.Alternatively, binding of the anti-native hpHAse antibody can be detected using anantibody that binds the anti-native hpHAse antibody. Binding of anti-nativehpHAse antibody to a tissue sample can then be compared to anti-native hpHAseW0 98ll66551015202530CA 02264484 1999-03-03PCT/U S97/ 1808929antibody binding to a control sample (e.g., a normal sample having no LuCa-1/hpHAse defect and/or a sample containing tissue associated with aLuCa—1/hpH'Ase defect) and the antibody binding correlated with the presence orabsence of a LuCa—1/hpHAse defect in the patient.The aaHAse immunodetection assays can be used in a variety of differentways with a variety of samples, as will be apparent to one of ordinary skill in theart upon reading the disclosure provided herein. For example, the aaHAseimmunodetection assays can be used to detect hpHAse in serum or plasma samplesof patient receiving hpHAse therapy and/or to correlate hpHAse in the serum orplasma of a patient with tumor progression in the patient, responsiveness totherapy, and/or uptake of hpHAse by the patient’s body.Where hpHAse is detected in serum or urine, the levels of hpHAse can becorrelated with susceptibility to and/or the presence of a LuCa-1 defect associateddisease state and/or the severity of such disease. For example, hpHAse levels inpatients having lung cancer associated with a heterozygous defect in LuCa-1 areapproximately 50% of normal hpHAse levels, while patients having a homozygousdefect in LuCa—l exhibit hpHAse levels that are very low or undetectable. Thus,hpHAse levels, as detected in an aaHAse assay of the invention or detected usingan anti-native hpHAse antibody of the invention, can not only be correlated withtumor progression, but can also be directly correlated with a genetic defect inLuCa-1, thus allowing identification of patients susceptible to conditions associatedwith LuCa-1 defects, e.g., cancer. Thus, the present invention allows one todirectly correlate hpHAse levels with the number of functional alleles in a patientand to detect a genetic defect by simply ascertaining the level of hpHAse in blood,plasma, serum or urine.Methods of Making hpHAseIn addition to the purification procedure outlined above, hpHAsehyaluronidase polypeptides can be made by standard synthetic techniques, or byusing recombinant DNA technology and expressed in bacterial, yeast, ormammalian cells using standard techniques. As used herein, the term "hpHAse"W0 98/166551015202530CA 02264484 1999-03-03PCT/US97/1808930includes natural, recombinant, and modified forms of the protein unless the contextin which the term is used clearly indicates otherwise.Chemical SynthesishpHAse polypeptides can be synthesized based on the amino acid sequencesdescribed herein and variations thereof by standard solid-phase methods using thetert—butyloxy-carbonyl and benzyl protection strategy described in Clark-Lewiset al., P.N.A.S., USA, $13574-3577 (1993) and Clark-Lewis et al., Biochemistry,§(_):3l28-3135 (1991). After deprotection with hydrogen fluoride, the proteins arefolded by air oxidation and purified by reverse-phase HPLC. Purity is determinedby reverse-phase HPLC and isoelectric focusing. Amino acid incorporation ismonitored during synthesis, and the final composition is determined by amino acidanalysis. The correct covalent structure of the protein can be confirmed using ion-spray mass spectrometry (SCIEX APIII).Recombinant DNA Techniques for Synthesis of hpHAse PolypeptidesAs discussed in the examples below, LuCa—l (SEQ ID NO:3)and hpHAse(SEQ ID N021) are identical. The only variation between the amino acid sequenceof hpHAse (SEQ ID NO:l) and the amino acid sequence of LuCa—l (SEQ IDNO:3) is a substitution of Val for Leu in the N-terminus at the 27th amino acidresidue (where the Met at the N—terrninus of the signal sequence is counted as thefirst amino acid residue); however, the two proteins are otherwise identical inThenucleotide sequence encoding hpHAse is identical to the sequence encoding LuCa—l(SEQ ID NO:4), except that the cytosine of the third residue in the codoncorresponding to the 27th amino acid residue (Leu in LuCa—l; Val in HpHAse) isamino acid sequence and immunological and biochemical characteristics.substituted with guanine. Thus, the nucleotide sequence encoding LuCa—l (SEQID NO:4) is the nucleotide sequence encoding hpHAse. The LuCa-l/hpHAse genehas been isolated and sequenced (Bader et al. GenBank accession no. UO3056, NIDG532973, submitted Nov. 1, 1993; see also GenBank Accession No. U96078).The amino acid and nucleotide sequences of LuCa—l as described by Bader et al.are provided below.WO 98116655101520253035404550CA 02264484 1999-03-03PCTIUS97/1808931MAGH LLPICALFLTLLDMAQGFRGPLVPNRPFTTVWNANTQWCLERHGVDVDVSVFDVVANPGQTFRGPDMTIFYSSQLGTYPYYTPTGEPVFGGLPQNASLIAHLARTFQDILAAIPAPDFSGLAVIDWEAWRPRWAFNWDTKDIYRQRSRALVQAQHPDWPAPQVEAVAQDQFQGAARAWMAGTLQLGGALRPRGLWGFYGFPDCYNYDFLSPNYTGQCPSGIRAQNDQLGWLWGQSRA LYPSIYM PAVLEGTGKSQMYVQHRVAEAFRVAVAAGDPNLPVLPYVQIFYDTTNHFLPLD ELEH SLGESAAQGAAGVVLWVSWENTRTKESCQAIKEYMDTTLGPFILNVTSGALLCSQALCSGI-IGRCVRRTSHPKALLLLNPASFSIQLTPGGGPLSLRGALSLEDQAQMAVEFKCRCYPGWQAPWCERKSMW (SEQ ID N023)1 ttcctccagg agtctctggt gcagctgggg tggaatctgg ccaggccctg cttaggcccc61 catcctgggg tcaggaaatt tggaggataa ggcccttcag ccccaaggtc agcagggacg121 agcgggcaga ctggcgggtg tacaggaggg ctgggttgac ctgtccttgg tcactgaggc181 cattggatct tcctccagtg gctgccagga tttctggtgg aagagacagg aaggcctccc241 ccccttggtc gggtcagcct gggggctgag ggcctggctg tcagccactc ttcccagaac301 atatgtcatg gcctcagtgg ctcatgggga agcaggggtg ggcgagctta ggctagagca361 agtcctgtgg gagatggcag aggcctggtc tgagaggcaa ctcggatgtg ccctccagtg421 gccatgctcc cctccatgcg tctcccctgc cctcctggag ccctgcaggt caatgtttaa481 cagaaaccag agcagcggtg gattaatgcg caagggctca gccccccagc cctgagcagt541 gggggaatcg gagactttgc aacctgttct cagctctgcc tcccctgggc aggttgtcct601 cgaccagtcc cgtgccatgg caggccacct gcttcccatc tgcgccctct tcctgacctt661 actcgatatg gcccaaggct ttaggggccc cttggtaccc aaccggccct tcaccaccgl721 ctggaatgca aacacccagt ggtgcctgga gaggcacggt gtggacgtgg atgtcagtgt781 cttcgatgtg gtagccaacc cagggcagac cttccgcggc cctgacatga caattttcta841 tagctcccag ctgggcacct acccctacta cacgcccact ggggagcctg tgtttggtgg901 tctgccccag aatgccagcc tgattgccca cctggcccgc acattccagg acatcctggc961 tgccatacct gctcctgact tctcagggct ggcagtcatc gactgggagg catggcgccc1021 acgctgggcc ttcaactggg acaccaagga catttaccgg cagcgctcac gggcactggt1081 acaggcacag caccctgatt ggccagctcc tcaggtggag gcagtagccc aggaccagtt1141 ccagggagct gcacgggcct ggatggcagg caccctccag ctgggggggg cactgcgtcc1201 tcgcggcctc tggggcttct atggcttccc tgactgctac aactatgact ttctaagccc1261 caactacacc ggccagtgcc catcaggcat ccgtgcccaa aatgaccagc tagggtggct1321 gtggggccag agccgtgccc tctatcccag catctacalg cccgcagtgc tggagggcac1381 agggaagtca cagatgtatg tgcaacaccg Igtggccgag gcattccgtg tggctgtggc1441 tgctggtgac cccaatctgc cggtgctgcc ctatgtccag atcttctatg acacgacaaa1501 ccactttctg cccctggatg agctggagca cagcctgggg gagagtgcgg cccagggggc1561 agctggagtg gtgctctggg tgagctggga aaatacaaga accaaggaat catglcaggc1621 catcaaggag tatatggaca ctacactggg gcccttcatc ctgaacgtga ccagtggggc1681 ccttctctgc agtcaagccc tgtgctccgg ccatggccgc tgtgtccgcc gcaccagcca1741 ccccaaagcc ctcctcctcc ttaaccctgc cagtttctcc atccagctca cgcctggtgg1801 tggccccctg agcctgcggg gtgccctctc acttgaagat caggcacaga tggctgtgga1861 gttcaaatgt cgatgctacc ctggctggca ggcaccgtgg tgtgagcgga agagcatgtg1921 gtgattggcc acacactgag ttgcacatat tgagaaccta atgcactctg ggtctggcca1981 gggcttcctc aaatacatgc acagtcatac aagtcatggt cacagtaaag agtacactca2041 gccactgtca caggcatatt ccctgcacac acatgcatac ttacagactg gaatagtggc2101 ataaggagtt agaaccacag cagacaccat tcattcctgc tccatatgca tctacttggc2161 aaggtcatag acaattcctc cagagacact gagccagtct ttgaactgca gcaatcacaa2221 aggctgacat tcactgagtg cctactcttt gccaatcccc gtgctaagcg ttttatgtgg2281 acttattcat tcctcacaat gaggctatga ggaaactgag tcactcacat tgagagtaag2341 cacgttgccc aaggttgcac agcaagaaaa gggagaagtt gagattcaaa cccaggctgt2401 ctagctccgg gggtacagcc cttgcactcc tactgagttt gtggtaacca gccctgcacg2461 acccctgaat ctgctgagag gcaccagtcc agcaaataaa gcagtcatga tttactt (SEQ ID N024)WO 981166551015202530CA 02264484 1999-03-03PCT/US97/1808932The nucleotide sequence encoding hpHAse can be isolated according to anyone of a variety of methods well known to those of ordinary skill in the art. Forexample, DNA encoding hpHAse can be isolated from either a cDNA library orfrom a genomic DNA library by hybridization methods. Alternatively, the DNAcan be isolated using standard polymerase chain reaction (PCR) amplification ofsynthetic oligonucleotide primers, e.g., as described in Mullis et al., U.S. PatentNo. 4,800,159, or expression cloning methods well known in the art (see, e.g.,Sambrook et al.Sambrook et al. 1989 Molecular Cloning: A Laboratory Manual,2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Wherehybridization or PCR is used to identify DNA encoding hpHAse, the sequence ofthe oligonucleotide probes or primers can be based upon the amino acid orThe sequence of isolated hpHAsepolypeptide—encoding DNA can be determined using methods well known in the artnucleotide sequence of LuCa—1 provided above.(see, for example, Sambrook et al., gipg). Following sequence confirmation, theresulting clones can be used to for example, identify homologs of a hpHAse (e.g.,other human alleles encoding hpHAse or an acid active serum hyaluronidase ofanother mammalian species (e.g., dog, rat, mouse, primate, cow), and/or totransform a target cell in a host for expression of the hpHAse—encoding DNA (e.g.,as in anti—cancer therapy through hpHAse polypeptide expression in host cells,preferably cancerous cells of a patient). .Production of hpHAse-Encoding Constructs and Expression of hpHAsePolypeptidesNumerous vectors are available for production of hpHAse-encodingconstructs and hpHAse expression (see, e.g., the American Type CultureCollection, Rockville, MD). Preferably the vector is capable of replication in botheukaryotic and prokaryotic hosts, and are generally composed of a bacterial originof replication and a eukaryotic promoter operably linked to a DNA of interest,thereby allowing production of hpHAse-encoding constructs. Suitable host cells,as well as methods for constructing stably-transforrned host cell lines, are alsopublicly available, e.g., Pouwels et al., 1985, Cloning Vectors: A LaboratoryW0 98/1615551015202530CA 02264484 1999-03-03PCT/U S97/ 1808933Manual, Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley& Sons, New York; Sambrook et al., supra; Kormal et al., 1987, Proc. Natl.Acad. Sci. USA, 84:2150—2154; each of which are hereby incorporated by referencewith respect to methods and compositions for manipulation of a DNA of interest).Expression of an hpHAse polypeptide is accomplished by inserting anucleotide sequence encoding an hpHAse polypeptide into a nucleic acid vectorsuch that a promoter in the construct is operably linked to the hpHAse—encodinghpHAseexpression can be accomplished in a mammalian cell line by either transient,sequence, which construct is then used to transform a host cell.constitutive, or inducible expression. In one preferred embodiment, the host cellis a mammalian cell, preferably a cos-7 cell line, or a cos-7 cell-derived cell line.More preferably, the host cell for hpHAse production is a mammalian cell that isprotected against hpHAse expression (i.e., the cell can produce high levels ofhpHAse without adverse affects upon the host cell (e.g., slow cell growth, celldeath)). In one embodiment, the hpHAse—resistant cell is an SV-transformed cellline or an adenovirus-transformed cell line (i.e., a cell line transformed withsheared adenovirus), preferably the HEK cell line (HEK 293) For example,transformation of HEK cells with hpHAse-encoding DNA driven by a strongpromoter (e. g., CMV) provides surprisingly high hpHAse expression and secretioninto the culture medium (approximately 2.9 x 10'” mg/cell/24 hrs.) (> 100 rTRU).Use of such cell lines allows for production of sufficient quantities of hpHAse foruse in, e.g, protein therapy.Where expression is desired in a tumor cell line that is not resistant tohpHAse, the hpHAse-encoding DNA is preferably under control of an induciblepromoter, preferably an inducible promoter responsive to an inducing agent thatdoes not significantly affect other mammalian cells in the vicinity of thetransformed, hpHAse construct-containing cells. For example, the hpHAse-encoding DNA can be under the control of a steroid inducible promoter (e.g., inthe ecdysone expression system, which is inducible by muristerone). The targetcells are transformed and, upon exposure to the inducing agent, express hpHAse.WO 98/166551015202530CA 02264484 1999-03-03PCT/US97/1808934Where the hpHAse-encoding DNA is used to transform tumor cells fortreatment of a tumor—bearing patient, the hpHAse—encoding DNA may betransiently, constitutively, or inducibly expressed, preferably transiently orconstitutively expressed, more preferably constitutively expressed. In addition oralternatively, peritumor cells (e.g., cells adjacent the tumor cells) or other cellscapable of expressing and secreting hpHAse (e.g., liver cell or monocyte) can betransformed.Recombinant hpHAse polypeptide expression (e.g. , produced by any of theexpression systems described herein) can be assayed by immunological procedures,such as Western blot or immunoprecipitation analysis of recombinant cell extracts,or by the HAse activity assay of the invention as described herein. For example,hpHAse polypeptides according to the invention can be produced by transformationof a suitable host cell with an hpHAse polypeptide—encoding nucleotide sequencein a suitable expression vehicle, and culturing the transformed cells underconditions that promote expression of the encoded polypeptide, and preferablysecretion of hpHAse into the culture medium. The method of transformation andthe choice of expression vehicle will depend on the host system selected. Those— skilled in the field of molecular biology will understand that any of a wide varietyof prokaryotic and eukaryotic expression systems may be used to produce hpHAsepolypeptides of the invention.Identification of Biologically Active hpHAse PolypgpfideshpHAse polypeptide—encoding DNAs can encode all or a portion of anhpHAse. Preferably, the hpHAse polypeptide is biologically active, e.g., exhibitsacid active hyaluronidase activity in the cleavage of hyaluronan and/or can bebound by an anti—native hpHAse antibody. In general, once information regardingthe ability of a protein to elicit antibodies and/or information regarding anenzymatic or other biological activity of a protein of interest is known, methodsfor identification of biologically active polypeptides of the ful1—length protein areroutine to the ordinarily skilled artisan, particularly where the nucleotide sequenceW0 98/166551015202530CA 02264484 1999-03-03PCT/US97l 1808935and/or amino acid sequence encoding the protein of interest (here hpHAse) isprovided as in the present case.Biologically active hpHAse polypeptides can be identified by using theHAse activity assay of the invention, or by using conventional HAse activity assays(e.g., the ELISA-like hyaluronan assay (Stern et al., 1992, Matrix _l_Z:391-403) orsubstrate gel zymography (Guentenhoener et al., 1992, Matrix l;:388—396)).Alternatively, biologically active hpHAse polypeptides can be detected by bindingof an anti-native hpHAse antibody to a component of the transformed host cellsupernatant and/or lysate. hpHAse polypeptides preferably exhibit at least 25%,more preferably 50%, still more preferably 75 % , even more preferably 95 % of theactivity of native hpHAse.Identification of Hyaluronidases Homologous to hpHAseDNA encoding hyaluronidases homologous to hpHAse (e.g., containconservative amino acid substitutions relative to a native hpHAse) can beaccomplished by screening various cDNA or genomic DNA libraries byhybridization or PCR using oligonucleotides based upon the DNA sequence and/oramino acid sequence of an hpHAse (e.g., a LuCa-1/hpHAse sequence).Alternatively the oligonucleotides used may be degenerate, e. g., based upon aselected amino acid sequence of hpHAse or designed so as to allow detection oramplification of DNA encoding an hpHAse-like amino acid sequence havingconservative amino acid substitutions and/or to take into account the frequency ofcodon usage in the mammalian species DNA to be screened. Such "degenerateoligonucleotide probes" can be used in combination in order to increase thesensitivity of the hybridization screen, and to identify and isolate hpHAse analogsMethods fordesigning and using degenerate oligonucleotide probes to identify a protein forin other species or variant alleles encoding hpHAse in humans.which an amino acid and/or nucleotide sequence, as well as methods forhybridization and PCR techniques for screening and isolation of homologousDNAs, are routine and well known in the art (see, for example, Sambrook et al.supra) .W0 98/ 166551015202530CA 02264484 1999-03-03PCT/US97/ 1808936Alternatively, the DNA encoding hpHAse may be isolated by expressioncloning methods well known in the art (see, for example, Sambrook et al., §1_1p_ra).For example, mammalian cells can be transformed with a cDNA expressionlibrary, i.e., a collection of clones containing various cDNA fragments operablylinked to a eukaryotic promoter. Expression of an hpHAse homology or abiologically active fragment thereof can be detected by assaying the culturesupernatant and/or cell lysates using the HAse activity assay of the invention.Therapies Using hpHAse PolypeptidesThe substantially pure native hpHAse polypeptides (e.g., hpHAsepolypeptides that are not associated with the components of plasma from whichhpHAse is purified) of the invention can be used in a variety of applicationsincluding human and veterinary therapies, either alone or in combination with othertherapeutic agents. Purified hpHAse of the invention can generally be used inplace of neutral HAse formulations or WYDASETM, where the condition to betreated is associated with excess hyaluronic acid and/or therapy is designed toincrease HAse activity generally (i.e., the conventional neutral hyaluronidase-containing formulation is not used to treat a specific defect in neutral HAseactivity, but rather provides a HAse (neutral or acid active) activity). Use of acidactive hpHAse is preferred to use of neutral HAses since acid active hpHAse canprovide controlled degradation of HA substrate and does not degrade allcomponents of the extracellular matrix in the patient.hpHAse can be used in the treatment of diseases associated with excesshyaluron, to enhance circulation of physiological fluids at the site of administration(e.g., as a spreading agent, e.g., by subcutaneous or topical application (e.g., incosmetic formulations such as cosmetic creams), and/or as an anti—cancer agenteither alone or in combination with chemotherapeutic agents. For example,hpHAse can be administered to a patient to facilitate clysis, particularlyhypoderrnoclysis. hpHAse can also be administered to patients suffering fromPreferably, hpHAse isadministered in the absence, or at very low levels, of heparin, a powerful inhibitorstroke or a myocardial infarction (e.g., by infusion).WO 981166551015202530CA 02264484 1999-03-03PCT/US97/1808937of hyaluronidase. Methods for administration, and amounts of hpHAseadministered, for treatment of myocardial infarction can be based upon methodsof administration of bovine testicular hyaluronidase and amounts administered (see,e.g., Wolf et al. 1982 J. Pharmacol. Exper. T herap. 2_2_Z:331-7; Braunwald et al.1976 Am. J. Cardiol. 3_7:550-6; DeGiovanni et al. 1961 Br. Heart J. _4_5_:350;DeO1iveira et al. 1959 Am. Heart J. 12 :712-22; Kloner et al. 1978 Circulation_5_8:220-6; Kloner et al. 1977 Am. J. Cardiol. 5l_(_)_:43-9; Koven et al. 1975 J.Trauma 1§:992—8; Maclean et al. 1978 J. Clin. Invest.§1:541-51; Maclean et al.1976 Science 1_94:199-200; Maroko et a1. 1975 Ann. Intern. Med. §;:516-20;Maroko et al. 1977 N. Engl. J. Med. 2fi:896-903; Maroko et al. 1972 Circulation4§:430-7; Salete 1980 Clin. Biochem. Q92-94; Snell et al. 1971 J. Clin.Invest. $9614-25; Wolf et al. 1981 Circ. Res. fl:88-95).Furthermore, hpHAse can also have therapeutic effects when administeredto patients having certain lysosomal storage diseases associated with a defect inhyaluronidase (see, e.g., Natowicz et al. 1996 N. Engl. J. Med. 3i:1029—33).hpHAse can used therapeutically by direct administration of hyaluronidase (e.g.,intracellularly or intravenously) as a form of shunt pathway and/or by gene therapy(e.g., to replace defective copy(ies) of the LuCa—1 gene). Lysosomal storagedisease amenable to hpHAse therapy are those diseases that result in accumulationof [GlcNAcB1-4GlcUAB1—3],, (GAGS) due to a defective mannose—6—phosphatepathway. hpHAse can degrade these accumulated GAGs under a nonfunctionalcellular system since HAse activity does not depend upon the mannose—6—phosphatepathway (Herd et al. 1976 Proc. Soc. Experim. Biol. Med. _1_§_1_:642-9).hpHAse can also be used in the treatment of edema associated with braintumors, particularly that associated with glioblastoma multiforrn. The edemaassociated with brain tumors results from the accumulation of hyaluronan in thenon-cancerous portions of the brain adjacent the tumor. Administration ofhyaluronidase to the sites of hyaluronan accumulation (e.g., by intravenousinjection or via a shunt) can relieve the edema associated with such malignanciesby degrading the excess hyaluronan at these sites. Thus, hyaluronidase issuccessful in the treatment of brain tumors not only in the reduction of the tumorW0 98/166551015202530CA 02264484 1999-03-03PCT/US97/1808938mass and inhibition of tumor growth and/or metastasis, but it also is useful inrelieving edema associated with the malignancy. hpHAse can be administered fortreatment of edema in a manner similar to that for administration of bovinetesticular hyaluronidase to treat edema (see, e.g. , SaEarp Arq. Braz. Med. 51 :217-20).Of particular interest is the use of hpHAse polypeptides’ in the treatment ofmetastatic and non-metastatic cancers, particularly metastatic cancers, havingdecreased to undetectable hpHAse activity relative to non-cancerous (normal) cells.hpHAse can be used as a chemotherapeutic agent (alone or in combination withother chemotherapeutics) in the treatment of any of a variety of cancers,particularly invasive tumors. For example, hpHAse polypeptides can be used inthe treatment of small lung cell carcinoma, squamous lung cell carcinoma, as wellas cancers of the breast, ovaries, head and neck, or any other cancer associatedwith depressed levels of hpHAse or with a defective LuCa-l (hpHAse) gene (e.g.,a LuCa-l gene that does not provide for expression of adequate hpHAse levels orencodes a defective hpHAse that does not provide for an adequate level ofhyaluronidase activity) or other defect associated with decreased hpHAse activity.hpHAse can also be used to increase the sensitivity of tumors that areresistant to conventional chemotherapy. In one embodiment, hpHAse isadministered to a patient having a tumor associated with a LuCa-l defect in anamount effective to increase diffusion around the tumor site (e.g., to increasecirculation of chemotherapeutic factors (e.g., to facilitate circulation and/orconcentrations of chemotherapeutic agents in and around the tumor site), inhibittumor cell motility (e.g., by HA degradation) and/or to lower the tumor cell(s)threshold of apoptosis (i.e., bring the tumor cell(s) to a state of anoikis), a statethat renders the tumor cell(s) more susceptible to the action of chemotherapeuticagents or other agents that may facilitate cell death, preferably preferentiallyfacilitate programmed cell death of cells in anoikis. Chemotherapeutic agents asused herein is meant to encompass all molecules, synthetic (e.g, cisplatin) as wellas naturally-occurring (e.g. , tumor necrosis factor (TNF)), that facilitate inhibitionW0 98/ 166551015202530CA 02264484 1999-03-03PCT/US97/1808939of tumor cell growth, and preferably facilitate, more preferably preferentiallyfacilitate tumor cell death.Patients having or susceptible to a disease or condition that is amenable totreatment with hpHAse can be identified using a variety of conventional methods,or by using an assay device of the invention having bound anti—native hpHAseantibodies as described above to determine blood, plasma, serum, or urine hpHAselevels, preferably blood, plasma, or serum, and correlate such levels with a LuCa-1 defect. For example, where the patient is suspected of having or of beingsusceptible to a condition associated with decreased hpHAse activity, a biologicalsample (e.g., blood, serum, or plasma) can be obtained from the patient andassayed using the HAse assay and/or immunoassays using anti—native hpHAseantibodies as described above.Alternatively, particularly where the patient has or is suspected of havinga cancer associated with a LuCa-1/hpHAse defect, anti—native hpHAse antibodiescan be used to determine the levels of hpHAse in a tissue sample (e.g., hpHAsepresent in the plasma membrane of, inside of, or surrounding cells associated withthe tumor tissue). Decreased levels of hpHAse in the tissue sample relative tolevels of hpHAse associated with a normal (i.e., non—cancerous) tissue sample isindicative of a LuCa-1/hpHAse defect-associated cancer.The route of administration and amount of hpHAse administered will varywidely according to the disease to be treated, and various patient variablesincluding size, weight, age, disease severity, and responsiveness to therapy.Methods for determining the appropriate route of administration and dosage aregenerally determined on a case-by-case basis by the attending physician. Suchdeterminations are routine to one of ordinary skill in the art (see, for example,Harrison's Principles of Internal Medicine, 11th Ed., 1987). For example, wherehpHAse is used to facilitate hypoderrnoclysis, a solution containing hpHAse isadministered by subcutaneous injection to facilitate absorption of the solution (e.g. ,nutrient, body fluid replacement, or blood pressure increasing solution).Preferably, hpHAse is administered by injection, e. g., parenteral injectionincluding subcutaneous, intramuscular, intraorbital, intracapsular, peritumoral, andWO 98/166551015202530CA 02264484 1999-03-03PCT/US97/1808940intravenous injection.In a preferred embodiment, hpHAse polypeptide is delivered to the patienthaving an hpHAse-treatable tumor by delivery of hpHAse polypeptide directly tothe tumor site, e.g., by peritumoral injection of hpHAse, injection or hpHAsedirectly into the tumor mass, and/or introduction of hpHAse-encoding DNA in atumor cell, peritumor cell, or other cell capable of expressing and secretinghpHAse (e.g., liver cell or monocyte). Because hpHAse has a fatty acidmodification (e.g., a lipid moiety), hpHAse may be readily incorporated into theplasma membrane of cells following injection at the site where hpHAse action isdesired (see, e.g., 1995 J. Cell Bio. ;3_l(3):669-77). Further, hpHAse may alsocomplex with LDL (Low Density Lipoprotein) receptors and be internalized intothe cell by receptor-mediated endocytosis.In one preferred embodiment, hpHAse is delivered to the patient within aliposome formulation to provide intracellular delivery of hpHAse to the target cell(e.g., a cancerous cell having a LuCa-1/hpHAse defect) and/or incorporatinghpHAse into the plasma membrane of such target cell. Preferably, the liposomeformulation is prepared to provide for specific, targeted delivery of hpHAseincorporated within the liposome to a target cell. Because hpHAse is fairlyinsoluble in aqueous solution (e.g., hpHAse precipitates in aqueous solution whenthe N aCl concentration is less than 50 mM), liposome incorporation creates a moresoluble preparation due to the biochemical characteristics of hpHAse associatedwith its post-translational fatty acid chain modification. Furthermore, introductionof hpHAse into the circulation would likely result in association of hpHAse withhigh density lipoprotein (HDL) complexes, since hpHAse co-purifies with theselipid fractions of plasma.The biochemical characteristics identified by the present inventors indicatethat hpHAse can be easily incorporated into liposomes. Methods for preparationof liposomes and administration of same are well known in the art. For example,hpHAse-containing liposomes can be prepared by combining detergent—freeimmunoaffinity purified (IAP) hpHAse and liposome formulation as described inLiposome Technology, G. Gregoriadis, ed., 1984, CRC Press, Boca Raton,W0 98/166551015202530CA 02264484 1999-03-03PCT/U S97] 1808941Florida. Preferably the liposome formulation of the invention comprises acompound(s) that enhance hpHAse activity. In a preferred embodiment, thehpHAse liposome formulation comprises cholesterol and/or cardiolipin, morepreferably, cardiolipin. Without being held to theory, cardiolipin enhanceshpHAse activity by preventing the denaturation of hpHAse, thus maintaining thestability of the enzyme and enhancing its incorporation into the plasma membranesof target cells. As discovered by the inventors, hpHAse activity is not enhancedby the presence of phosphatidyl—ethanolamine or phosphatidyl-choline. Liposomeformulations comprising cholesterol and cardiolipin are especially preferred.Especially where hpHAse is to be used in therapy, e. g., chemotherapy, itmay be desirable to modify hpHAse to provide one or more desirablecharacteristics. For example, although hpHAse is a serum protein and thus shouldinherently have a substantial half-life in serum, it may be desirable to increase itsbiological half—1ife (e.g., serum half-life) by, for example, modification of thepolypeptide. Various methods for increasing the half-life of a protein are wellknown in the art and include, for example, conjugation of the protein topolyethylene glycol moieties, i.e. , PEGy1ation (see, for example, USPN 4,179,337;USPN 5,166,322; USPN 5,206,344; Nucci et al., 1991, Adv. Drug Delivery Rev.4:133-151; Zalipsky et al., 1991, "Polymeric Drugs and Drug Delivery Systems,"ACS) conjugation of the protein to dextran (Maksimenko, 1986, Bull. Exp. Biol.Med. (Russian) 2:567-569), and deglycosylation of the protein by treatment withendoglycosidase F (Lace et al., 1990, Carbohydrate Res. g9§:306-311).In general, these methods are designed to increase the molecular weight ofthe protein, decrease the sensitivity of the protein to proteinases, and/or decreasethe rate of clearance of the protein from the subject to be treated. The increasedhalf-life of modified hpHAse polypeptides decreases the amount of protein neededfor an effective dosage, reduces the number and frequency of administrationsrequired, and decreases the patient’s exposure to the protein, thus decreasing theThesecharacteristics of modified hpHAse polypeptides having an increased half-life alsopotential for allergic reactions, toxic effects, or other side effect.allow for long-term use of the protein with less potential for undesirable sideW0 98/ 166551015202530CA 02264484 1999-03-03PCT/U S97/ 1808942effects related to protein immunogenicity and/or toxicity. Preferably these methodscan be used so that the half—life of the protein is increased without substantiallycompromising the protein’s biological activity. The enzymatic activity of hpHAsecan be assayed by, for example, using the HAse activity assay of the invention.Methods for testing the biological half-life of a proteins are well known in the art.The specific dosage appropriate for administration can be readilydetermined by one of ordinary skill in the art according to the factors discussedabove (see, for example, Harrison’s Principles of Internal Medicine, 11th Ed.,1987). In addition, the estimates for appropriate dosages in humans may beextrapolated from determinations of the level of enzymatic activity of hpHAseFor example, 70-300 TRUhyaluronidase is effective in reducing the tumor load in a scid mouse. Given thisin vitro and/or dosages effective in animal studies.information, the corresponding dosages in the average 70 kg human would rangefrom about 250,000-1,200,000 TRU hyaluronidase. The amount of hpHAsepolypeptide administered to a human patient is generally in the range of 1 TRU to5,000,000 TRU of enzymatic activity, preferably between about 1,000 TRU to2,500,000 TRU, more preferably between about 100,000 TRU to 1,500,000 TRU,normally between about 250,000 TRU and 1,200,000 TRU, with about725,000 TRU representing average prescribed doses.In one embodiment, an hpHAse polypeptide is formulated in a 0.15 Msaline solution containing hpHAse at a concentration of about 150,000 TRU/cc.The formulation is then injected intravenously at 15,000 TRU/kg body weight ofthe patient. Alternatively, the enzyme formulation may also be injectedsubcutaneously to allow the hyaluronidase to perfuse around the tumor site. In apreferred embodiment, hpHAse is injected peritumorally or into the tumor mass.In another preferred embodiment, hpHAse is formulated as a liposome and isdelivered by injection either intravenously or at or near the site of cancerous cellsassociated with a defect in the LuCa—1 (hpHAse) gene. Injection of hpHAseintravenously results in hpHAse in the tumor site.W0 98/ 166551015202530CA 02264484 1999-03-03PCTIUS97/1808943Anti-cancer therapy by expression of hpHAse in cancerous or pre-cancerous cellsThe association of cancer with defects in the human chromosome at 3p21 .3The LuCa—l gene (SEQ IDN024) is one of several candidate tumor suppressor genes at 3p21.3. The identityhas been described in several tissues (see, e.g., ).of LuCa—1 and hpHAse, as well as the observations that hyaluronidase expressionis associated with inhibition of tumor progression and tumor formation in mice(De Maeyer et al. supra; Pawlowski et al. supra), indicate that the LuCa—1 gene,which encodes hpHAse, has activity as a tumor suppressor gene. Therefore,introduction of a nucleotide sequence(s) encoding LuCa-1/phase into the genomeof cells having a LuCa-1/phase defect can repair the defect and thus prevent orinhibit tumor development and/or progression (e.g., replacement gene therapy).The introduced hpHAse-encoding sequence can be either constitutively or induciblyexpressed upon transformation of the target cell. Alternatively, the LuCa-1/phasedefect can be repaired by introduction of genetic elements that may enhanceexpression of hpHAse (e.g., introduce an inhibitor of a factor that is responsiblefor inhibition of hpHAse transcription or translation). This latter approach may beuseful where the hpHAse coding sequence itself is not defective, but rather thedefect results from decreased expression of hpHAse. For example, transformationof tumor cells or cells in the vicinity of tumors cells (e.g., which can expresshpHAse which in turn is exposed to the tumor cells) can be used in the treatmentof small lung cell carcinoma, squamous lung cell carcinoma, as well as cancers ofthe breast, ovaries, head and neck, or any other cancer associated with depressedlevels of hpHAse or with a defective LuCa-l (phase) gene (e.g., a LuCa-1 genethat does not provide for expression of adequate hpHAse levels or encodes adefective hpHAse that does not provide for an adequate level of hyaluronidaseactivity) or other defect associated with decreased hpHAse activity.Methods for introduction of a sequence of interest into a host cell toaccomplish gene therapy are known in the art. In general, such gene therapymethods include ex vivo and in vivo methods. Ex vivo gene therapy according tothe present invention involves, for example, transformation of cells isolated fromthe patient with an hpHAse polypeptide encoding sequence, and implantation of the, _.4 .._....4.......»....u...........-....-........—.... ..... ,W0 98/166551015202530CA 02264484 1999-03-03PCT/U S97] 1808944transformed cells in the patient to provide a reservoir of hpHAse production,preferably at a site within or near the tumor. The implanted cells thus providesecreted hpHAse to combat the progression of tumor(s) in the patient. Methodsfor accomplishing ex vivo gene therapy are well known in the art (see, e.g.,Morgan et al. 1987 Science B_7:1476; Gerrard et al. 1993 Nat. Genet. _3_:180). Invivo gene therapy according to the present invention is used accomplished repairof the hpHAse defect within the patient by delivery of hpHAse polypeptide-encoding nucleic acid, introduction of the nucleic acid into the cells (e.g., intocancerous cells, pre-cancerous cells, peritumoral cells, or cells capable ofexpressing and secreting hpHAse, preferably into cancerous or pre-cancerouscells), and expression of hpHAse by the transformed cells in the patient, therebyrepairing the hpHAse defect.Several different methods for transforming cells can be used in accordancewith either the ex vivo or in vivo transfection procedures. For example, variousmechanical methods can be used to deliver the genetic material, including the useof fusogenic lipid vesicles (liposomes incorporating cationic lipids such asNatl. Acad. Sci. U.S.A. 8427413-7417(1987)); direct injection of DNA (Wolff, et al., Science (1990) 247:l465—l468);lipofection; see Felgner et al., Proc.pneumatic delivery of DNA—coated gold particles with a device referred to as thegene gun (Yang et al., Proc. Natl. Acad. Sci. U.S.A. 1990; 8721568-9572); andvarious viral vectors (e.g., non-replicative mutants/variants of adenovirus,retrovirus, adeno-associated virus, herpes simplex virus (HSV), cytomegalovirus(CMV), vaccinia virus, and poliovirus). A review of the different techniques alongwith a citation of numerous publications in each area is contained within a recentarticle on human gene therapy (see Morsy et al. 1993 JAMA _{7_Q:2338—2345).The formulation to accomplish gene therapy will vary with the gene therapymethod used, the route of administration (e.g., administration for systemic genetherapy or administration for transformation of specifically targeted cells), and theThe hpHAsepolypeptide-encoding nucleic acid sequence may be naked (i.e. , not encapsulated),site targeted for transformation (e.g., lung, breast, or ovaries).provided as a formulation of DNA and cationic compounds (e.g., dextran sulfate),WO 981166551015202530CA 02264484 1999-03-03PCT/US97I1808945or may be contained within liposomes. Alternatively, the DNA of interest can bepneumatically delivered using a “gene gun” and associated techniques which arewell known in the art (Fynan et al. 1993 Proc. Natl. Acad. Sci. USA $211478-11482). The various elements of the construct for hpHAse expression in the targetcell (e.g., the promoter selected, the presence of elements to enhance expression)will also vary according to the cell type and level of hpHAse expression desired.The amount of DNA administered will vary greatly according to a numberof factors including the susceptibility of the target cells to transformation, the sizeand weight of the subject, the levels of hpHAse expression desired, and thecondition to be treated. For example, the amount of DNA injected into a humanbreast tumor is generally from about 1 pg to 200 mg, preferably from about 100pg to 100 mg, more preferably from about 500 ug to 50 mg, most preferably about10 mg. Generally, the amounts of DNA for human gene therapy can beextrapolated from the amounts of DNA effective for gene therapy in an animalmodel. For example, the amount of DNA for gene therapy in a human is roughly100 times the amount of DNA effective in gene therapy in a rat. The amount ofDNA necessary to accomplish cell transformation will decrease with an increasein the efficiency of the transformation method used.Numerous other uses for phase, anti-phase antibodies, and hpHAsepolypeptide-encoding nucleotide sequences are readily apparent to one of ordinaryskill in the art. Nucleotide sequences encoding hpHAse polypeptides can be usedin hybridization screening methods to detect other hyaluronidases having homologyto hpHAse.DEPOSITSThe hybridoma cell lines l7E9 and 4D5, which produces an anti-hpHAseantibody that binds native hpHAse, has been deposited on behalf of The Regentsof the University of California, 300 Lakeside Drive, 22nd Floor, Oakland,California 94612 with the American Type Culture Collection (ATCC), Rockville,W0 98/ 166551015202530CA 02264484 1999-03-03PCT/US97l1808946Maryland, U.S.A. for patent purposes. The deposit of the hybridoma cell line17E9 was received by the ATCC on October 17, 1996, ATCC Designation ATCCHB~12213. The deposit of the hybridoma cell line 4D5 was received by the ATCCon October 17, 1996, ATCC Designation ATCC HB-12214. The hybridoma cellswere deposited under the conditions specified by the Budapest Treaty on theinternational recognition of the deposit of microorganisms (Budapest Treaty).Availability of the deposited material is not to be construed as a license topractice the invention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.EXAMPLESThe following examples are put forth so as to provide those of ordinary skill in theart with a complete disclosure and description of how to carry out the inventionand is not intended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect to numbersused (e. g. , amounts, temperatures, etc.), but some experimental error and deviationshould be accounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.Example 1: Hyaluronidase (HAse) Activitv Assav Using Covalentlv~BoundBiotinylated Hyaluronic AcidHuman umbilical cord hyaluronic acid (HA; ICN) was biotinylated through freecarboxyl groups with biotin hydrazide (Pierce), coupled using1-ethyl-dimethylaminopropyl carbodiamide (EDC, Sigma). 100 mg of hyaluronicacid (HA) was dissolved in 0.1 M Mes pH 5.0, and biotin hydrazide (Pierce),dissolved in DMSO, was added to a final concentration of 1 mM. EDC wasdissolved into the HA/biotin solution to a final concentration of 0.03 mM and leftstirring overnight at 4°C. Uncoupled biotin was then removed through exhaustivedialysis against distilled water (dH20) and was stored at -20°C at a finalconcentration of 1 mg/ml.WO 9811665510152025CA 02264484 1999-03-03PCTIUS97/1808947Up to about 10 days before performing the hyaluronidase assay, thebiotinylated hyaluronic acid (bHA) was then covalently coupled to Covalink-NHmicrotiter plates (NUNC, Placerville, NJ) at a final concentration of 5 pg ofbHA/well with 9.2 ug/well Sulfo-NHS (Pierce) and 6 pg/well fresh EDC overnightat 4°C. Unbound HA was removed through washing plates with wash buffer (PBSwith 2 M NaCl, 50 mM MgSO,,, 0.05% Tween-20).hyaluronidase activity were performed by diluting human plasma 1:400 in assaybuffer (0.1 M Formate pH 3.7, 0.1 M NaCl, 1% Triton X-100, 0.02% Azide,Incubations for plasma5 mM CaCl2 and 5 mM sacharrolactone (to inhibit exoglycosidase activity)) andincubating 100 pl samples in the bHA plates for 30 min at 37°C. The reactionwas terminated through the addition of 6 M Guanidine HCl and washed three timeswith PBS wash buffer.Residual undigested hyaluronic acid remaining covalently bound to the platewas reacted with avidin peroxidase(ABC-kit Vectastain) using o-phenylendiamineas a substrate. Fluorescence associated with bHA was detected using amicrotiterplate reader at 492 nm. A standard curve was generated throughco-incubation of serial dilutions of bovine testicular hyaluronidase (WYDASETM)and activity of unknown samples were interpolated through a four parameter curvefit to yield values given in relative Turbidity Reducing Units (rTRU)/ml related tothe standardized commercial preparations of hyaluronidase.The bHA hyaluronidase activity assay is approximately 1,000 times moresensitive than the conventional colorimetric assay (Afify et al. supra) and requiresonly 60 min to complete. Moreover, the assay does not require the tediouspreparation of a biotinylated—HA binding aggrecan peptide from bovine nasalcartilage as used in conventional ELISA-like assays (Stern et al. supra; Delpechet al. supra). The assay can also be used to detect HAse activity from cell cuturesthat produce very low levels of activity by using longer incubation periods.WO 98/1665510152025CA 02264484 1999-03-03PCT/US97/1808948Example 2: Purification of Human Plasma Hvaluronidase Using the BiochemicalPurification Method of the Invention hpHAse was purified in a three-step biochemical procedure: 1) temperature-induced detergent hpHAse extraction of human plasma; 2) Fast Flow-S cationexchange chromatography; and 3) hydroxyl-apatite resin. Hyaluronidase activityand protein concentration were determined at each stage of purification, and thespecific hyaluronidase activity calculated. Hyaluronidase activity was determinedusing the assay in Example 1. Protein concentration was determined by bothabsorbance at 280 nm and with the Biorad (Burlingame, CA) protein microassaykit (Tengblad 1979 Biochim. Biophys. Acta 57§:28l-289) in 96 well plates, usingcrystallized bovine serum albumin as a standard and read at 595 nM.1) Temperature—induced detergent phase extractionTwo liters of outdated human plasma, obtained from either UCSF or IrwinMemorial blood donor centers, were routinely used for enzyme purifications. Twoliters of chilled human plasma were dissolved in a solution 0.02% Sodium Azide,50 mM NaCl, 5% sucrose and 7.5% Triton X-114 (Boehringer Mannheim) weredissolved at 4°C with stirring for 90 min followed by centrifugation at 10,000 x gfor 30 min to remove insoluble material. The plasma was then subjected to— temperature-induced phase extraction at 37°C to separate the detergent-rich anddetergent-poor phases. The extract was centrifuged at 10,000 x g for 30 min at37°C to clarify the two phases. The detergent-rich phase was removed and dilutedto 2 liters with cold 50 mM Hepes, pH 7.5, 0.15 M NaCl. The solution was thenallowed to mix thoroughly on ice followed by repartitioning at 37°C withcentrifugation. This was repeated three times in order to increase the specificactivity of the hyaluronidase that partitioned into the detergent phase. The finaldetergent-rich phase was enriched 60-fold for hpHAse specific activity relative tothe human plasma starting material as determined by the HAse activity assay ofExample 1.W0 98/16655101520CA 02264484 1999-03-03PCT/US97/18089492) Fast Flow-S cation exchange chromatographyThe final detergent-rich phase from step 1) was diluted six-fold with 20 mlof equilibrated SP—Sepharose cation exchange resin (Pharmacia) in 25 mM Mes,pH 6.0 and stirred overnight at 4°C. The beads were collected throughcentrifugation and washed extensively with 25 mM Mes, pH 6.0, containing46 mm octylglucoside (Boehringer Mamiheim). Hyaluronidase was eluted from thebeads through the addition of 0.3 M NaCl in Mes pH 6.0 buffer with severalwashes. The SP-Sepharose eluant was concentrated on a YM3 (Amicon)membrane and desalted into 10 mM P04 pH 7.4 with 25 mM NaCl, 46 mMoctylglucoside on a f.p.l.c. Fast-Desalting column.3) Hydroxyl-apatite resinThe hyaluronidase preparation from step 2) was combined with 10 ml ofequilibrated hydroxylapatite resin (Biorad) and left on a rocker overnight at 4°C.hpHAse did not adsorb to the resin under these conditions and was recovered inthe supernatant. hpHAse recovered in the supernatant was purified to homogeneityas determined by electrophoretic analysis on silver-stained 12.5% polyacrylamidegels on a Pharmacia Phast Gel System.Table 1 is a summary of the hyaluronidase activity, protein concentration,and specific activities of the hpHAse-containing fractions during each of thepurification steps described above.WO 98/1665510152025CA 02264484 1999-03-03PCT/US97/1808950Table 1. Biochemical Purification of hpHAsePurification Step Volume Activity Protein Specific X-Fold Purifi-(ml) (rTRU/ml) (mg/ml) Activity cation(rTRU/mg)Starting Material 2,100 35 86 0.406 1.0(human plasma)1) Final Detergent 650 33 1.3 25.4 63Phase2) Fast Flow-S Cation 60 298 0.85 350.6 875Exchange3) Hydroxyl-Apatite 15 907 0.0015 6.0 x 10’ 1.5 x 10‘(unbound)Summary of results of biochemical hpHAse purificationPurification of hpHAse using the biochemical method described aboverevealed that hpHAse has the unique property of partitioning into the temperature-induced detergent phase. hpHAse partitioning into the detergent-rich phase isusually observed with certain integral membrane or lipid-anchored proteins(Bordier 1981 J. Biol. chem. $11604-7).phosphatidylinositol specific phospholipase-C (which cleaves glycosyl—phosphatidylTreatment of hpHAse withinositol (GPI) anchors), phospholipase-D (which cleaves GPI anchors), orN—glycosidase—F (which cleaves N—linked glycosylated moieties) did not alter thehpHAse partitioning properties of hpHAse (although N—glycosidase—F treatmentcombined with gel zymography did show that hpHAse has at least two N—linkedglycosylation sites).These experiments suggested that hpHAse is an integral membrane proteinor has a phospholipase—resistant anchor, similar to the GPI anchor of humanerythrocyte acetylcholinesterase (Roberts et al. 1988 J. Biol. Chem. E: 18766-75).Although GPI anchors are normally associated with proteins that reside on theextracellular domain of the plasma membrane, there are examples of GPI anchoredWO 98/166551015202530CA 02264484 1999-03-03PCT/US97/ 1808951proteins in serum (e. g., CD59, Vakeva et al. 1994 Immunology Q:28—33).Furthermore, several of the neutral PH2O sperm hyaluronidases have GPI—specificphospholipase-C-susceptible anchors (Gmachl et al. 1993 FEBS Lett. §3§:545-8;Thaler et al. 1995 Biochemistry 337788-95), but no GPI—anchor-like post-translation modifications have been described in an acid active hyaluronidases.Moreover, such post-translational modification would not be obvious from theamino acid or nucleotide sequence encoding such acid active hyaluronidases (i.e.,no specific sites for post—translational modification by addition of a fatty acid orGPI anchor have been described). For example, there is no presently knownconsensus sequence for GPI-anchored proteins. The consensus sequence forisoprenylated proteins (CAAX; SEQ ID NO:10) is known; however, hpHAse doesnot contain this sequence.hpHAse displayed other strongly amphiphilic characteristics. For example,hpHAse precipitated from solution when the ionic strength of the solution in whichhpHAse was dissolved was lowered through dialysis. hpHAse was enriched withinthe 0—30% ammonium sulfate fraction. The concavalin—A binding properties ofhpHAse revealed that hpHAse is a mannose—containing glycoprotein.Gel filtration chromatography on an S200-f.p.l.c. column (Pharmacia) atneutral pH resulted in elution of the HAse activity peak within the void volume atapproximately 120 kDa. When gel filtration chromatography was performed in theabsence of non-ionic detergents, the HAse activity peak eluted at approximately60 kDa. This difference in the molecular weight associated with the hpHAseactivity is likely due to hpHAse aggregation or oligimerzation (e.g., throughintramolecular bonds) in the presence of detergents. hpHAse is very stable at37°C in the presence of non-ionic detergents; thus, Triton X-114 phase extractionsare ideal as an initial fractionation step, particularly since few plasma proteinspartition into the detergent-rich phase.During cation exchange chromatography to remove detergent and furtherpurify hpHAse, it was found that the addition of non-ionic detergents andmaintenance of at least 50 mM NaCl was essential to prevent precipitation of theenzyme. Batch adsorption chromatography preserved activity in comparison toW0 98l166551015202530CA 02264484 1999-03-03PCT/US97/1808952column chromatography. Interestingly, hpHAse activity was not retained whenconcentrating hpHAse—containing preparations on 10 kDa cut off membrane in thepresence of 60 mM octylglucoside; however, hpHAse activity was retained byusing a 3 kDa cutoff membrane. These results suggest that the Stokes radius forhpHAse deviates substantially from a spherical model. These data show thathpHAse does not have a spherical shape in the presence of octyl glucosidedetergent as assumed for most globular proteins, but rather likely has more rod—likeshape. hpHAse in one dimension appears to have a molecular weight of 6-7kDaprotein rather than a 57kDa protein. The protein can thus in one dimension passthrough barriers reserved to small proteins and peptides, which would explain thefinding of the plasma enzyme in human urine (see infra, usually the kidneyexcludes proteins <50-60kDa). Thus, patients may excrete a significant amountof hpHAse into their urine if hpHAse is not incorporated into liposome or1iposome—like structures.The post SP-Sepharose preparation of hpHAse was purified to homogeneitythrough adsorbtion of contaminating proteins with hydroxyl apatite resin, resultingin an overall purification of 1.5 million fold. SDS-PAGE electrophoresis andsilver staining of an hpHAse sample from this final purification step revealed asingle band, thus indicating that hpHAse had been purified to electrophoretichomogeneity. The specific activity of the purified enzyme (600,000 rTRU/mg astested using the assay described in Example 1) was roughly 6-fold that of thereported values for the sperm hyaluronidase, PHZO. The purified enzyme migratedon SDS electrophoresis gels with a relative molecular mass of 57 kDa, thoughlevels of nonionic detergents in the final preparation made molecular mass analysisvariable.Example 3: Screening Assay for Anti—Native Acid Active HAse (aaHAse)AntibodiesBiotinylated HA (bHA) was prepared as described in Example 1.Approximately 5 pg of bHA/well and 1.25 pg/well of goat anti—mouse IgG(Jackson Immunolabs) were covalently coupled to Covalink-NH microtiter platesWO 981166551015202530CA 02264484 1999-03-03PCT/US97/1808953(NUNC, Placerville, NJ) with 9.2 pg/wel1Sulfo—NHS (Pierce) and 6 pg/well freshEDC overnight at 4°C. Unbound HA and goat antibody were removed throughwashing plates with wash buffer (PBS with 2M NaCl, 50mM MgSO4, 0.05%Tween~20).Anti-native acid active HAse (anti—native aaHAse) antibodies were screenedby incubating candidate antibodies with a sample containing an acid active HAsein a neutral pH buffer composed of 1% Triton X-100 in phosphate—buffered saline(PBS). If the sample was a diluted sample (e. g., a diluted plasma sample), theneutral pH buffer additionally contained 5 mg/ml bovine serum albumin (BSA).The aaHAse-containing sample was incubated with the candidate antibodies toallow for formation of native aaHAse-antibody complexes. The sample was thenplaced into bHA/anti-mouse antibody wells and incubated to allow for formationof anti-mouse antibody/anti—native aaHAse/aaHAse antibodies in the neutral pHbuffer. A sample containing antibodies that do not bind aaHAse can be used as anegative control.Detection of antibodies bound to native aaHAse was accomplished by firstwashing away unbound material with neutral pH buffer, then replacing the neutralpH buffer with the acidic assay buffer used in the HAse activity assay describedin Example 1 (0.1 M Formate pH 3.7, 0.1 M NaCl, 1% Triton X-100, 0.02%Azide, 5 mM CaCl2 and 5mM sacharrolactone). The shift in the pH from neutralto acidic allows for the HAse activity of any bound aaHAse to degrade thecovalently bound bHA. The reaction was terminated by addition of 6 M guanidineHCL and washed three times with PBS wash buffer (PBS, 2 M NaCl, 50 mMMgSO4, 0.05% Tween-20). Degradation of bHA was detected by reaction of thewells with avidin peroxidase using o-phenylendiamine as a substrate and readingthe plates at 492 nm as described in Example 1. Anti-native aaHAse antibodieswere identified by degradation of bHA. The assay takes advantage of the aaHAsecharacteristic that aaHAses have no affinity for the HA substrate above pH 4.5 (asdetermined through HA-Sepharose affinity chromatography with hpHAse, data notshown).W0 98/ 166551015202530CA 02264484 1999-03-03PCT/US97/1808954Example 4: Generation and Identification of Anti-hpHAse Monoclonal AntibodieshpHAse isolated from the post-hydroxyl apatite step in the purificationmethod of Example 2 was used to irnmunize five 6 week old female Balb—c miceaccording to methods well known in the art (see, e.g., Harlow and Lane, supra).Briefly, the hpHAse was combined with Freund’s complete adjuvant and injectedintraperitoneally into the mice. At 21 day intervals, the animals were boosted withprotein plus Freund’s incomplete adjuvant. The fourth and final boost containedhpHAse in Freund’s incomplete adjuvant was injected intravenously. Serumsamples were obtained, the mice sacrificed, and spleen cells from two of the fiveisolated for preparation of hybridoma cell lines according to methods well knownin the art (see, e.g., Harlow and Lane, supra; Schrier et al. supra). Fusion of thebalb/c cells was performed with sp2/0 myelomas.Antibody-secreting hybridomas were screened using the anti-native aaHAseantibody assay of the invention as described Example 3. Briefly, hybridomasupematants from 20 fusion plates were incubated with diluted human plasma for60 min at 37°C followed by incubation in the bHA/anti-mouse—IgG plates for60 min at 37°C. The plates were washed five times with PBS containing 1%Triton X-100 and BSA tohyaluronidase. The acidic forrnate assay buffer (pH 3.7) was then added to the10 mg/ml remove non-immunoprecipitatedwells and incubated at 37°C for 60 min. After stopping the reaction with 6 Mguanidine, undegraded bHA remaining in the wells was detected by reaction of thewells with avidin peroxidase using 0-phenylendiamine as a substrate and readingthe plates at 492 nm as described in Example 3.Eight clones were identified from the original 20 hybridoma fusion plates.Two clones, l7E9 (producing IgG2,, class antibody; kappa chain) and 4D5(producing IgG1 class antibody; kappa chain), were used to generate ascites.Immunoglobulin from ascites of single-cell cloned hybridoma lines were generatedin Balb/c mice and purified through protein-A affinity chromatography. Neitherthe l7E9 or 4D5 antibodies blocked hpHAse activity. The l7E9 antibody ispreferred for use in immunoaffinity purification and imrnunoprecipitation; the 4D5antibody is preferred for use in irnmunohistochemistry.W0 98/ 166551015202530CA 02264484 1999-03-03PCTIUS97/1808955Example 5: Immunoprecipitation of hpHAse using the 17E9 Monoclonal AntibodyhpHAse was immunoprecipitated from human plasma using either purifiedIgG2a from the l7E9 anti~native hpHAse clone or a control IgG2a non-specificantibody. Human plasma was diluted in R.I.P.A. buffer (1% NP40, 1%Deoxycholate, 1% Triton X-100, 5 mM EDTA in PBS).conjugated to serial dilutions of either control IgG2a antibody or 17E9 antibodyProtein-A Sepharosewas added to the sample. Unbound material was separated from the beads bycentrifugation and the HAse activity remaining in the supernatant detected usingthe assay described in Example 1. As shown in Fig. 4, immunoprecipitation withthe 17E9 antibody resulted in removal of essentially all of the detectable acid activehyaluronidase activity. Further, the 17E9 antibody did not bind bovine testicularhyaluronidase (PH20), suggesting that 17E9 does not bind a carbohydrate moietyor other moiety that may be shared by PH20 and hpHAse.Example 6: Immunoaffinity Purification of hpHAsehpHAse was purified to homogeneity from raw human plasma in a singlestep using the 17E9 antibody bound to an immunoaffinity chromatography column.Approximately 3 mg of purified IgG from the 17E9 hybridoma clone ascites wascoupled to a lml High Trap—NHS activated column (Pharmacia) as per themanufacturer’s instructions. Human plasma was diluted 1:2 with R.I.P.A. buffer,and passed over the 17E9 IgG antibody column. The column was then washedwith PBS containing 2M NaCl, 100 mM octylglucoside followed by washing with100 mM citrate pH 4Ø hpHAse was eluted with 100mM citrate, pH 3.0, with150 mM NaCl. hpHAse eluted as a sharp, homogenous peak at pH 3Ø Thespecific activity of immunoaffinity purified hpHAse ranged from about 4 x 105rTRU/mg. Thus very little, if any, activity is lost in the immunoaffinitypurification process relative to the biochemical purification process. Moreover,irnmunoaffinity purification resulted in a yield of hpHAse approximately 100% ofthe hpHAse in the sample; HAse assay detected no HAse activity remaining in theunbound fraction.W0 98/166551015202530CA 02264484 1999-03-03PCT/US97/1808956Example 7: Amino Acid Sequencing of hpHAseApproximately 50 pg of immunoaffinity purified hpHAse from Example 6was digested with Lys-C (to generate N-terminAl fragments) or trypsin (to generateinternal fragments) and chromatographed on a Vydac C-18 reverse phase columnto separate fragments. Purified peptides were sequenced on a gas phase Edmansequencer. For N—tenninal amino acid sequencing, the purified protein wasimmobilized on a Prosorb membrane (ABI). N—terminal amino acid sequencingwas accomplished by automated protein sequence analysis using gas phase Edmandegradation. Fasta queries (Pearson et al. 1988 Proc. Natl. Acad. Sci. USA§§ 12444-8) of N —teiminal and 5 separate internal amino acid sequence derived fromthe Lys-C digests of purified hpHAse (SEQ ID N021) revealed identity with thepredicted amino acid sequence of the LuCa-1 gene (SEQ ID N023; GenBankaccession no. uO3056).Example 8: Characterization of LuCa-1/hpHAseAs described in the Example 7 above, hpHAse is identical to LuCa—1. TheLuCa-1 gene is one of a group of 17 sequences located on chromosome 3p.21.3that display a 100% loss of heterozygosity in small cell lung carcinoma linesDietrich et al. 1966 Clin. Chim. Acta Q2746-52). 3p.21 deletions spanning theregion containing LuCa-1 have also been described in non-small cell lungcarcinomas, mammary carcinoma, carcinoma of the prostate and head and neckcarcinoma. Chromatofocusing of hpHAse on a MON 0-P f.p.l.c. system providedadditional data regarding the identity of hpHAse (SEQ ID N021) and LuCa-1 (SEQID NO:3).theoretical isoelectric point of 6.58.hpHAse eluted at pH 6.5, very close to the LuCa-l’s calculatedPearson Lipman alignment (Pearson et al. supra) of LuCa—1 (SEQ ID NO:3)and human testicular hyaluronidase PH20 (SEQ ID NO:1l) amino acid sequencesrevealed that LuCa—1 and PH20 share over 40% sequence identity and 60% percenthomology (Fig. 5). PH20 is a predominantly sperm-specific neutral hyaluronidaseand shares considerable homology with the venom hyaluronidases found in bee andyellow jacket vespid venoms (Gmachl et al. 1993 Proc. Natl. Acad. Sci. USAW0 98/ 16655202530CA 02264484 1999-03-03PCT/U S97! 18089572_(_)_:3569-73).hyaluronidase (LuCa-1/hpHAse) and PH20 is quite surprising given their veryThe striking homology between a strictly acid active plasmadifferent pH optima, suggesting that all mammalian B,1—4 hyaluronidases, bothneutral and acidic, are in members of a highly conserved family of enzymes.Hydropathy plots of LuCa-1/hpHAse (Fig. 6) did not reveal any richhydrophobic domains (e.g., such as those associated with integral membraneproteins) that would explain the phase partitioning properties of hpHAse describedin Example 2. These data thus suggest that partitioning of LuCa-1/hpHAse intothe detergent-rich phase is due to a post-translational modification (e. g., fatty acidchain modification or phospholipase C/phospholipase D/N-glycosidase F-resistantGPI anchor.Example 9: Isolation of LuCa-1/hpHAse-encoding DNAA cDNA encoding the LuCa—1 coding sequence was isolated using two roundsof nested PCR reactions with a >\gt10 5’—STRETCH PLUS Human Liver cDNAlibrary (Clontech Laboratories, Inc., Palo Alto, California, USA). In the firstround of PCR, which amplified nucleotides 590 to 1948 of the LuCa-l cDNA, thefollowing primers were used: LuCaF1 (5’- CAGGTTGTCCTGCACCAGTC' -3’)(SEQ ID NO:5), and LuCaR1 (5’- ATGTGCAACTCAGTGTGTGGC —3’)(SEQID NO:6). The PCR reaction was performed in a total reaction volume of 50 mlcontaining 1 ml of liver cDNA library, 1 ml each of 25 mM primers, 1 ml of10 mM dNTPs (Gibco BRL, Grand Island, New York, USA), 2 units of Pfu DNApolymerase (Stratagene Cloning Systems, La Jolla, California, USA), buffered with10 mM Tris—HCl, 50 mM KCl, and 2 mM MgCl,. The PCR reaction conditionsincluded a 1 min denaturation step at 95°C, 1 min annealing step at 60°C, and a1 min extension at 74°C for 37 cycles, followed by a final 7 min extension at74°C.A second round of PCR amplification was used to amplify nucleotides 612to 1925 of the Luca-1-encoding CDNA with the nested PCR primers: LuCaF2 (5’—GTGCCATGGCAGGCCACC -3’)(SEQ ID NO:7) and LuCaR2 (5’-ATCACCACATGCTCTTCCGC -3’)(SEQ ID NO:8). Primer LuCaF2 wasW0 98/ 166551015202530CA 02264484 1999-03-03PCT /US97ll808958phosphorylated prior to the PCR reaction in order to facilitate subsequentunidirectional cloning of the PCR product. Phosphorylation was carried out byincubating 2 ml of 25 mM LuCaF2 with 10 units of T4 Polynucleotide Kinase,1 ml of 10 mM ATP buffered in 50 mM Tris—HCl pH 7.5, 10 mM MgCl2, 5 mMdithiothreitol, and 0.1 mM spermidine in a final volume of 9 ml. The reaction wascarried out in a total volume of 50 ml containing 1 ml of the final product from thefirst PCR reaction as template, 4 ml of phosphorylated LuCaF2, 1 ml of 25 mMLuCaR2, 1 ml 10 mM dNTPs (Gibco BRL), buffered with 10 mM Tris-HCl, 50mM KCl, and 1.5 mM MgCl2 and containing 0.5 units of Taq DNA polymerase(Gibco BRL). The second PCR reaction consisted of 7 cycles of a 1 mindenaturation at 95 °C, a 1 min annealing at 58°C, and a 1 min extension at 72°C,followed by a final extension at 72°C for 7 min.The product of the PCR reaction was ligated into 2 pl of the unidirectionalTA—expression vector pCR3.1—Uni (Invitrogen, San Diego, CA, USA) with T4DNA ligase. The ligated vector was used to transform One Shot” TOPIOF’(Invitrogen) competent cells , which were plated out onto LB agar plates containing50 ;tg/ ml ampicillin and incubated overnight at 37°C. Colonies were tested for thepresence of LuCa-l inserts by growing overnight colonies in 10 ml LB broth with50 pg/ml ampicillin, and by plasmid purification with the Wizard” Plus MiniprepDNA Purification System (Promega Corporation, Madison, WI , USA), followedby restriction mapping of the vector with Dra III endonuclease (BoehringerManheirn, Indianapolis, IN, USA ) and automated fluorescent sequencing on anABI Prism” DNA sequencer.Restriction mapping with DraIII and sequencing of the plasmid showed thatthe cloned sequence was identical to the’LuCa-1 sequence available in the GenBankdatabase except for a Val”-Leu substitution found in the N-terminus. Sequencingof the active PCR product from the liver >\gt1O cDNA library revealed a G—Cdiscrepancy between the sequence obtained and the published LuCa-l sequence atThus, either thepublished Luca—1 sequence contains an error or the clone sequenced here is anthe position corresponding to the 27th amino acid residue.allelic variant.W0 98/ 166551015202530CA 02264484 1999-03-03PCT/US97/1808959Example 10: Expression of recombinant LuCa-1' in Cos-7 cellsThe purified LuCa-1-encoding cDNA in the pCR3. 1-Uni expression plasmidwas transfected into 50% confluent cos—7 cells in T75 flasks for five hours using9 pg of DNA with 60 pl of Lipofectamine in 20 ml of DME/F12 50/50 mix withnon- essential amino acids (UCSF cells culture facility) containing insulin,transferrin and selenium (Gibco BRL). The transfected cells were then grown foran additional 48 hr in DME/F12 50/50 mix containing 10% fetal bovine serum.Example 11: Purification and Characterization of Recombinant LuCa—l Expressedin Cos-7 CellsRecombinant LuCa—l was further characterized to further verify that LuCa—lis identical to hpHAse by virtue of its ability to bind anti-native hpHAse antibodiesand exhibit acid active HAse activity. Recombinant LuCa—l was expressed in cos-7 cells as described above and subjected to the following experiments: 1) HAseactivity assays using anti-hpHAse antibodies; 2) immunoprecipitation of LuCa—lusing anti-native hpHAse antibodies; 3) gel zymography of anti-native hpHAseantibody—precipitated LuCa—l; and 4) determination of the pH optima of the acidactive HAse activity associated with LuCa—l. Cells transfected with 9 pg of thepCR3.l-Uni vector containing the chloramphenicol transferase gene served as anegative control (mock—transfected cells).HAse activity of recombinant LuCa—l using anti-hpHAse antibodies in theenzyme capture assayCos-7 cells expressing hpHAse and their conditioned media were separatelyextracted with 2% Triton X-114 followed by temperature-induced detergent phaseseparation as described in Example 2. The detergent-rich phase extracts wereanalyzed for HAse activity using the enzyme irnmuno—capture assay of Example 1.Triton X-114 detergent phase extracts of both the cell layer and conditioned mediacontained an acid active HAse activity as detected in the assay of the invention(Fig. 7A). Mock-transfected control cells secreted a detectable, low-level, acidactive HAse activity (Fig. 7A).W0 98/1665510152025CA 02264484 1999-03-03PCT/US97/ 1808960To further assess the HAse activity of recombinant hpHAse, HEK 293 cellswere stably transfected with hpHAse-encoding DNA in a manner similar to thatdescribed above for transformation of Cos-7 cells. Briefly, the hpHAse-encodingconstruct was transfected into HEK 293 cells using 9 pg of DNA with 60 ;/.l ofLipofectin. After 48 hrs, the cells were plated out using the limiting dilutionmethod into 24 well plates with 500 pg/ml G418. After 14 days, the conditionsmedia of resistant colonies was assayed for hyaluronidase activity as describedabove. Colonies with high level expression were expanded for furthercharacterization. Analysis of the recombinant hpHAse was carried out by groringan HEK-293 cell line overexpressing the hpHAse-encoding construct for 48 hrs inserum free media. The condition media was passed through a l7E9 anti-hpHAseimmunoaffintiy column. Recombinant hpHAse was eluted using the protocolabove. As shown in Fig. 7B, both the cell layer and conditioned media containedan acid active hpHase activity; mock-transfected cells secreted no detectablehyaluronidase activity.These data show that LuCa-1 exhibits an acid active HAse activity, thuslending further support to the observation that hpHAse and LuCa-1 are identical.Immunoprecipitation of recombinant LuCa-1 with anti-hpHAse antibodiesBinding of anti—native hpHAse antibodies to LuCa-1 was tested in theirnmunoprecipitation method described in Example 5 using the l7E9 anti-hpHAsemonoclonal antibody described in Example 4 bound to protein A Sepharose. Thel7E9 antibodies immunoprecipitated LuCa-1 from both the LuCa-1 expressing-cellsand from the conditioned media. However, the acid active HAse activity of mocktransfected cells was not irnmunoprecipitated with the 17139 antibody—protein ASepharose beads. These experiments show that hpHAse and LuCa-1 share theantigenic epitope bound by the l7E9 anti—native hpHAse antibody. Moreover, thisepitope is unique to hpHAse and LuCa-1, since the l7E9 did not bind an acidactive HAse expressed by mock transfected cos-7 cells.W0 98/166551015202530CA 02264484 1999-03-03PCT/US97l1808961Gel zymography of LuCa-1 immunoprecipitated with anti—hpHAseantibodiesRecombinant LuCa-1 was immunoprecipitated with the l7E9 anti-nativehpHAse antibody was also tested for acid active HAse activity in substrate gelzymography experiments according to methods known in the art (Afify et al.supra). Briefly, cell lysates and conditioned media from LuCa-1-transfected cells(test samples) and mock transfected cells (chloramphenicol-transferase encodingplasmid; negative control) were immunoprecipitated with anti—native hpHAse boundto protein A Sepharose beads. The samples were suspended in SDS sample bufferand electrophoresed in 10% polyacrylamide gels containing 40 pg/ml HA.Samples containing immunoprecipitated hpHAse (Example 5) and bovine testicularhyaluronidase (WYDASE"' 150 rTRU/ml) served as positive controls. Incubationswere performed as described by Afify et al. (supra) and digested HA detected bystaining with Alcian blue/Acetic acid followed by Commassie staining to enhancethe Alcian/carbohydrate stain and visualize protein.The region of clearing the SDS-HA gel corresponded with the same relativemolecular mass as the irnrnunoaffinity purified hpHAse preparation. No zone ofclearing, and thus no immunoprecipitated acid active hyaluronidase activity wasobserved in the mock-transfected cell samples.pH optima of HAse activity of recombinant LuCa-1The HAse activity assay described in Example 1 was used to determine thepH optimum of the recombinant LuCa-1 HAse activity. The HAse activity assayof Example 1 was performed as described, except that the assay buffer wascomposed of 0.1 M Formate pH 3-4.5, Acetate pH 5.0, Mes pH 6.0, Hepes pH7.0-8.0, 0.1 M NaCl, 1% Triton X-100, 0.02% Azide, 5 mM CaCl?. and 5 mMsacharrolactone. Sigma Type VI-S testicular hyaluronidase (3 ,000 TRU/mg solid),which contains the neutral HAse PH20 (maximal HAse activity at about pH 7.5),was used as a comparison. The Type VI-S sample was treated identically, exceptthat the assay buffer for this sample was prepared without NaCl (NaCl inhibitsneutral HAse activity). As shown in Fig. 8, the pH optima curve of recombinantLuCa—1 displays the same strictly acid active profile as immunoaffinity purifiedW0 98/166551015202530CA 02264484 1999-03-03PCT/US97/ 1808962hpHAse. Neither LuCa-1 nor hpHAse display HAse activity above pH 4.5; incontrast, Type VI—S testicular hyaluronidase exhibited HAse activity only above pH7.5.In summary, biochemical, molecular, and immunological criterion stronglyindicate that LuCa-1 and hpHAse are identical.Example 12: Organ Survey of LuCa-1/hpHAse and Transient Expression of LuCa-1/ hpHAseThe primers described in Example 11 that were used to amplify the 1.3kbcoding region of the LuCa-1 CDNA were used to analyze the tissue distribution ofLuCa-1/hpHAse transcripts according to methods well known in the art. AmplifiedPCR products from )\gt10 CDN A libraries of various tissues were detected in heart,kidney, liver, lung, placenta, and skeletal muscle, but were not detected in brain.Heart tissue exhibited one of the highest levels of LuCa-1/hpHAse transcriptproduction.Following procedures similar to those described above, other hyaluronidaseshaving substantially the same sequence as hpHAse of the invention can be purified,cloned, and expressed.Example 13: Biochemical Purification of a Form of hpHAse from UrineThe urine form of hpHAse was purified from human urine according to thebiochemical purification method described in Example 2, except that concentratedurine was used as the sample in lieu of outdated human plasma. Urine hpHAsepartitioned into the detergent-rich Triton X~l14 phase in a manner similar to thatof hpHAse, suggesting the urine hpHAse contains a lipid modification similar tothat of hpHAse. The isoelectric point of urine hpHAse is 6.5 as determined byelution in chromatofocusing on Mono—P f.p.l.c. Human urine hpHAseimmunoprecipitated with the anti-native hpHAse antibody 17E9.Gel zymography revealed two bands of HAse activity in crude plasma andurine samples. In plasma, HAse activity was detected in two bands correspondingto 57 kDa and 46-47 kDa; the 57 kDa band was the predominant species. In urine,W0 98/166551015202530CA 02264484 1999-03-03PCT/US97I1808963HAse activity was also detected in two bands corresponding 57 kDa and 46-47 kDa; however, neither species was predominant (i.e., the bands appeared in theurine sample with equal intensity). These data suggest that hpHAse and urinehpHAse are present in plasma and urine, respectively, in two distinct modifiedforms (e.g., as a holoprotein and a proteolytic or otherwise modified fragment),or that there are two distinct acid active HAse proteins present in urine and inplasma.Example 14: Expression of hpHAse in Metastasis—Derived CarcinomasSeveral carcinoma lines (SCC 10A, SCC 10B, HSC-3, NIC H740, andDMS 153) were examined for the production of human plasma hyaluronidase bycharacterizing the levels of 17E9 immunoreactive hpHAse activity using theimmmunoprecipitation assay described in Example 5 above. hpHAse levels wereexamined in both the conditioned media and in the cell layer itself. Normalforeskin human keratinocytes served as a positive control. The SCC 10 A cell lineis derived from a primary tumor of a laryngeal carcinoma; the SCC 10B cell lineis from a lymph node metastasis from the same patient. The HSC-3 cell line isderived from a lymph node metastasis. The NIC H740 cell line is a small cell lungcarcinoma containing a homozygous deletion of the region of chromosome 3p21.3encoding hpHAse. The DMS 153 cell line is a classic small lung cell carcinomacell line (as opposed to a small lung cell carcinoma variant cell line).As shown in Fig. 9, the SCC 10A cell lines produces hpHAse activitylevels comparable to that of normal human keratinocytes, but the levels of activityin the lymph node metastasis from the same patient (SCC 10B) is completelyThe HSC-3 derived cell lineexhibited no detectable hpHAse activity (the hpHAse activity assay used canabsent in the cell layer or conditioned media.accurately detect at least 1/ 100th of the activity found in normal keratinocytes).Neither of the small cell lung carcinoma lines NIC H740 or DMS 153 havedetectable activity. NIC H740 and DMS 153 cells also did not generate anydetectable hpHAse-encoding transcripts (data not shown). These data suggest thatloss of hpHAse expression is strongly correlated with tumorigenesis. The presentW0 98/16655101520CA 02264484 1999-03-03PCT/U S97/ 1808964inventors have identified no metastasis—derived carcinoma that produces afunctional plasma hyaluronidase gene product. Some non—metastatic lines derivedfrom primary carcinomas do have functional activity, suggesting that complete lossof enzyme function is associated with tumor metastasis.Example 14: Recombinant hpHAse Expression Svstem Having Improved YieldA recombinant hpHAse expression system to provide high yields of hpHAsein the culture supernatant was developed. hpHAse-encoding DNA was operablyinserted into the CMV-promoter driven PCR3.1 Uni vector (Invitrogen). ThishpHAse-encoding construct was then used to transform HEK cells according tomethods well known in the art. The HEK cells were then grown in a T225 cm’flask at 37°C, 5% CO2 in DME H21 (4.5 g/l glucose), 10% FBS media.Conditioned culture media was collected from the cells and the levels of hpHAseassessed.While there are often inherent difficulties generating recombinant plasmaproducts with competitive yields to raw plasma, the recombinant hpHAseexpression system described herein produces over 30-fold the amount ofhyaluronidase present in raw human plasma Cohn fraction-I paste (prepared by coldalcohol precipitation according to methods well known in the art) (Table 1). Incomparison, transient expression of hpHAse in Cos-7 cells expressed about 10%of the levels achieved with HEK cells. Without being held to theory, mammaliancell lines that are adenovirus transformedTable 1 Levels of hpHAse in various pre arationsEnzyme SourceHuman Plasma 3-6Hyaluronidase ActivityrTRU/ mlRecombinant Plasma HA’se 150.0 rTRU/mlConditioned MediaWO 981166551015202530CA 02264484 1999-03-03PCT/U S97/ 1808965The recombinant hpHAse from the mammalian expression system isindistinguishable from the biochemically purified enzyme as determined by usingseveral criteria. Immunoaffinity purified recombinant hpHAse migrates with thesame molecular mass as biochemically purified hpHAse. The amino acid sequenceof immunoaffinity purified recombinant hpHAse has the same processed N-terminus as the native plasma enzyme (FRGPLLVP)(SEQ ID N029). Moreover,N terminal sequencing indicates that recombinant hpHAse is processed properly inour expression system. Recombinant hpHAse has a specific activity equivalent tobiochemically purified hpHAse as determined by gel zymography. IOU samplesof recombinant hpHAse, biochemically purified hpHAse, and the “purest”commercial preparation of testicular hyaluronidase can depolymerize lO0pg of highmolecular weight HA in 10 min. In addition, recombinant hpHAse displays thesame detergent—phase partitioning properties as the biochemically purified hpHAse,suggesting that the expression system facilitates the correct post—translationprocessing of recombinant hpHAse.Example 15: Activity of hpHAse in Inhibition of Tumor Growth in an AnimalModelThe ability of recombinant hpHAse to inhibit tumor growth was examinedin the HSC-3 head and neck squamous cell carcinoma model, an orthotopic tumorxenografts (transplanted tumors into organ/tissue of origin). Briefly, Nu/Nu micewere orthotopically implanted with 5 x 106 HSC-3 human squamous cell carcinomacells in Matrigel into the floor of the mouth. The cells were allowed to grow for7 days. Animals were then segregated randomly into two groups.Recombinant hpHASe was prepared from HEK-conditioned mediaoverexpressing hyal-1 under a CMV promoter using an immunoaffinity columnhaving the 17E9 IgG2a antibody coupled to NHS sepharose. Eluted hpHAse wasexchanged into bovine HDL complex (Sigma) and dialyzed against Biobeads. HDLcomplex carrier without hpHAse served as a negative control. The material wasinjected into the mice peritumorally (approximately 10 mm from the primary tumorsite) in a 100[l.1 volume. Approximately 400 rTRU hpHAse was administered perW0 98/166551015202530CA 02264484 1999-03-03PCT/U S97/ 1808966injection. Injections were repeated every 48 hours. Tumor volume was measuredin 3 dimensions using a spherical model. As shown in Fig. 10, administration ofhpHAse significantly reduced tumor volume.Example 16: Inhibition of Tumor Growth by Expression of hpHAse-EncodingDNA in an Animal ModelThe effect of expression of hpHAse in tumor cells upon tumor growth wasexamined by introducing a functional hpHAse gene into a carcinoma line, wherethe hpHAse-encoding sequence is under control of an inducible promoter. Thesteroid-responsive expression plasmid ecdysone was chosen for this study sinceonly those mammalian cells that contain the appropriate steroid-responsive plasmidresponded to injection of the stimulating hormone (muristerone). This system thuspermits examination of the effects of specific reactivation of the hpHAse-encodinggene within the tumor cells and assessment of in vivo effects.HSC—3 oral squamous cell carcinoma cells were transfected with Lipofectin(Gibco) at 60 ul lipofectin, 9 pg. plasmid per T75 flask with a muristeroneinducible gene construct (Ecdysone system, Invitrogen) containing the ecdysonereceptor plasmid and encoding hpHAse (under inducible steroid control), as wellas G418S and zeocin resistance. Cells transformed with the muristerone inducibleconstruct without hpHAse-encoding DNA served as a control. The transformedcells were injected in the floor of the mouth of nu/nu mice at 5 x 10° cells perinjection as described in Example 15. After 7 days of tumor growth, the micereceived an intraperitoneal injection of muristerone (5 mg) every 72 hours toinduce expression of the constructs. Tumor volumes were measured as describedabove in Example 15. In addition, the time to cachexia (defined by a drop inanimal mass 15% below starting weight). Survival curves were generated usinga Kaplan Meyer analysis using percent cachexic rather than percent surviving.As shown in Fig. 11, mice having HSC—3 cells transformed with thehpHAse-encoding construct were significantly reduced in tumor growth relative tocontrol mice. Moreover, significantly fewer mice bearing the hpHAse-expressingtumor cells progressed to cachexia over the course of 39 days.CA 02264484 1999-03-03WO 98/16655 PCT/US97/ 1808967The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can be made theretowithout departing from the spirit or scope of the appended claims.CA 02264484 1999-03-03W0 98/16655 PCT/US97l 1808968SEQUENCE LISTING(1) GENERAL INFORMATION:(i) APPLICANT: The Regents of the University of California(ii) TITLE OF INVENTION: Human Plasma Hyaluronidase(iii) NUMBER OF SEQUENCES: 10(iv) CORRESPONDENCE ADDRESS:(A) ADDRESSEE: Robbins, Berliner & Carson, LLP(B) STREET: 201 N. Figueroa Street, 5th Floor(C) CITY: Los Angeles(D) STATE: CA(E) COUNTRY: USA(F) ZIP: 90012-2628(V) COMPUTER READABLE FORM:(A) MEDIUM TYPE: Floppy disk(3) COMPUTER: IBM PC compatible(C) OPERATING SYSTEM: PC-DOS/MS-DOS(D) SOFTWARE: Patentln Release #1.0, Version #1.25(vi) CURRENT APPLICATION DATA:(A) APPLICATION NUMBER:(B) FILING DATE:(C) CLASSIFICATION:(viii) ATTORNEY/AGENT INFORMATION:(A) NAME: Berliner, Robert(8) REGISTRATION NUMBER: 20,121(C) REFERENCE/DOCKET NUMBER: 5555-458C1-XPC(ix) TELECOMMUNICATION INFORMATION:(A) TELEPHONE: (213) 977-1001(B) TELEFAX: (213) 977-1003(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 435 amino acids(8) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:Met Ala Gly His Leu Leu Pro Ile Cys Ala Leu Phe Leu Thr Leu Leu1 5 10 15Asp Met Ala Gln Gly Phe Arg Gly Pro Leu Leu Pro Asn Arg Pro Phe20 25 30Thr Thr Val Trp Asn Ala Asn Thr Gln Trp Cys Leu Glu Arg His Gly35 40 45Val Asp Val Asp Val Ser Val Phe Asp Val Val Ala Asn Pro Gly Gln50 55 60Thr Phe Arg Gly Pro Asp Met Thr Ile Phe Tyr Ser Ser Gln Leu Gly65 70 75 80Thr Tyr Pro Tyr Tyr Thr Pro Thr Gly Glu Pro Val Phe Gly Gly Leu85 90 95W0 98/ 16655ProlleAspAsp145AspGlyLeuAsnlle225AlaLysAlalleHis305TrpLysSerCysAla385ArgLysSET‘GlnLeuTrp130lleTrpAlaArgTyr210ArgLeuSerValPhe290SerValGluGlyVal370SerGlyCysMetAsnAla115GluTyrProAlaPro195AspAlaTyrGlnAla275TyrLeuSerTyrAla355ArgPheAlaArgTrp435Ala100AlaAlaArgAlaArg180ArgPheGlnProMet260AlaAspGlyTrpMet340LeuArgSerLeuCys420SerIleTrpGlnPro165AlaGlyLeuAsnSer245TyrGlyThrGluGlu325AspLeuThrlleSer405TyrCALeuProAC9Arg150GlnTrpLeuSerAsp230ValAspThrSer310AsnThrCysSerGln390LeuProlleAlaPro135SerValMetTrpPro215GlnTyrGlnProAsn295AlaThrThrSerHis375LeuGluGly(2) INFORMATION FOR SEQ ID NO:2:Ala His105Pro120AspArg TrpArg AlaGlu AlaAla Gly185Gly Phe200Asn TyrLeu GlyMet ProHis Arg265Asn Leu280His PheAla GlnArg ThrLeu Gly345Gln360AlaPro LysThr ProAsp GlnTrp Gln425(1) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids(3) TYPE: amino acid(0) TOPOLOGY: linear LeuPheAlaLeuVal170ThrTyrThrTrpAla250ValProLeuGlyLys330ProLeuAlaGlyAla410Ala69AlaSerPheVal155AlaLeuGlyGlyLeu235ValAlaValProAla315GluPheCysLeuGly395GlnPro02264484 1999-03-03Arg ThrLeu125GlyAsn140TrpGln AlaGln AspGln LeuPhe Pro205Gln220CysTrp GlyLeu GluGlu AlaPro285LeuLeu300AspAla GlySer Cyslle LeuSer Gly365Leu380LeuGly ProMet AlaTrp CysPhe110AlaAspGlnGlnGly190AspProGlnGlyPhe270TyrGluValGlnAsn350HisLeuLeuValGlu430GlnValThrHisPhe175GlyCysSerSerThr255ArgValLeuValAla335ValGlyAsnSerGlu415ArgAsplleLysPro160GlnAlaTyrGlyArg240GlyValGlnGluLeu320lleThrArgProLeu400PheLysPCT /U S97 I 18089 W0 98/16655CA02264484 1999-03-03(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:Met Ala Gly His Leu Leu Pro lle Cys Ala Leu Phe Leu Thr Leu Leu15Asp Met Ala Gln Gly20(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 435 amino acids(8) TYPE: amino acid(D) TOPOLOGY:(ii)(xi)MetAspThrValThr65ThrProlleAspAsp145AspGlyLeuAsnIle225AlaLyslinearMOLECULE TYPE: protein10SEQUENCE DESCRIPTION: SEQ ID NO:3:Ala Gly His Leu Leu Pro lle Cys AlaMetThrAsp50PheTyrGlnLEUTrp130IleTrpAlaArgTyr210ArgLeuSerAlaVal35ValArgProAsnAla115GluTyrPFOAlaPro195AspAlaTyrGlnGln20TrpAspGlyTyrAla100AlaAlaArgAlaArg180ArgPheGlnProMet5GlyAsnValProTyr85SerlleTrpGlnPro165AlaGlyLeuAsnSer245TyrPheAlaSerAsp70ThrLeuProArgArg150GlnTrpLeuSerAsp230lleValArgAsnVal55MetPPOlleAlaPro135SerValMetTrpPro215GlnTyrGlnGlyThr40PheThrThrAlaPro120ArgArgGluAlaGly200AsnLeuMetHisPro25GlnAsplleGlyHis105AspTrpAlaAlaGly185PheTyrGlyProArg10LeuTrpValPheGlu90LeuPheAlaLeuVal170ThrTyrThrTrpAla250Val70LeuValCysValTyrProAlaSerPheVal155AlaLeuGlyGlyLeu235ValAlaPheProLeuAla60SerValArgGlyAsn140GlnGlnGlnPheGln220TrpLeuGluLeuAsnGlu45AsnSerPheThrLeu125TrpAlaAspLeuPro205CysGlyGluAlaThrArgArgProGlnGlyPhe110AlaAspGlnGlnGly190AspPFOGlnGlyPhe15Leu15ProHisGlyLeuGlyGlnValThrHisPhe175GlyCysSerSerThr255ArgLeuPheGlyGlnGly80LeuAsplleLysPro160GlnAlaTyrGlyArg240GlyValPCT/US97/ 18089CA 02264484 1999-03-03W0 98l16655 PCT /US97/1808971260 265 270Ala Val Ala Ala Gly Asp Pro Asn Leu Pro Val Leu Pro Tyr Val Gln275 280 285Ile Phe Tyr Asp Thr Thr Asn His Phe Leu Pro Leu Asp Glu Leu Glu290 295 300His Ser Leu Gly Glu Ser Ala Ala Gln Gly Ala Ala Gly Val Val Leu305 310 315 320Trp Val Ser Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gln Ala Ile325 330 335Lys Glu Tyr Met Asp Thr Thr Leu Gly Pro Phe Ile Leu Asn Val Thr340 345 350Ser Gly Ala Leu Leu Cys Ser Gln Ala Leu Cys Ser Gly His Gly Arg355 360 365Cys Val Arg Arg Thr Ser His Pro Lys Ala Leu Leu Leu Leu Asn Pro370 375 380Ala Ser Phe Ser Ile Gln Leu Thr Pro Gly Gly Gly Pro Leu Ser Leu385 390 395 400Arg Gly Ala Leu Ser Leu Glu Asp Gln Ala Gln Met Ala Val Glu Phe405 410 415Lys Cys Arg Cys Tyr Pro Gly Trp Gln Ala Pro Trp Cys Glu Arg Lys420 425 430Ser Met Trp435(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2517 base pairs(8) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: CDNA(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:TTCCTCCAGG AGTCTCTGGT GCAGCTGGGG TGGAATCTGG CCAGGCCCTG CTTAGGCCCC 60CATCCTGGGG TCAGGAAATT TGGAGGATAA GGCCCTTCAG CCCCAAGGTC AGCAGGGACG 120AGCGGGCAGA CTGGCGGGTG TACAGGAGGG CTGGGTTGAC CTGTCCTTGG TCACTGAGGC 180CATTGGATCT TCCTCCAGTG GCTGCCAGGA TTTCTGGTGG AAGAGACAGG AAGGCCTCCC 240CCCCTTGGTC GGGTCAGCCT GGGGGCTGAG GGCCTGGCTG TCAGCCACTC TTCCCAGAAC 300ATATGTCATG GCCTCAGTGG CTCATGGGGA AGCAGGGGTG GGCGAGCTTA GGCTAGAGCA 360AGTCCTGTGG GAGATGGCAG AGGCCTGGTC TGAGAGGCAA CTCGGATGTG CCCTCCAGTG 420GCCATGCTCC CCTCCATGCG TCTCCCCTGC CCTCCTGGAG CCCTGCAGGT CAATGTTTAA 480CAGAAACCAG AGCAGCGGTG GATTAATGCG CAAGGGCTCA GCCCCCCAGC CCTGAGCAGT 540GGGGGAATCG GAGACTTTGC AACCTGTTCT CAGCTCTGCC TCCCCTGGGC AGGTTGTCCT 600CGACCAGTCC CGTGCCATGG CAGGCCACCT GCTTCCCATC TGCGCCCTCT TCCTGACCTT 660ACTCGATATG GCCCAAGGCT TTAGGGGCCC CTTGGTACCC AACCGGCCCT TCACCACCGT 720W0 98/16655CTGGAATGCA AACACCCAGTCTTCGATGTG GTAGCCAACCTAGCTCCCAG CTGGGCACCTTCTGCCCCAG AATGCCAGCCTGCCATACCT GCTCCTGACTACGCTGGGCC TTCAACTGGGACAGGCACAG CACCCTGATTCCAGGGAGCT GCACGGGCCTTCGCGGCCTC TGGGGCTTCTCAACTACACC GGCCAGTGCCGTGGGGCCAG AGCCGTGCCCAGGGAAGTCA CAGATGTATGTGCTGGTGAC CCCAATCTGCCCACTTTCTG CCCCTGGATGAGCTGGAGTG GTGCTCTGGGCATCAAGGAG TATATGGACACCTTCTCTGC AGTCAAGCCCCCCCAAAGCC CTCCTCCTCCTGGCCCCCTG AGCCTGCGGGGTTCAAATGT CGATGCTACCGTGATTGGCC ACACACTGAGGGGCTTCCTC AAATACATGCGCCACTGTCA CAGGCATATTATAAGGAGTT AGAACCACAGAAGGTCATAG ACAATTCCTCAGGCTGACAT TCACTGAGTGACTTATTCAT TCCTCACAATCACGTTGCCC AAGGTTGCACCTAGCTCCGG GGGTACAGCCACCCCTGAAT CTGCTGAGAGCAGGTGCCTGGACAGGGCAGACACCCCTACTATGATTGCCCATCTCAGGGCTACACCAAGGAGGCCAGCTCCGGATGGCAGGATGGCTTCCCCATCAGGCATTCTATCCCAGTGCAACACCGCGGTGCTGCCAGCTGGAGCATGAGCTGGGACTACACTGGGTGTGCTCCGGTTAACCCTGCGTGCCCTCTCCTGGCTGGCATTGCACATATACAGTCATACCCCTGCACACCAGACACCATCAGAGACACTCCTACTCTTTGAGGCTATGAAGCAAGAAAACTTGCACTCCGCACCAGTCC(2) INFORMATION FOR SEQ ID N0:5:GAGGCACGGTCTTCCGCGGCCACGCCCACTCCTGGCCCGCGGCAGTCATCCATTTACCGGTCAGGTGGAGCACCCTCCAGTGACTGCTACCCGTGCCCAACATCTACATGTGTGGCCGAGCTATGTCCAGCAGCCTGGGGAAATACAAGAGCCCTTCATCCCATGGCCGCCAGTTTCTCCACTTGAAGATGGCACCGTGGTGAGAACCTAAAGTCATGGTACATGCATACTCATTCCTGCGAGCCAGTCTGCCAATCCCCGGAAACTGAGGGGAGAAGTTTACTGAGTTTAGCAAATAAA(1) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(8) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: CDNA02264484 1999-03-0372GTGGACGTGGCCTGACATGAGGGGAGCCTGACATTCCAGGGACTGGGAGGCAGCGCTCACGCAGTAGCCCCTGGGGGGGGAACTATGACTAATGACCAGCCCCGCAGTGCGCATTCCGTGATCTTCTATGGAGAGTGCGGACCAAGGAATCTGAACGTGATGTGTCCGCCATCCAGCTCACAGGCACAGATGTGAGCGGAATGCACTCTGCACAGTAAAGTTACAGACTGTCCATATGCATTGAACTGCAGTGCTAAGCGTCACTCACATGAGATTCAAAGTGGTAACCAGCAGTCATGAATGTCAGTGTCAATTTTCTATGTTTGGTGGACATCCTGGCCATGGCGCCCGGGCACTGGTAGGACCAGTTCACTGCGTCCTTCTAAGCCCTAGGGTGGCTTGGAGGGCACTGGCTGTGGCACACGACAAACCCAGGGGGCCATGTCAGGCCCAGTGGGGCGCACCAGCCACGCCTGGTGGTGGCTGTGGAAGAGCATGTGGGTCTGGCCAAGTACACTCAGAATAGTGGCTCTACTTGGCGCAATCACAATTTTATGTGGTGAGAGTAAGCCCAGGCTGTGCCCTGCACGTTTACTT78084090096010201080114012001260132013801440150015601620168017401800186019201980204021002160222022802340240024602517PCTIUS97/18089CAW0 98/1665573(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:CAGGTTGTCC TGCACCAGTC(2) INFORMATION FOR SEQ ID NO:6:(I) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: CDNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:ATGTGCAACT CAGTGTGTGG C(2) INFORMATION FOR SEQ ID NO:7:(I) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: CDNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:GTGCCATGGC AGGCCACC(2) INFORMATION FOR SEQ ID NO:8:(I) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: CDNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:ATCACCACAT GCTCTTCCGC(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(8) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:Phe Arg Gly Pro Leu Leu Val Pro1 5(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:02264484 1999-03-03PCTIU S97/ 1808920211820W0 98l16655CA02264484 1999-03-03(A) LENGTH: 4 amino acids(3) TYPE: amino acid(D) TOPOLOGY:linear(ii) MOLECULE TYPE: peptide(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:Cys Ala Ala Xaa1MetSerCysPheAsp65IleGlyGlyLysIle145LysValGluLeuTyr225ValThrAlaSerArg305LeuValLeuVal(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 509 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY:linear(xi) SEQUENCE DESCRIPTION: SEQ IDGlyGluLeuLeu50GluAsnTyrIleAsp130AspAspGlnLysLeu210AsnGluAlaAlaLys290IleValIleLeuThrVal Leu Lys Phe Lys His Ile Phe5GlyThr35TrpProAlaTyrPro115IleTrpValLeuAla195ArgHisIleLeuThr275IleValTyrTrpVal20LeuAlaLeuThrPro100GlnThrGluTyrSer180GlyProHisLysTyr260LeuProPheThrGly340Asp Asn355SerAsnTrpAspGly85TyrLysPheGluLys165LeuLysAsnTyrArg245ProTyrAspThrPhe325ThrTyrLeu Ala AlaGlnPheAsnMet70GlnIleIleTyrTrp150AsnThrAspHisLys230AsnSerValAlaAsp310GlyLeuMetLYSIleArgAla55SerGlyAspSerMet135ArgArgGluPheLeu215LysAspIleArgLys295GlnGluSerGluMetValAla40ProLeuValSerLeu120ProProSerAlaLeu200TrpProAspTyrAsn280SerValThrIleThr360CysPhe25ProSerPheThrIle105GlnValThrIleThr185ValGlyGlyLeuLeu265ArgProLeuValMet345IleSer10ThrProGluSerIle90ThrAspAspTrpGlu170GluGluTyrTyrSer250AsnValLeuLysAla330ArgLeuGln74N0:11:Phe Arg SerPheValPhePhe75PheGlyHisAsnAla155LeuLysThrTyrAsn235TrpThrArgProPhe315LeuSerAsnValLeuIleCys60IleTyrValLeuLeu140ArgValAlaIleLeu220GlyLeuGlnGluVal300LeuGlyMetProLeuLeuPro45LeuGlyValThrAsp125GlyAsnGlnLysLys205PheSerTrpGlnAla285PheSerAlaLysTyr365CysPheIle30AsnGlySerAspVal110LysMetTrpGlnGln190LeuProCysAsnSer270IleAlaGlnSerSer350IleGlnVal15ProValLysProArg95AsnAlaAlaLysGln175GluGlyAspPheGlu255ProArgTyrAspGly335CysIleGluLysCysAlaPheArg80LeuGlyLysValPro160AsnPheLysCysAsn240SerValValThrGlu320IleLeuASHGlnPCT/US97/ 18089W0 98/ 16655Gly385AsnValPheVallle465PheSer370Val CysPro AspArg GlyTyr Cys435Lys Asp450Asp AlaTyr Asnlle LeuIteAsnLys420SerThrPheAlaPhe500Arg Lys390Phe Ala405Pro ThrCys TyrAsp AlaLeu Lys470Ser Pro485Leu IleCA375AsnIleLeuSerVal455ProSerIle02264484 1999-03-03Trp AsnGln LeuGlu Asp425Thr Leu440Asp ValPro MetThr LeuSer505SerSerGlu410LeuSerCysGluSer490ValSer395LysGluCysXleThr475AlaAla75380AspGlyGlnLysAla460GluThrSerTyr LeuGly LysPhe Ser430Glu Lys445Asp GlyGlu ProMet PheLeuHisPhe415GluAlaValGlnlle495Leu400ThrLysAspCyslle480ValPCT/U S97/ 18089

Claims (64)

What is claimed is:

1. An isolated antibody characterized by its ability to bind specifically to native human plasma hyaluronidase (hpHAse), wherein the antibody binds to native hpHAse with a binding affinity K a of 10 7 l/mole or more.

2. An antibody that specifically binds to native human acid active plasma hyaluronidase (aaHAse), wherein said antibody is produced by a process comprising the steps of (a) incubating a candidate antibody with a sample comprising native aaHAse, said incubating being at a neutral pH and for a time for formation of antibody-aaHAse complexes;
(b) contacting the antibody-aaHAse complex in the sample with an insoluble support having anti-antibody and detectably-labeled hyaluronic acid bound thereto at a neutral pH and for a time for formation of anti-antibody-candidate antibody-aaHAse complexes; and (c) exposing the anti-antibody-candidate antibody-aaHAse complexes in the sample to an acidic pH of about 3.4 to 3.7, thereby allowing aaHAse in the antibody-aaHAse complex to degrade the detectably labeled hyaluronic acid;
wherein hyaluronic acid degradation indicates the presence of an anti- native aaHAse antibody in the sample.

3. The antibody of claim 2, wherein the native aaHAse is native human plasma hyaluronidase (hpHAse), and wherein the antibody is specific for native acid active hpHAse.

4. The antibody of claim 2 or 3, wherein the antibody binds specifically to native hpHAse with a binding affinity K a of 10 7 l/mole or more.

5. The antibody of any one of claims 1 to 4, wherein the antibody is a monoclonal antibody.

6. A hybridoma cell line 17E9 having ATCC accession number ATCC HB-12213.

7. A hybridoma cell line 4D5 having ATCC accession number ATCC HB-12214.

8. A device for immunopurification of native human plasma hyaluronidase (hpHAse) comprising:
an insoluble support; and an anti-hpHAse antibody characterized by an ability to bind native hpHAse with a binding affinity K a of 10 7 l/mole or more.

9. The device of claim 8, wherein the antibody is characterized by an ability to bind 50% or more of native hpHAse in a liquid flowable sample.

10. The device of claim 8 or 9, wherein a plurality of different antibodies are bound to the support surface and each antibody has a K a of 10 7 l/mole or more relative to native hpHAse.

11. A method of purifying native human plasma hyaluronidase (hpHAse) from a sample, the method comprising:
contacting a sample comprising hpHAse with an anti-hpHAse antibody in the device according to any one of claims 8, 9, and 10, said contacting being for a time for formation of anti-hpHase antibody-hpHAse complexes;
isolating hpHAse from the complexes.

12. A method for identifying a patient having or susceptible to a condition associated with a LuCa-1 defect, the method comprising the steps of:
contacting a sample from the patient with an anti-native hpHAse antibody, the sample being selected from the group consisting of tissue, blood, plasma, serum, and urine, said contacting being for a time for formation of anti-native hpHAse antibody-hpHAse complexes;

detecting the amount of native hpHAse present in the anti-native hpHAse antibody-hpHAse complexes in the sample; and comparing the amount of native hpHAse detected in the sample with an amount of native hpHAse in a control sample;
wherein detection of an amount of native hpHAse in the patient sample that is less than the amount of native hpHAse in the control sample is indicative of a LuCa-1 defect in the patient.

13. A method of purifying a native human acid active hyaluronidase (aaHAse) from a sample, the method comprising the steps of:
(a) dissolving a sample suspected of containing native aaHAse in a solution at a temperature less than room temperature, the solution comprising a non-ionic detergent;
(b) raising the temperature of the solution to a temperature above 25°C, said raising resulting in the formation of a detergent-rich phase comprising native aaHAse and a detergent-poor phase; and (c) isolating native aaHAse from the detergent-rich phase.

14. The method of claim 13, wherein steps (a)-(c) are repeated twice.

15. An expression system for production of recombinant hpHAse, the system comprising a transformed cell containing a nucleic acid construct comprising hpHAse-encoding nucleic acid operably linked to a eukaryotic promoter.

16. A formulation for administration of human plasma hyaluronidase (hpHAse) to a patient having a condition associated with a LuCa-1 gene defect comprising:
(a) a therapeutically effective amount of a substantially pure recombinant human plasma hyaluronidase polypeptide that is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated, wherein said polypeptide is glycosylated, and wherein said hpHAse polypeptide partitions into a non-ionic detergent-rich phase at a temperature above 25°C; and (b) a pharmaceutically acceptable carrier.

17. The formulation of claim 16, wherein the carrier comprises a liposome.

18. A pharmaceutical composition comprising a therapeutically effective amount of. i) a substantially pure recombinant human plasma hyaluronidase polypeptide that is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated, wherein said polypeptide is glycosylated, and wherein said hpHAse polypeptide partitions into a non-ionic detergent-rich phase at a temperature above 25°C; and ii) a pharmaceutically acceptable carrier, for use in treatment of a condition associated with a LuCa-1 gene defect.

19. The pharmaceutical composition of claim 18, wherein the carrier comprises a liposome.

20. Use of a pharmaceutical composition comprising: i) a therapeutically effective amount of a substantially pure recombinant human plasma hyaluronidase polypeptide that is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated, wherein said polypeptide is glycosylated, and wherein said hpHAse polypeptide partitions into a non-ionic detergent-rich phase at a temperature above 25°C; and ii) a pharmaceutically acceptable carrier, in treatment of a condition associated with a LuCa-1 gene defect.

21. The use according to claim 20, wherein the carrier comprises a liposome.

22. Use of a substantially pure, enzymatically active human plasma hyaluronidase (hpHAse) that is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated, wherein said polypeptide is glycosylated, and wherein said hpHAse polypeptide partitions into a non-ionic detergent-rich phase at a temperature above 25°C, for treatment of cancer associated with a LuCa-1 defect.

23. Use of substantially pure, enzymatically active human plasma hyaluronidase (hpHAse) that is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated, wherein said polypeptide is glycosylated, and wherein said hpHAse polypeptide partitions into a non-ionic detergent-rich phase at a temperature above 25°C, for preparation of a medicament for treatment of cancer associated with it LuCa-1 defect.

24. The use according to claim 22 or 23, wherein the hpHAse is for administration by peritumoral injection.

25. The use according to claim 22 or 23, wherein the hpHAse is for administration by intratumoral injection.

26. Use of a construct comprising a nucleotide sequence encoding a human plasma hyaluronidase polypeptide and a eukaryotic promoter sequence operably linked thereto for the treatment of cancer associated with a defective LuCa-1 gene.

27. Use of a construct comprising a nucleotide sequence encoding a human plasma hyaluronidase polypeptide and a eukaryotic promoter sequence operably linked thereto for preparation of a medicament for treatment of cancer associated with a defective LuCa-1 gene.

28. The use according to any one of claims 22 to 27, wherein the cancer is a metastatic cancer.

29. A substantially pure, enzymatically active human plasma hyaluronidase (hpHAse) that is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated, wherein said polypeptide is glycosylated, and wherein said bpHAse polypeptide partitions into a non-ionic detergent-rich phase at a temperature above 25°C, for use in preparation of a medicament for treatment of cancer associated with a LuCa-1 defect.

30. The human plasma hyaluronidase (hpHAse) of claim 29, wherein the hpHAse is formulated for administration by peritumoral administration.

31. The human plasma hyaluronidase (hpHAse) of claim 29, wherein the hpHAse is formulated for administration by intratumoral administration.

32. The human plasma hyaluronidase (hpHAse) according to any one of claims 29, 30, and 31, wherein the cancer is a metastatic cancer.

33. A construct comprising a nucleotide sequence encoding a human plasma hyaluronidase polypeptide and a eukaryotic promoter sequence operably linked thereto for use in treatment of cancer associated with a LuCa-1 defect.

34. A construct comprising a nucleotide sequence encoding a human plasma hyaluronidase polypeptide and a eukaryotic promoter sequence operably linked thereto for use in preparation of a medicament for treatment of cancer associated with a LuCa-1 defect.

35. The construct of claim 33 or 34, wherein the cancer is a metastatic cancer.

36. A method of making a recombinant native human plasma hyaluronidase polypeptide, the method comprising the steps of:
introducing into a host cell a nucleotide sequence encoding a human plasma hyaluronidase polypeptide;
culturing the host cell so as to allow for expression of the polypeptide from the introduced sequence; and isolating human plasma hyaluronidase polypeptide.

37. The isolated recombinant native human plasma hyaluronidase polypeptide produced by the method of claim 36.

38. A substantially pure, enzymatically active, human plasma hyaluronidase (hpHAse) polypeptide, wherein said polypeptide is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated, wherein said polypeptide is glycosylated, and wherein said hpHAse polypeptide partitions into a non-ionic detergent-rich phase at a temperature above 25°C.

39. The polypeptide of claim 38, wherein said polypeptide is sensitive to N-glycosidase-F treatment.

40. The polypeptide of claim 38 or 39, wherein said polypeptide comprises a mannose residue.

41. The polypeptide of any one of claims 38, 39, and 40, wherein said polypeptide further comprises a fatty acid modification.

42. The polypeptide of claim 41, wherein said fatty acid modification is resistant to phospholipase-C, phospholipase-D, and N-glycosidase-F.

43. The polypeptide of any one of claims 38 to 42, wherein said polypeptide exhibits a specific activity of at least 6 x 10 5 relative turbidity reducing units per mg protein.

44. The polypeptide of any one of claims 38 to 42, wherein said polypeptide exhibits a specific activity of at least 2 x 10 5 relative turbidity reducing units per mg protein.

45. The polypeptide of any one of claims 38 to 44, wherein said polypeptide has a relative molecular mass of about 57 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

46. The polypeptide of any one of claims 38 to 45, wherein the polypeptide is at least 75% pure.

47. The polypeptide of any one of claims 38 to 45, wherein the polypeptide is at least 90% pure.

48. The polypeptide of any one of claims 38 to 45, wherein the polypeptide is at least 99% pure.

49. The polypeptide of any one of claims 38 to 48, wherein the polypeptide is recombinant.

50. The polypeptide of any one of claims 38 to 48, wherein the polypeptide is naturally occurring.

51. A formulation comprising:
(a) a therapeutically effective amount of a substantially pure, enzymatically active, human plasma hyaluronidase polypeptide, wherein said polypeptide is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated, wherein said polypeptide is glycosylated, and wherein said polypeptide partitions into a non-ionic detergent-rich phase at a temperature above 25°C; and (b) a pharmaceutically acceptable carrier.

52. The formulation of claim 51, wherein said polypeptide exhibits a specific activity of at least 2 x 10 5 relative turbidity reducing units per mg protein.

53. The formulation of claim 51, wherein said polypeptide exhibits a specific activity of at least 6 x 10 5 relative turbidity reducing units per mg protein.

54. The formulation of any one of claims 51, 52, and 53, wherein said polypeptide is present at a concentration of about 1.5 x 10 5 turbidity reducing units per milliliter of formulation.

55. The formulation of any one of claims 51 to 54, wherein said polypeptide is sensitive to N-glycosidase-F treatment.

56. The formulation of any one of claims 51 to 55, wherein said polypeptide comprises a mannose residue.

57. The formulation of any one of claims 51 to 56, wherein said polypeptide further comprises a fatty acid modification.

58. The formulation of claim 57, wherein said fatty acid modification is resistant to phospholipase-C, phospholipase-D, and N-glycosidase-F.

59. The formulation of any one of claims 51 to 58, wherein said polypeptide has a relative molecular mass of about 57 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

60. The formulation of any one of claims 51 to 59, wherein the polypeptide is at least 75% pure.

61. The formulation of any one of claims 51 to 59, wherein the polypeptide is at least 90% pure.

62. The formulation of any one of claims 51 to 59, wherein the polypeptide is at least 99% pure.

63. The formulation of any one of claims 51 to 62, wherein the polypeptide is recombinant.

64. The formulation of any one of claims 51 to 62, wherein the polypeptide is naturally occurring.