HUE033055T2 - Kombinációk és eljárások immunglobulin és hialuronidáz szubkután beadására - Google Patents

Description

(12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 391395 <2m01> A61K 38147 <200601> 03.05.2017 Bulletin 2017/18 C07K16106<200e 01> C12N 9I26<2006 01> A61P 37100<200e 01> (21) Application number: 09721669.1 (86) International application number: (22) Date of filing: 16.03.2009 PCT/US2009/001670 (87) International publication number: WO 2009/117085 (24.09.2009 Gazette 2009/39)

(54) COMBINATIONS AND METHODS FOR SUBCUTANEOUS ADMINISTRATION OF IMMUNE GLOBULIN AND HYALURONIDASE

KOMBINATIONEN UND VERFAHREN ZUR SUBKUTANEN VERABREICHUNG VON IMMUNGLOBULIN UND HYALURONIDASE

COMBINAISONS ET PROCEDES POUR L’ADMINISTRATION SOUS-CUTANEE DE GAMMAGLOBULINE ET D’HYALURONIDASE (84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR WO-A1-2006/091871

HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR · MELAMED I R ET AL: "Recombinant human

Designated Extension States: hyaluronidase facilitates dispersion of AL BA RS subcutaneously administered Gammagard liquid and enables administration of afull monthly bose (30) Priority: 17.03.2008 US 69841 P in a single site to patients with immunodeficiency

diseases" JOURNAL OF ALLERGY AND (43) Date of publication of application: CLINICAL IMMUNOLOGY, vol. 121, no. 2, Suppl. 12.01.2011 Bulletin 2011/02 1, February 2008 (2008-02), page S83,

XP002541721 &amp; 64TH ANNUAL MEETING OF THE (73) Proprietors: AMERICAN-ACADEMY-OF-ALLERGY-ASTHMA- • Baxalta Incorporated AND-IMM UNOLOGY; PHILADELPHIA, PA, USA;

Bannockburn, IL 60015 (US) MARCH 14 -18, 2008 ISSN: 0091-6749 • Baxalta GmbH · BOOKBINDER LHETAL: "A recombinant human 8152 Glattpark, Opfikon (CH) enzyme for enhanced interstitial transport of

therapeutics" JOURNAL OF CONTROLLED (72) Inventors: RELEASE, ELSEVIER, AMSTERDAM, NL, vol. • SCHIFF, Richard 114, no. 2, 28 August 2006 (2006-08-28), pages

Eastsound WA 98245 (US) 230-241,XP024957594ISSN:0168-3659 [retrieved • LEIBL, Heinz on 2006-08-28] A-1120 Vienna (AT) • FROST, Gregory, I.

Del Mar, CA 92014 (US) (74) Representative: Boult Wade Tennant Verulam Gardens 70 Gray’s Inn Road London WC1X8BT (GB) • ANN GARDULFETAL: "Rapid Subcutaneous IgG · JOSEPH A CHURCH ETAL: "Efficacy, Safety and

Replacement Therapy is Effective and Safe in Tolerability of a New 10% Liquid Intravenous

Children and Adults with Primary Immune Globulin [IGIV 10%] in Patients with

Immunodeficiencies-A Prospective, Primary Immunodeficiency" JOURNAL OF

Multi-National Study" JOURNAL OF CLINICAL CLINICAL IMMUNOLOGY, KLUWER ACADEMIC IMMUNOLOGY, KLUWER ACADEMIC PUBLISHERS-PLENUM PUBLISHERS, NE, vol. PUBLISHERS-PLENUM PUBLISHERS, NE, vol. 26, no. 4, 17 May 2006 (2006-05-17), pages 26, no. 2, 26 April 2006 (2006-04-26), pages 388-395, XP019400139 ISSN: 1573-2592 177-185, XP019400116 ISSN: 1573-2592 cited in · ANDERSON ET AL: "Guidelines on the Use of the application Intravenous Immune Globulin for Hematologic

• HANS D OCHS ET AL: "Safety and Efficacy of Conditions", TRANSFUSION MEDICINE

Self-Administered Subcutaneous REVIEWS, GRUNE AND STRATTON, ORLANDO,

Immunoglobulin in Patients with Primary FL, US, vol. 21,28 March 2007 (2007-03-28), pages

Immunodeficiency Diseases" JOURNAL OF S9-S56, XP005937815, ISSN: 0887-7963, DOI: CLINICAL IMMUNOLOGY, KLUWER ACADEMIC 10.1016/J.TMRV.2007.01.001 PUBLISHERS-PLENUM PUBLISHERS, NE, vol. · FEASBY ET AL: "Guidelines on the Use of 26,no.3,2June2006(2006-06-02),pages265-273, Intravenous Immune Globulin for Neurologic

XP019400135 ISSN: 1573-2592 cited in the Conditions", TRANSFUSION MEDICINE application REVIEWS, GRUNE AND STRATTON, ORLANDO, • LEESCH VW ET AL: "30-Day Pharmacokinetic FL, US, vol. 21,28 March 2007 (2007-03-28), pages

Evaluation of IV versus Subcutaneous S57-S107, XP005937816, ISSN: 0887-7963, DOI:

Administration of Immunoglobulin with and 10.1016/J.TMRV.2007.01.002 without Recombinant Human Hyaluronidase in · PRINS CHRISTA ET AL: "Intravenous

Dogs" JOURNAL OF ALLERGY AND CLINICAL immunoglobulin: properties, mode of action and

IMMUNOLOGY, vol.123, no. 2, Suppl.S, February practical use in dermatology.", ACTA 2009 (2009-02), page S10, XP002541722 &amp; 65TH DERMATO-VENEREOLOGICA 2007, vol. 87, no. ANNUAL MEETING OF THE 3, 2007, pages 206-218, ISSN: 0001-5555 AMERICAN-ACADEMY-OF-ALLERGY-ASTHMA- AND-IMM UNOLOGY; WASHINGTON, DC, USA; MARCH 13 -17, 2009 ISSN: 0091-6749 • Anonymous: "GAMMAGARD LIQUID [Immune Globulin Intravenous (Human)] 10%" Baxter April 2005 (2005-04), pages 1-4, XP002542175 Retrieved from the Internet: URL: http://www.baxter.com/products/biophar maceuticals/downloads/gamliquid_Pl.pdf> [retrieved on 2009-08-20]

Description

FIELD OF THE INVENTION

[0001] Provided are combinations containing an immune globulin (IG) composition and a soluble hyaluronidase composition formulated for subcutaneous administration. Such products are for use in methods of treating IG-treatable diseases or conditions.

BACKGROUND

[0002] The intravenous (IV) administration of immune globulin (IVIG) is the primary treatment of individuals with immune deficiencies. Although the initial IVIG preparations caused severe side effects, the IVIG preparations available at the present time are well tolerated in the majority of immune deficient patients. Nonetheless, a small proportion of patients continue to have unpleasant, even disabling, reactions such as headache, fatigue, and myalgia. Fever and chills remains a problem, especially when patients have intercurrent infections. The reactions often persist despite trying other IVIG preparations or pre-medicating with acetaminophen, diphenhydramine, and corticosteroids. Further, due to the requirement for IV administration, there are issues with patient compliance.

[0003] Subcutaneous (SQ) administration of immune globulin is an alternative to intravenous administration. Compared to IV infusions, SQ administration of immune globulin has several advantages. For example, it reduces the incidence of systemic reactions, does not require sometimes-difficult IV access, improves trough levels, and gives patients more independence. Because of the difficulty in administering large quantities of fluid in a single site, it is necessary to do SQ infusions once or twice a week, using two to as many as 5 sites at a time. Thus, unlike IVIG, which is given once a month, subcutaneous administration is usually done weekly. Hence, there is a need for alternative methods for administering immune globulin.

[0004] Melamed IR et al (Journal of Allergy and Clinical Immunology, vol. 121, no. 2, Suppl. 1, February 2008, page S83, and 64th Annual Meeting of the American Academy of Allergy Asthma and Immunology; Philadelphia, PA, USA, March 14-18,2008) describes subcutaneous infusion of Gammagard™ and hyaluronidase into immunodeficient subjects.

SUMMARY

[0005] Provided herein are combinations for subcutaneous administration of immune globulin and for use in treating IG-treatable diseases or conditions.

[0006] In particular, provided herein is a combination of compositions of immune globulin (IG) and a soluble hyaluronidase for use for treating an IG-treatable disease or condition in a subject, wherein: the IG and soluble hyaluronidase are formulated separately for subcutaneous administration; the IG and soluble hyaluronidase are formulated for single dosage administration once a month; the soluble hyaluronidase is administered separately from the IG prior to administration of the IG and at the same site as the IG; the concentration of IG is 5 to 15% w/v and the amount of IG in the composition is 0.5 grams (g) to 70 g; the IG is formulated in a volume of liquid that is 50 mL to 700 mL; the soluble hyaluronidase is formulated for administration as a ratio of hyaluronidase to IG of 10 U/gram (g) to 500 U/g of IG, whereby the amount of hyaluronidase in the composition is sufficient to effect increased bioavailability of the IG when administered in combination with the IG to at least 90% of the bioavailability of the same single dosage of IG administered via intravenous administration for treatment of the same IG-treatable disease or condition; the soluble hyaluronidase is formulated in a volume of liquid that is 5 to 30 mL; and the IG-treatable disease or condition is selected from among immunodeficiency; acquired hypogammaglobulinemia secondary to hematological malignancies; Kawasaki’s disease; chronic inflammatory demyelinating polyneuropathy (CIDP); Guillain-Barre Syndrome; Idiopathic thrombocytopenic purpura; inflammatory myopathies; Lambert-Eaton myasthenic syndrome; multifocal motor neuropathy; Myasthenia Gravis; Moersch-Woltmann syndrome; secondary hypogammaglobulinaemia, including iatrogenic immunodeficiency; specific antibody deficiency; Acute disseminated encephalomyelitis; ANCA-positive systemic necrotizing vasculitis; Autoimmune haemolytic anaemia; Bullous pemphigoid; Cicatricial pemphigoid; Evans syndrome, including autoimmune haemolytic anaemia with immune thrombocytopenia; Foeto-maternal/neonatal alloimmune thrombocytopenia (FMAIT/NAIT); Haemophagocytic syndrome; High-risk allogeneic haemopoietic stem cell transplantation; IgM paraproteinaemic neuropathy; kidney transplantation; multiple sclerosis; Opsoclonus myoclonus ataxia; Pemphigus foliaceus; Pemphigus vulgaris; Post-transfusion purpura; Toxic epidermal necrolysis/Steven Johnson syndrome (TEN/SJS); Toxic shock syndrome; Alzheimer’s Disease; Systemic lupus erythematosus; multiple myeloma; sepsis; B cell tumors; trauma; and a bacterial, viral or fungal infection.

[0007] Also described are methods for treating an IG-treatable disease or condition in a subject by subcutaneously administering to the subject soluble hyaluronidase and an immune globulin (IG) for treating the disease or condition. Co-administration of IG and hyaluronidase increases the bioavailability of subcutaneously administered IG to permit administration of IG subcutaneously using a dosing regime substantially the same as intravenous IG (IVIG) administration for the particular disease or condition. The administration of the IG is effected such that the amount administered and frequency of administration is substantially the same as for IV (intravenous) administration of the same amount via IV for the same disease or condition. The amount administered via IV can be predetermined or is known for a particular disease or condition. Typically, in the presence of a soluble hyaluronidase, for subcutaneous administration, the rate of administration can be greater than for IV administration. Hence, the time for administration of a particular dose can be reduced. Rate of administration can be controlled, such as by a pump, or can rely on gravity.

[0008] Thus, among the methods described are methods for treating an IG-treatable disease or condition in a subject in need of such treatment by subcutaneously administering to the subject a soluble hyaluronidase and an amount of immune globulin (IG) effective for treating the disease or condition. The administration is performed with a dosage regimen in which: (a) a quantity of IG; and (b) a dosing frequency for successive administrations of IG to the subject are selected such that the therapeutic effect of the subcutaneous IG administration upon the subject is at least substantially equivalent to intravenous administration of the IG to the subject using the same dosing regimen.

[0009] In some examples of the methods described herein, bioavailability of the subcutaneously administered IG is at least about 90% of the bioavailability of the same dosage administered via IV administration. The amount of soluble hyaluronidase administered can be sufficient to effect subcutaneous administration of the IG at a dosage administered no more than once a month. In other examples, administration of IG is no more than once monthly.

[0010] The soluble hyaluronidase used in the methods and uses described herein can be PH20 or a truncated form thereof. For example, the soluble hyaluronidase can be an ovine or bovine or truncated human PH20. In instances where a truncated human PH20 is used in the methods and uses described herein, the truncated human PH20 can be selected from among polypeptides having a sequence of amino acids set forth in any of SEQ ID NOS:4-9, or allelic variants or other variants thereof. In one example, the soluble hyaluronidase is rHuPH20.

[0011] The IG administered using the methods and uses herein can be purified from human plasma, such as by alcohol fractionation. In some examples, the IG is further purified by any one or more of a chemical modification, incubation at pH 4.0 with or without pepsin, polyethylene glycol (PEG) precipitation, ion-exchange chromatography, enzymatic cleavage, solvent detergent treatment, diafiltration or ultrafiltration. The methods and uses described herein can employ IG that contains IgG, IgA and IgM. In some examples, the IG contains greater than 95% IgG. Further, the IgG can be monomeric. Protein-stabilizing excipients, such as one or more of glycine, maltose, a polyol, human serum albumin, mannitol, and non-ionic detergent, also can be present in the IG. In some examples, the pH of the IG preparation is at or about 4.2 to 5.4, 4.6 to 5.1 or 4.8 to 5.0, and the protein concentration is or is about 5 to 15% w/v, 6 to 15% w/v, or 8 to 12% w/v of IG composition. In one example, the protein concentration of the IG is 10% w/v.

[0012] Described herein are methods and uses for treating an IG-treatable disease or condition, in which soluble hyaluronidase and an immune globulin (IG) for treating the disease or condition are subcutaneously administered, and the IG is infused at a rate of 10 ml/hr to 300 ml/hr, such as at or about 10 ml/hr, 20 ml/hr, 30 ml/hr, 40 ml/hr, 50 ml/hr, 60 ml/hr, 70 ml/hr, 80 ml/hr, 90 ml/hr, 100 ml/hr, 150 ml/hr, 200 ml/hr, 250 ml/hr and 300 ml/hr. The rate can be controlled by a pump or gravity. In some examples, the IG and hyaluronidase are administered separately, simultaneously or intermittently. For example, the hyaluronidase can be administered prior to administration of IG, such as 0.5 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes or 30 minutes prior to administration of IG. In other examples, the IG and hyaluronidase are in a single composition. In further examples, about or 5 grams (g), 10g, 15g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g, 31g, 32g, 33g, 34g, 35g, 36g, 37g, 38g, 39g or 40g of IG is administered, and the hyaluronidase is administered at a ratio (units hyaluronidase/grams of IG) at or about 10 U/ gram (g), 20 U/g, 30 U/g, 35 U/g, 40 U/g, 50 U/g, 60 U/g, 70 U/g, 80 U/g, 90 U/g, 100 U/g, 150 U/g, or 300 U/g.

[0013] Soluble hyaluronidase and IG can be administered using the methods and uses described herein to treat, for example, immunodeficiency; acquired hypogammaglobulinemia secondary to hematological malignancies; Kawasaki’s disease; chronic inflammatory demyelinating polyneuropathy (CIDP); Guillain-Barre Syndrome; Idiopathic thrombocytopenic purpura; inflammatory myopathies; Lambert-Eaton myasthenic syndrome; multifocal motor neuropathy; Myasthenia Gravis; Moersch-Woltmann syndrome; secondary hypogammaglobulinaemia (including iatrogenic immunodeficiency); specific antibody deficiency; Acute disseminated encephalomyelitis; ANCA-positive systemic necrotizing vasculitis; Autoimmune haemolytic anaemia; Bullous pemphigoid; Cicatricial pemphigoid; Evans syndrome (including autoimmune haemolytic anaemia with immune thrombocytopenia); Foeto-maternal/neonatal alloimmune thrombocytopenia (FMAIT/NAIT); Haemophagocytic syndrome; High-risk allogeneic haemopoietic stem cell transplantation; IgM parapro-teinaemic neuropathy; kidney transplantation; multiple sclerosis; Opsoclonus myoclonus ataxia; Pemphigus foliaceus;

Pemphigus vulgaris; Post-transfusion purpura; Toxic epidermal necrolysis/Steven Johnson syndrome (TEN/SJS); Toxic shock syndrome; Alzheimer’s Disease; Systemic lupus erythrematosus; multiple myeloma; sepsis; B cell tumors; trauma; and a bacterial, viral orfugal infection. In instances where the IG and hyaluronidase are administered to treat an immunodeficiency, the immunodeficiency can be selected from among common variable immunodeficiency (CVID), congenital agammaglobulinemia, Wiskott-Aldrich syndrome, severe combined immunodeficiency (SCID), primary hypogammaglobulinemia, primary immunodeficiency diseases with antibody deficiency, X-linked agammaglobulinemia (XLA), hypogammaglobulinemia of infancy, and paraneoplastic cerebellar degeneration with no antibodies.

[0014] In instances where the IG-treatable disease or condition is acquired hypogammaglobulinemia secondary to hematological malignancies, and the hematological malignancy can be chronic lymphocytic leukemia (CLL), multiple myeloma (MM) or non-Hodgkin’s lymphoma (NHL). In instances where the IG-treatable disease or condition is an inflammatory myopathy, the inflammatory myopathy can be polymyositis, dermatomyositis or inclusion body myositis.

[0015] In some examples, soluble hyaluronidase and IG is administered subcutaneously to treat a bacteria, viral or fungal condition, such as, for example, Haemophilus influenzae type B, Psuedomonas aeruginosa types A and B, Staphylococcus aureus, Group B Streptococcus, Streptococcus pneumoniae types 1,3, 4, 6, 7, 8, 9,12,14,18,19, and 23, Adenovirus types 2 and 5, Cytomegalovirus, Epstein Barr virus VCA, Hepatitis A virus, Hepatitis B virus, Herpes simplex virus-1, Herpes simplex virus-2, Influenza A, Measles, Parainfluenza types 1,2 and 3, Polio, Varicella zoster virus, Apergillus and Candida albicans.

[0016] Provided herein are combinations of compositions for use in treating an IG-treatable disease or condition in a subject, containing a first composition containing IG formulated for subcutaneous single dosage administration no more than once per month, and a second composition containing a soluble hyaluronidase formulated for single dosage administration no more than once per month. The compositions can be in a dual chamber container or in single container separated from each other. In some examples, the hyaluronidase is positioned in the container to be administered before the IG. The container can be a syringe, tube or bottle, and can further contain a needle for injection.

[0017] The combinations of compositions provided herein can contain PH20, ora truncated form thereof. For example, ovine, bovine or truncated human PH20, such as a polypeptide having a sequence of amino acids set forth in any of SEQ ID NOS:4-9, or allelic variants or other variants thereof, can be included in the combinations of compositions provided herein. In some examples, the soluble hyaluronidase in the combination is rHuPH20. Further, the IG in the combinations of compositions can be purified from human plasma, and can be lyophilized or a liquid.

[0018] In some examples, the volume of liquid in the combinations of compositions provided herein is or is about 100 ml, 150 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml or 700 ml. The IG in the combinations of compositions can have a protein concentration that is or is about 5 to 15% w/v, 6 to 15% w/v, or 8 to 12% w/v of IG composition, such as, for example, 10% w/v. In some examples, the IG in the composition is or is about 5 grams (g), 10g, 15g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g, 31g, 32g, 33g, 34g, 35g, 36g, 37g, 38g, 39g or40g. The hyaluronidase can be a liquid. In some examples, the volume of the hyaluronidase liquid is or is about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml or 30 ml, and the hyaluronidase is at or about 10 Units to 500,000 Units, 100 Units to 100,000 Units, 500 Units to 50,000 Units, 1000 Units to 10,000 Units, 5000 Units to 7500 Units, 5000 Units to 50,000 Units, or 1,000 Units to 10,000 Units.

[0019] Described herein are compositions containing immune globulin (IG) and a soluble hyaluronidase formulated for single dosage administration once a month. The soluble hyaluronidase contained in the composition can be PH20, or a truncated form thereof. For example, ovine, bovine or truncated human PH20, such as a polypeptide having a sequence of amino acids set forth in any of SEQ ID NOS:4-9, or allelic variants or other variants thereof, can be included in the com positions formulated for single dosage administration provided herein. In some examples, the soluble hyaluronidase in the composition is rHuPH20. The IG in the compositions can be purified from human plasma, and can be a liquid.

[0020] In exemplary embodiments, the IG in the compositions formulated for single dosage administration provided herein has a protein concentration that is or is about 5 to 15% w/v, 6 to 15% w/v, or 8 to 12% w/v of IG composition, such as, for example, 10% w/v. The IG in the composition is or is about 5 grams (g), 10g, 15g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g, 31g, 32g, 33g, 34g, 35g, 36g, 37g, 38g, 39g or40g, and the hyaluronidase is at or about 10 Units to 500,000 Units, 100 Units to 100,000 Units, 500 Units to 50,000 Units, 1000 Units to 10,000 Units, 5000 Units to 7500 Units, 5000 Units to 50,000 Units, or 1,000 Units to 10,000 Units. The volume of liquid in the composition can be at or about 100 ml, 150 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml or 700 ml.

[0021] Described herein are kits containing combinations of compositions, containing a first composition containing IG formulated for subcutaneous single dosage administration no more than once per month, and a second composition containing a soluble hyaluronidase formulated for single dosage administration no more than once per month. Also described herein are compositions containing immune globulin (IG) and a soluble hyaluronidase formulated for single dosage administration once a month. Optionally, instructions can be included in the kits. DETAILED DESCRIPTION Outline [0022] A. Definitions B. Subcutaneous Administration of Immune Globulin (IG) C. Immune Globulin D. Hyaluronidase

Soluble Hyaluronidase Soluble Human PH20

Soluble Recombinant Human PH20 (rHuPH20) E. Methods of Producing Nucleic Acids encoding a soluble Hyaluronidase and Polypeptides Thereof 1. Vectors and Cells 2. Expression a. Prokaryotic Cells b. Yeast Cells c. Insect Cells d. Mammalian Cells e. Plants 3. Purification Techniques F. Preparation, Formulation and Administration of Immune Globulins and Soluble Hyaluronidase Polypeptides 1. Formulations Lyophilized powders 2. Dosage and Administration G. Methods of Assessing Activity, Bioavailability and Pharmacokinetics 1. Pharmacokinetics and tolerability 2. Biological Activity a. Immune globulin b. Hyaluronidase H. Therapeutic Uses 1. Primary immune deficiency with antibody deficiency 2. Acquired hypogammaglobulinemia secondary to hematological malignancies 3. Kawasaki’s disease 4. Chronic inflammatory demyelinating polyneuropathy 5. Guillain-Barre Syndrome 6. Idiopathic thrombocytopenic purpura 7. Inflammatory myopathies: polymyositis, dermatomyositis and inclusion body myositis 8. Lambert-Eaton myasthenic syndrome 9. Multifocal motor neuropathy 10. Myasthenia Gravis 11. Moersch-Woltmann syndrome 12. Alzheimer’s Disease 13. Other diseases and conditions I. Articles of manufacture and kits J. Examples

A. DEFINITIONS

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0024] As used herein, "immunoglobulin," "immune globulin,""gamma globulin" refer to preparations of plasma proteins derived from the pooled plasma of adult donors. IgG antibodies predominate; other antibody subclasses, such as IgA and IgM are present. Therapeutic immune globulin can provide passive immunization by increasing a recipient’s serum levels of circulating antibodies. IgG antibodies can, for example, bind to and neutralize bacterial toxins; opsonize pathogens; activate complement; and suppress pathogenic cytokines and phagocytes through interaction with cytokines and receptors thereof, such as CD5, interleukin-la (IL-1 a), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and T-cell receptors. Therapeutic immune globulin can inhibit the activity of autoantibodies. Immune globulin preparations also include, but are not limited to, immune globulin intravenous (IGIV), immune globulin IV, therapeutic immunoglobulin. Immune globulin preparation are well known, and include brand names, such as BayGam®, Gamimune® N, Gammagard® S/D, Gammar®-P, Iveegam® EN, Panglobulin®, Polygam® S/D, Sandoglobulin®, Venoglobulin®-!, Venoglobulin®-S, WinRho® SDF and others. Immune globulin preparations can be derived from human plasma, or are recombinantly produced.

[0025] As used herein, IG-treatable diseases or conditions refer to any disease or condition for which immune globulin preparations are used. Such diseases and conditions, include, but are not limited to, any disease in which an increase in circulating antibodies is ameliorative, such as, for example, immunodeficiency; acquired hypogammaglobulinemia secondary to hematological malignancies; Kawasaki’s disease; chronic inflammatory demyelinating polyneuropathy (CIDP); Guillain-Barre Syndrome; Idiopathic thrombocytopenic purpura; inflammatory myopathies; Lambert-Eaton myasthenic syndrome; multifocal motor neuropathy; Myasthenia Gravis; Moersch-Woltmann syndrome; secondary hy-pogammaglobulinaemia (including iatrogenic immunodeficiency); specific antibody deficiency; Acute disseminated encephalomyelitis; ANCA-positive systemic necrotizing vasculitis; Autoimmune haemolytic anaemia; Bullous pemphigoid; Cicatricial pemphigoid; Evans syndrome (including autoimmune haemolytic anaemia with immune thrombocytopenia); Foeto-maternal/neonatal alloimmune thrombocytopenia (FMAIT/NAIT); Haemophagocytic syndrome; High-risk allogeneic haemopoieticstem cell transplantation; IgM paraproteinaemic neuropathy; kidney transplantation; multiple sclerosis; Opsoclonus myoclonus ataxia; Pemphigus foliaceus; Pemphigus vulgaris; Post-transfusion purpura; Toxic epidermal necrolysis/Steven Johnson syndrome (TEN/SJS); Toxic shock syndrome; Alzheimer’s Disease; Systemic lupus erythematosus; multiple myeloma; sepsis; B cell tumors; trauma; and a bacterial viral orfugal infection.

[0026] As used herein, dosing regime refers to the amount of immune globulin administered and the frequency of administration. The dosing regime is a function of the disease or condition to be treated, and thus can vary.

[0027] As used herein, "substantially the same as an intravenous IG (IVIG) dosing regime" refers to a regimen in which the dose and/or frequency is within an amount that is effective for treating a particular disease or condition, typically is about or 10%, of the IV dose or frequency. Amounts of IVIG that are effective for treating a particular disease or condition are known or can be empirically determined by one of skill in the art. For example, as exemplified below, 300 mg/kg (i.e. 21 grams assuming the average adult weighs 70 kg) to 600 mg/kg (i.e. 42 grams) is the typical monthly dose of IVIG administered to patients having primary immunodeficiency diseases. Hence, IG, when administered in combination with hyaluronidase, is administered subcutaneously at doses that are or are about 300 mg/kg to 600 mg/kg for treatment of primary immunodeficiency diseases.

[0028] As used herein, frequency of administration refers to the time between successive doses of immune globulin. For example, frequency can be one, two, three, four weeks, and is a function of the particular disease or condition treated. Generally, frequency is a least every two or three weeks, and typically no more than once a month.

[0029] As used herein, hyaluronidase refers to an enzyme that degrades hyaluronic acid. Hyaluronidases include bacterial hyaluronidases (EC 4.2.99.1), hyaluronidases from leeches, other parasites, and crustaceans (EC 3.2.1.36), and mammalian-type hyaluronidases (EC 3.2.1.35). Hyaluronidases also include any of non-human origin including, but not limited to, murine, canine, feline, leporine, avian, bovine, ovine, porcine, equine, piscine, ranine, bacterial, and any from leeches, other parasites, and crustaceans. Exemplary non-human hyaluronidases include, hyaluronidases from cows (SEQ ID NO:10 and 11), yellow jacket wasp (SEQ ID NOS:12 and 13), honey bee (SEQ ID NO:14), white-face hornet (SEQ ID NO:15), paper wasp (SEQ ID NO:16), mouse (SEQ ID NOS:17-19, 32), pig (SEQ ID NOS:20-21), rat (SEQ ID NOS:22-24,31), rabbit (SEQ ID NO:25), sheep (SEQ ID NO:26 and 27), orangutan (SEQ ID NO:28), cynomolgus monkey (SEQ ID NO:29), guinea pig (SEQ ID NO:30), Staphylococcus aureus (SEQ ID NO:33), Streptococcus pyogenes (SEQ ID NO:34), and Clostridium perfringens (SEQ ID NO:35). Hyaluronidases also include those of human origin. Exemplary human hyaluronidases include HYAL1 (SEQ ID NO:36), HYAL2 (SEQ ID NO:37), HYAL3 (SEQ ID NO:38), HYAL4 (SEQ ID NO:39), and PH20 (SEQ ID NO: 1). Also included amongst hyaluronidases are soluble hyaluronidases, including, ovine and bovine PH20, soluble human PH20 and soluble rHuPH20.

[0030] Reference to hyaluronidases includes precursor hyaluronidase polypeptides and mature hyaluronidase polypeptides (such as those in which a signal sequence has been removed), truncated forms thereof that have activity, and includes allelic variants and species variants, variants encoded by splice variants, and other variants, including polypeptides that have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the precursor polypeptides set forth in SEQ ID NOS: 1 and 10-39, or the mature form thereof. For example, reference to hyaluronidase also includes the human PH20 precursor polypeptide variants set forth in SEQ ID NOS: 50-51. Hyaluronidases also include those that contain chemical or posttranslational modifications and those that do not contain chemical or posttranslational modifications. Such modifications include, but are not limited to, pegylation, albumination, glycosylation, farnesylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art.

[0031] As used herein, a soluble hyaluronidase refers to a polypeptide characterized by its solubility under physiologic conditions. Soluble hyaluronidases can be distinguished, for example, by its partitioning into the aqueous phase of a Triton X-114 solution warmed to 37°C (Bordier et al., (1981) J. Biol. Chem., 256:1604-7). Membrane-anchored, such as lipid anchored hyaluronidases, will partition into the detergent rich phase, but will partition into the detergent-poor or aqueous phase following treatment with Phospholipase-C. Included among soluble hyaluronidases are membrane anchored hyaluronidases in which one or more regions associated with anchoring of the hyaluronidase to the membrane has been removed or modified, where the soluble form retains hyaluronidase activity. Soluble hyaluronidases include recombinant soluble hyaluronidases and those contained in or purified from natural sources, such as, for example, testes extracts from sheep or cows. Exemplary of such soluble hyaluronidases are soluble human PH20. Other soluble hyaluronidases include ovine (SEQ ID NO:27) and bovine (SEQ ID NO: 11) PH20.

[0032] As used herein, soluble human PH20 or sHuPH20 include mature polypeptides lacking all or a portion of the glycosylphospatidylinositol (GPI) attachment site at the C-terminus such that upon expression, the polypeptides are soluble. Exemplary sHuPH20 polypeptides include mature polypeptides having an amino acid sequence setforth in any one of SEQ ID NOS: 4-9 and 47-48. The precursor polypeptides for such exemplary sHuPH20 polypeptides include a signal sequence. Exemplary of the precursors are those setforth in SEQ ID NOS:3 and 40-46, each of which contains a 35 amino acid signal sequence at amino acid positions 1 -35. Soluble HuPH20 polypeptides also include those degraded during or after the production and purification methods described herein.

[0033] As used herein, soluble recombinant human PH20 (rHuPH20) refers to a soluble form of human PH20 that is recombinantly expressed in Chinese Hamster Ovary (CHO) cells. Soluble rHuPH20 is encoded by nucleic acid that includes the signal sequence and is setforth in SEQ ID NO: 49. Also included are DNA molecules that are allelic variants thereof and other soluble variants. The nucleic acid encoding soluble rHuPH20 is expressed in CHO cells which secrete the mature polypeptide. As produced in the culture medium there is heterogeneity at the C-terminus so that the product includes a mixture of species that can include any one or more of SEQ ID NOS: 4-9 in various abundance. Corresponding allelic variants and other variants also are included, including those corresponding to the precursor human PH20 polypeptides setforth in SEQ ID NOS:50-51. Other variants can have 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ ID NOS:4-9 and 47-48 as long they retain a hyaluronidase activity and are soluble.

[0034] As used herein, activity refers to a functional activity or activities of a polypeptide or portion thereof associated with a full-length (complete) protein. Functional activities include, but are not limited to, biological activity, catalytic or enzymatic activity, antigenicity (ability to bind or compete with a polypeptide for binding to an anti-polypeptide antibody), immunogenicity, ability to form multimers, and the ability to specifically bind to a receptor or ligand for the polypeptide.

[0035] As used herein, hyaluronidase activity refers to the ability of hyaluronidase to cleave hyaluronic acid. In vitro assays to determine the hyaluronidase activity of hyaluronidases, such as soluble rHuPH20, are known in the art and described herein. Exemplary assays include the microturbidity assay described below (see e.g. Example 3) that measures cleavage of hyaluronic acid by hyaluronidase indirectly by detecting the insoluble precipitate formed when the uncleaved hyaluronic acid binds with serum albumin.

[0036] As used herein, the residues of naturally occurring α-amino acids are the residues of those 20 α-amino acids found in nature which are incorporated into protein by the specific recognition of the charged tRNA molecule with its cognate mRNA codon in humans.

[0037] As used herein, nucleic acids include DNA, RNA and analogs thereof, including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single or double-stranded. When referring to probes or primers, which are optionally labeled, such as with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that their target is statistically unique or of low copy number (typically less than 5, generally less than 3) for probing or priming a library. Generally a probe or primer contains at least 14,16 or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids long.

[0038] As used herein, a peptide refers to a polypeptide that is from 2 to 40 amino acids in length.

[0039] As used herein, the amino acids which occur in the various sequences of amino acids provided herein are identified according to their known, three-letter or one-letter abbreviations (Table 1). The nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art.

[0040] As used herein, an "amino acid" is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids include the twenty naturally- occurring amino acids, non-natural amino acids and amino acid analogs (i.e., amino acids wherein the α-carbon has a side chain).

[0041] As used herein, "amino acid residue" refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are presumed to be in the "L" isomeric form. Residues in the "D" isomeric form, which are so designated, can be substituted for any L-amino acid residue as long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), and adopted 37 C.F.R. □§§ 1.821-1.822, abbreviations for amino acid residues are shown in Table 1:

Table 1 - Table of Correspondence

(continued)

[0042] It should be noted that all amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase "amino acid residue" is broadly defined to include the amino acids listed in the Table of Correspondence (Table 1) and modified and unusual amino acids, such as those referred to in 37 C.F.R. §§ 1.821-1.822. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, to an amino-terminal group such as NH2 or to a carboxyl-terminal group such as COOH.

[0043] As used herein, "naturally occurring amino acids" refer to the 20 L-amino acids that occur in polypeptides.

[0044] As used herein, "non-natural amino acid" refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non-naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally-occurring amino acids and include, but are not limited to, the D-isostereomers of amino acids. Exemplary non-natural amino acids are described herein and are known to those of skill in the art.

[0045] As used herein, a DNA construct is a single or double stranded, linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.

[0046] As used herein, a DNA segment is a portion of a larger DNA molecule having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5’ to 3’ direction, encodes the sequence of amino acids of the specified polypeptide.

[0047] As used herein, the term polynucleotide means a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5’ to the 3’ end. Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in terms of nucleotides (abbreviated "nt") or base pairs (abbreviated "bp"). The term nucleotides is used for single- and double-stranded molecules where the context permits. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term base pairs. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide can differ slightly in length and that the ends thereof can be staggered; thus all nucleotides within a double-stranded polynucleotide molecule can not be paired. Such unpaired ends will, in general, not exceed 20 nucleotides in length.

[0048] As used herein, "similarity" between two proteins or nucleic acids refers to the relatedness between the sequence of amino acids of the proteins or the nucleotide sequences of the nucleic acids. Similarity can be based on the degree of identity and/or homology of sequences of residues and the residues contained therein. Methods for assessing the degree of similarity between proteins or nucleic acids are known to those of skill in the art. For example, in one method of assessing sequence similarity, two amino acid or nucleotide sequences are aligned in a manner that yields a maximal level of identity between the sequences. "Identity" refers to the extent to which the amino acid or nucleotide sequences are invariant. Alignment of amino acid sequences, and to some extent nucleotide sequences, also can take into account conservative differences and/or frequent substitutions in amino acids (or nucleotides). Conservative differences are those that preserve the physico-chemical properties of the residues involved. Alignments can be global (alignment of the compared sequences over the entire length of the sequences and including all residues) or local (the alignment of a portion of the sequences that includes only the most similar region or regions).

[0049] "Identity" per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g.\ Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, FI.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exists a number of methods to measure identity between two polynucleotide or polypeptides, the term "identity" is well known to skilled artisans (Carillo, H. &amp; Lipton, D., SIAM J Applied Math 48:1073 (1988)).

[0050] As used herein, homologous (with respect to nucleic acid and/or amino acid sequences) means about greater than or equal to 25% sequence homology, typically greater than or equal to 25%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence homology; the precise percentage can be specified if necessary. For purposes herein the terms "homology" and "identity" are often used interchangeably, unless otherwise indicated. In general, for determination of the percentage homology or identity, sequences are aligned so that the highest order match is obtained (see, e.g.: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carillo et al. (1988) SIAM J Applied Math 48:1073). By sequence homology, the number of conserved amino acids is determined by standard alignment algorithms programs, and can be used with default gap penalties established by each supplier. Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.

[0051] Whether any two molecules have nucleotide sequences or amino acid sequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical" or "homologous" can be determined using known computer algorithms such as the "FASTA" program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S.F., et al., JMolec Biol 215:403 (1990)); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48:1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar "MegAlign" program (Madison, Wl) and the University of Wisconsin Genetics Computer Group (UWG) "Gap" program (Madison Wl). Percent homology or identity of proteins and/or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (e.g., Needleman et al. (1970) J. Mol. Biol. 48:443, as revised by Smith and Waterman ((1981) Adv. Appl. Math. 2:482). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids), which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCEAND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

[0052] Therefore, as used herein, the term "identity" or "homology" represents a comparison between a test and a reference polypeptide or polynucleotide. As used herein, the term at least "90% identical to" refers to percent identities from 90 to 99.99 relative to the reference nucleic acid or amino acid sequence of the polypeptide. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 100 amino acids are compared. No more than 10% (i.e., 10 out of 100) of the amino acids in the test polypeptide differs from that of the reference polypeptide. Similar comparisons can be made between test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed overthe entire length of a polypeptide or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of homologies or identities above about 85-90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often by manual alignment without relying on software.

[0053] As used herein, an aligned sequence refers to the use of homology (similarity and/or identity) to align corresponding positions in a sequence of nucleotides or amino acids. Typically, two or more sequences that are related by 50% or more identity are aligned. An aligned set of sequences refers to 2 or more sequences that are aligned at corresponding positions and can include aligning sequences derived from RNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.

[0054] As used herein, "primer" refers to a nucleic acid molecule that can act as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and a polymerization agent, such as DNA polymerase, RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. It will be appreciated that a certain nucleic acid molecules can serve as a "probe" and as a "primer." A primer, however, has a 3’ hydroxyl group for extension. A primer can be used in a variety of methods, including, for example, polymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, RNA PCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3’ and 5’ RACE, in situ PCR, ligation-mediated PCR and other amplification protocols.

[0055] As used herein, "primer pair" refers to a set of primers that includes a 5’ (upstream) primer that hybridizes with the 5’ end of a sequence to be amplified (e.g. by PCR) and a 3’ (downstream) primer that hybridizes with the complement of the 3’ end of the sequence to be amplified.

[0056] As used herein, "specifically hybridizes" refers to annealing, by complementary base-pairing, of a nucleic acid molecule (e.g. an oligonucleotide) to a target nucleic acid molecule. Those of skill in the art are familiar with in vitro and in vivo parameters that affect specific hybridization, such as length and composition of the particular molecule. Parameters particularly relevant to in vitro hybridization further include annealing and washing temperature, buffer composition and salt concentration. Exemplary washing conditions for removing non-specifically bound nucleic acid molecules at high stringency are 0.1 x SSPE, 0.1% SDS, 65°C, and at medium stringency are 0.2 x SSPE, 0.1% SDS, 50°C. Equivalent stringency conditions are known in the art. The skilled person can readily adjust these parameters to achieve specific hybridization of a nucleic acid molecule to a target nucleic acid molecule appropriate for a particular application. Complementary, when referring to two nucleotide sequences, means that the two sequences of nucleotides are capable of hybridizing, typically with less than 25%, 15% or 5% mismatches between opposed nucleotides. If necessary, the percentage of complementarity will be specified. Typically the two molecules are selected such that they will hybridize under conditions of high stringency.

[0057] As used herein, substantially identical to a product means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.

[0058] As used herein, it also is understood that the terms "substantially identical" or "similar" varies with the context as understood by those skilled in the relevant art.

[0059] As used herein, an allelic variant or allelic variation references any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and can result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides having altered amino acid sequence. The term "allelic variant" also is used herein to denote a protein encoded by an allelic variant of a gene. Typically the reference form of the gene encodes a wildtype form and/or predominant form of a polypeptide from a population or single reference member of a species. Typically, allelic variants, which include variants between and among species typically have at least 80%, 90% or greater amino acid identity with a wildtype and/or predominant form from the same species; the degree of identity depends upon the gene and whether comparison is interspecies or intraspecies. Generally, intraspecies allelic variants have at least about 80%, 85%, 90% or 95% identity or greater with a wildtype and/or predominant form, including 96%, 97%, 98%, 99% or greater identity with a wildtype and/or predominant form of a polypeptide. Reference to an allelic variant herein generally refers to variations n proteins among members of the same species.

[0060] As used herein, "allele," which is used interchangeably herein with "allelic variant" refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for that gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide or several nucleotides, and can include substitutions, deletions and insertions of nucleotides. An allele of a gene also can be a form of a gene containing a mutation.

[0061] As used herein, species variants refer to variants in polypeptides among different species, including different mammalian species, such as mouse and human.

[0062] As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.

[0063] As used herein, modification is in reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively. Methods of modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies.

[0064] As used herein, the term promoter means a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5’ non-coding region of genes.

[0065] As used herein, isolated or purified polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.

[0066] The term substantially free of cellular material includes preparations of proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the term substantially free of cellular material includes preparations of enzyme proteins having less that about 30% (by dry weight) of non-enzyme proteins (also referred to herein as a contaminating protein), generally less than about 20% of nonenzyme proteins or 10% of non-enzyme proteins or less that about 5% of non-enzyme proteins. When the enzyme protein is recombinantly produced, it also is substantially free of culture medium, i.e., culture medium represents less than about or at 20%, 10% or 5% of the volume of the enzyme protein preparation.

[0067] As used herein, the term substantially free of chemical precursors or other chemicals includes preparations of enzyme proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. The term includes preparations of enzyme proteins having less than about 30% (by dry weight) 20%, 10%, 5% or less of chemical precursors or non-enzyme chemicals or components.

[0068] As used herein, synthetic, with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and/or by chemical synthesis methods.

[0069] As used herein, production by recombinant means by using recombinant DNA methods means the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA.

[0070] As used herein, vector (or plasmid) refers to discrete elements that are used to introduce a heterologous nucleic acid into cells for either expression or replication thereof. The vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.

[0071] As used herein, an expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[0072] As used herein, vector also includes "virus vectors" or "viral vectors." Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

[0073] As used herein, operably or operatively linked when referring to DNA segments means that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.

[0074] As used herein the term assessing is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the activity of a protease, or a domain thereof, present in the sample, and also of obtaining an index, ratio, percentage, visual or other value indicative of the level of the activity. Assessment can be direct or indirect and the chemical species actually detected need not of course be the proteolysis product itself but can for example be a derivative thereof or some further substance. For example, detection of a cleavage product of a complement protein, such as by SDS-PAGE and protein staining with Coomasie blue.

[0075] As used herein, biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Biological activities can be observed in in vitro systems designed to test or use such activities. Thus, for purposes herein a biological activity of a protease is its catalytic activity in which a polypeptide is hydrolyzed.

[0076] As used herein equivalent, when referring to two sequences of nucleic acids, means that the two sequences in question encode the same sequence of amino acids or equivalent proteins. When equivalent is used in referring to two proteins or peptides, it means that the two proteins or peptides have substantially the same amino acid sequence with only amino acid substitutions that do not substantially alter the activity or function of the protein or peptide. When equivalent refers to a property, the property does not need to be present to the same extent (e.g., two peptides can exhibit different rates of the same type of enzymatic activity), but the activities are usually substantially the same.

[0077] As used herein, "modulate" and "modulation" or "alter" refer to a change of an activity of a molecule, such as a protein. Exemplary activities include, but are not limited to, biological activities, such as signal transduction. Modulation can include an increase in the activity (i.e., up-regulation or agonist activity) a decrease in activity (i.e., down-regulation or inhibition) or any other alteration in an activity (such as a change in periodicity, frequency, duration, kinetics or other parameter). Modulation can be context dependent and typically modulation is compared to a designated state, for example, the wildtype protein, the protein in a constitutive state, or the protein as expressed in a designated cell type or condition.

[0078] As used herein, a composition refers to any mixture. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.

[0079] As used herein, a combination refers to any association between or among two or more items. The combination can be two or more separate items, such as two compositions or two collections, can be a mixture thereof, such as a single mixture of the two or more items, or any variation thereof. The elements of a combination are generally functionally associated or related.

[0080] As used herein, a kit is a packaged combination that optionally includes other elements, such as additional reagents and instructions for use of the combination or elements thereof.

[0081] As used herein, "disease or disorder" refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms. Diseases and disorders of interest herein are those that are treatable by immune globulin.

[0082] As used herein, "treating" a subject with a disease or condition means that the subject’s symptoms are partially or totally alleviated, or remain static following treatment. Hence treatment encompasses prophylaxis, therapy and/or cure. Prophylaxis refers to prevention of a potential disease and/or a prevention of worsening of symptoms or progression of a disease. Treatment also encompasses any pharmaceutical use of an immune globulin preparation and compositions provided herein.

[0083] As used herein, a pharmaceutically effective agent, includes any therapeutic agent or bioactive agents, including, but not limited to, for example, anesthetics, vasoconstrictors, dispersing agents, conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.

[0084] As used herein, treatment means any manner in which the symptoms of a condition, disorder or disease or other indication, are ameliorated or otherwise beneficially altered.

[0085] As used herein therapeutic effect means an effect resulting from treatment of a subject that alters, typically improves or ameliorates the symptoms of a disease or condition or that cures a disease or condition. A therapeutically effective amount refers to the amount of a composition, molecule or compound which results in a therapeutic effect following administration to a subject.

[0086] As used herein, the term "subject" refers to an animal, including a mammal, such as a human being.

[0087] As used herein, a patient refers to a human subject.

[0088] As used herein, amelioration of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic, refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic.

[0089] As used herein, prevention or prophylaxis refers to methods in which the risk of developing disease or condition is reduced.

[0090] As used herein, a "therapeutically effective amount" or a "therapeutically effective dose" refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder.

[0091] As used herein, unit dose form refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.

[0092] As used herein, a single dosage formulation refers to a formulation for direct administration.

[0093] As used herein, an "article of manufacture" is a product that is made and sold. As used throughout this application, the term is intended to encompass IG and hyaluronidase compositions contained in articles of packaging.

[0094] As used herein, fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.

[0095] As used herein, a "kit" refers to a combination of compositions provided herein and another item for a purpose including, but not limited to, activation, administration, diagnosis, and assessment of a biological activity or property. Kits optionally include instructions for use.

[0096] As used herein, a cellular extract or lysate refers to a preparation or fraction which is made from a lysed or disrupted cell.

[0097] As used herein, animal includes any animal, such as, but are not limited to primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; ovine, such as pigs and other animals. Non-human animals exclude humans as the contemplated animal. The enzymes provided herein are from any source, animal, plant, prokaryotic and fungal. Most enzymes are of animal origin, including mammalian origin.

[0098] As used herein, a control refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be from a normal volunteer not affected with the condition of interest. A control also can be an internal control.

[0099] As used herein, the singularforms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a compound, comprising "an extracellular domain"" includes compounds with one or a plurality of extracellular domains.

[0100] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Hence "about 5 bases" means "about 5 bases" and also "5 bases." [0101] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally substituted group means that the group is unsubstituted or is substituted.

[0102] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726). B. SUBCUTANEOUS ADMINISTRATION OF IMMUNE GLOBULIN (IG) [0103] Described herein are methods and uses of treating IG-treatable diseases and conditions by subcutaneously administering immune globulin (IG) in combination with a soluble hyaluronidase. Hence, also provided are combinations of IG and a soluble hyaluronidase. By virtue of the ability of hyaluronidase to break down hyaluronic acid in the extracellular matrix, hyaluronidase facilitates subcutaneous infusions of therapeutic agents. Immune globulin is a therapeutic that is primarily given by intravenous administration to treat individuals with immune deficiencies, referred to as IVIG therapy. The bioavailability of IG in the presence of hyaluronidase is greater than 90% of the bioavailability of IG following IVIG treatment. Hence, in the methods and use described herein, the combination therapy of hyaluronidase and IG permits the subcutaneous administration of immune globulin at dosages and frequencies that are similar to IVIG treatment. Thus, for example, in the methods and uses described herein, IG, when administered subcutaneously in the presence of a soluble hyaluronidase, can be administered once monthly at prevailing IVIG doses for the particular indication. Further, because hyaluronidase acts to open flow channels in the skin it can speed infusion rates. Hence, methods of subcutaneously administering IG co-formulated and/or co-administered with hyaluronidase increases infusion rates, and thereby decreases time of delivery of IG therapy.

[0104] Defective antibody formation is the most common abnormality in the majority of primary immunodeficiency (PID) diseases; it is most often reflected by a decrease in serum immunoglobulins, which in turn leads to increased susceptibility to bacterial infections, especially of the sinopulmonary tract. Decreased immunoglobulin levels are found in individuals having antibody defects such as X-linked agammaglobulinemia, immunoglobulin heavy chain deletion, selective immunoglobulin G (IgG) subclass deficiency, common variable immunodeficiency, or X-linked hyperimmunoglobulin M syndrome. Decreased immunoglobulin levels also are found in individuals having combined immunodeficiencies due to defects in T and B cells, such as, but not limited to, severe combined immunodeficiency orWiskott Aldrich Syndrome (IUIS Scientific Committee, 1999).

[0105] Individuals with these diseases require replacement therapy with immunoglobulin products to prevent or reduce the severity of infections. Initially, immunoglobulin replacement therapy was given by the intramuscular route, but starting in 1981 the overwhelming majority of patients have been treated by the intravenous (IV) route. Currently, the majority of immunoglobulin products in the United States are for IV administration. Immune globulin preparations have, however, been developed more recently for subcutaneous administration (Gardulf etal. (2006) Curr. Opin. Allergy Clin. Immunol., 6: 434-42; Gardulf et al. (2006) J Clin. Immunol., 26: 177-85; Ochs et al. (2006) J Clin. Immunol., 26:265-73). At least one product, Vivaglobin®, is approved for subcutaneous administration.

[0106] All of the immunoglobulin preparations presently used are formulated at 16%, compared to IVIG preparations formulated at 5 to 12%. The higher concentration relative to IV preparations allows smaller infusion volumes; such preparations cannot be infused intravenously. Such subcutaneous methods of immunoglobulin replacement therapy is considered to be effective, safe and also highly appreciated by patients as it has a low risk of systemic adverse reactions and leads to higher trough serum IgG concentrations compared to monthly IV infusions (Gardulf et al. (1995) J Adv. Nurs., 21: 917-27; Gardulf et al. (1993) Clin. Exp. Immunol., 92: 200-4; Gardulf et al. (1991) Lancet, 338: 162-6).

[0107] The bioavailability of immunoglobulin administered subcutaneously generally is less than that infused intravenously. Immunoglobulin is immediately available in the blood, and slowly equilibrates to the extra-vascular compartment over3to5days(Schiffetal. (1986) J. Clin. Immunol., 6:256-64). Subcutaneously administered immunoglobulin is slowly absorbed from the subcutaneous space into the blood and at the same time equilibrates with the extra-vascular compartment; there is no high IV spike. The bioavailability has not been extensively studied, but in a recent trial of the ZLB-Behring preparation (i.e. Vivaglobin®), it was determined by measuring the area under the curve (AUC) that only 67% of the immunoglobulin was absorbed and thus, the recommended dose was 137% of the IV dose (Ochs et al. (2006) J Clin. Immunol., 26:265-73). Despite the technical difficulties of comparing AUC for 2 different routes and frequency of administration, studies ofintradermally administered immunoglobulin in rabbits suggests there isdecreased bioavailability through the subcutaneous route. This may be due to the mode of absorption of large protein molecules, which cannot readily diffuse through the capillary walls and must be absorbed via the lymphatics (Supersaxo et al. (1990) Pharm. Res., 7: 167-9).

[0108] In addition to problems with bioavailability associated with subcutaneous administration of IG, the primary disadvantage of SC administration is that only small volumes can be infused in each site, necessitating the use of multiple sites on a weekly or biweekly (ever other week) basis. Generally, using a 16% solution, approximately 20 ml can be infused per site; and adult patient receiving 400 mg/kg body weight (BW) thus would require at least 3 sites per week or 12 sites per month. Even though weekly or biweekly administration has the added advantage of maintaining better trough levels than monthly IV infusions, the requirement of multiple needle insertions has been a deterrent for many patients.

[0109] The SC space is formed by a collagen network filled with a gel-like substance, hyaluronic acid. Hyaluronic acid is replaced with a half-life of approximately 5 h, and is largely responsible for the resistance to fluid flow through the tissues. Hyaluronidase temporarily digests the hyaluronic acid, thereby facilitating infusions into the subcutaneous space. The hyaluronic acid is restored within 24 hours, leaving no observable changes. Thus, due to the ability of hyaluronidase to open channels in the interstitial space through degradation of glycosaminoglycans, administration of a soluble hyaluronidase permits the diffusion of molecules, thereby improving the bioavailability, pharmacokinetics and/or pharmacodynamic characteristics of such co-formulated or co-administered molecules.

[0110] In some examples, the bioavailability of IG co-administered subcutaneously with hyaluronidase is 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the bioavailability of IVIG. Typically, the bioavailability is greater than 90%. Further, co-administration with a soluble hyaluronidase permits infusion of large volumes at a single subcutaneous site. For example, volumes up to 600 ml or greater of IG can be administered at a single site in a single sitting, for example 200 ml, 300 ml, 400 ml, 500, ml, 600 ml or more can be administered at a single site in a single administration. Thus, an IG preparation formulated at or between 5-12% can be co-administered subcutaneously with a soluble hyaluronidase at dosages equivalent to once monthly IVIG doses, for example, at or about 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg or more. The dosages can be administered as a single dose or can be divided into multiple doses given daily or weekly, such as once a week or every two, three or four weeks or combinations thereof. Hence, by administering IG subcutaneously in the presence of a soluble hyaluronidase, one or all of the considerations and problems associated with subcutaneous administration of IG are addressed. Thus, by virtue of the dispersion properties of hyaluronidase, subcutaneously administering IG in the presence of a soluble hyaluronidase permits administration of IVIG doses at once monthly IVIG frequencies, while maintaining IVIG bioavailability.

[0111] The following sections describe exemplary immunoglobulins and soluble hyaluronidases in the combinations herein, methods of making them, and using them to treat IG-treatable diseases and conditions. C. Immune Globulin [0112] Provided herein are immune globulins (IG, also referred to as gamma globulin or IgG) that can be used for subcutaneous administration in combination with a soluble hyaluronidase. IG acts to strengthen the immune system by modulating the activity of complement, suppressing autoantibody production, saturating or blocking Fc receptors on macrophages and B lymphocytes, and suppressing the production of inflammatory mediators such as cytokines, chem-okines and metalloproteinases.

[0113] IG is a protein fraction found in the plasma of higher animals and contains a large number of antibodies having varying specificities. Generally, IG contains serum immunoglobulins that can be any idiotype, such as IgG and various subclasses, IgA, IgM, IgD, IgE. The various immunoglobulins or subclasses can be present at various concentrations and specificities, which can differ between immune globulin preparations depending, for example, on the plasma donor’s exposure to antigens (e.g., byway of vaccinations). Often, immunoglobulins are present in amounts normally found in the serum (see Table 2), although purification steps can be employed to alter ratios of particular immunoglobulin class or classes. Typically, IG preparations contain 90% or more IgG, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more IgG. The immunoglobulins can be polyclonal or monoclonal. Typically, preparations include a high percentage of monomeric IgG and a low IgA content.

[0114] Immune globulins can be isolated from human or animal blood or produced by other means, for example, by recombinant DNA technology or hybridoma technology. For example, immune globulin can be obtained from tissues, lymphocyte hybridoma cultures, blood plasma or serum, or recombinant cell cultures using any suitable fractionation procedure, e.g., alcohol precipitations or ion exchange separations. In general, IG is prepared from blood plasma by alcohol fractionation, such as was originally employed by Cohn and modified by Oncley (the Cohn-Oncley method, see e.g, Cohn et al. (1946) J. Am. Chem. Soc. 68: 459-475; Oncley et al. (1949) J Am. Chem. Soc., 71: 541-550). The use of alcohol in the purification can inactivate potentially contaminating viruses. The Cohn-Oncley method can result in denatured and aggregated proteins, which can result in high molecular weight forms that can act as antibody-antigen complexes having the capacity to freely fix complement.

[0115] To prevent such unwanted effects, modified Cohn-Oncley methods have been developed for the preparation and purification of IG. Various such procedures are known and can be adapted and modified for use of IG preparations herein. It is within the skill of the art to prepare IG preparations in view of the detailed methods known and available in the art. Typically, IG is manufactured using a primary cold ethanol fractionation and a secondary fractionation that can include any one or more of a chemical modification, incubation at pH 4.0 with or without pepsin, PEG precipitation, ion-exchange chromatography, enzymatic cleavage, solvent detergent treatment and diafiltration and ultrafiltration.

[0116] For exam pie, the separation of IG aggregates by conventional techniques, such as ultra-centrifuging or exclusion chromatography, makes it possible to obtain a product having a low anti-complementary activity. Other methods of IG preparation include, but are not limited to, a process forfractionating human plasma by means of ethyleneglycol polymers (Poison et al. (1964) Biochim. Biophys. Acta., 82: 463-475); incorporation of a polyethyleneglycol (PEG) as a purification agent starting from a material separated from the Cohn fractionation (fraction II or II + III, see e.g., U.S. Patent Nos. 4,093,606 and 4,165,370); and other similar methods of purification processes with polyethyleneglycol (EP 0246579). In addition, processes have been described for obtaining IG that exhibits low anticomplement activity by treatment with enzymes such as pepsin, plasmin, immobilized trypsin, treatment at a moderate acidic pH, B-propiolactone treatment, fractionation methods which use polyethylene glycol as a precipitating agent, and other techniques described in U.S. Pat Nos. 4,093,606, 4,126,605, 3,966,906, and 4,124,576. Other processes are based on chemically and partially modifying the IG molecules by treating them with reducing agents, alchoholization, alkylation and sulphonation (see e.g., U.S. Patent 6,875,848). Ion exchange chromatography can be used to eliminate undesirable contaminants from the starting materials used to obtain the IG preparations (see e.g., U.S. Patent No. 3,869,436, EP 91300790 and WO 94/29334). EP0440483 describes a combination of techniques useful for facilitating the intravenous preparation of the product based on ion exchange chromatography and diafiltration at a weak acid pH. Other methods also are described in the art and are known to one of skill in the art (see e.g., U.S. Patent 5,177,194 and 6,875,848).

[0117] The IG preparations should be treated to remove viral load. There are two methods of decreasing viral load: viral inactivation and viral partitioning or removal. Exemplary of viral inactivation methods include, but are not limited to, cold ethanol fractionation, heating (pasterurization), solvent/detergent and acidic environment (low pH). For example, solvent/detergent process has been demonstrated to have virucidal action against VSV (vesicular stomatitits virus), Sindbis virus, HIV, HBV (hepatitis B virus, and HCV (hepatic C virus). Exemplary of viral partitioning or removal include, but are not limited to, cold ethanol fractionation, phase partitioning or PEG precipitation, affinity chromatography, ion exchange or gel exclusion chromatography and filtration.

[0118] The final purified formulation also must be prepared to avoid excessive aggregation and to stabilize the protein. Aggregation of IG preparation can be minimized by preparing lyophilized preparations for improved stability on storage, for reconstitution with a diluent before use. Another way of increasing the stability of IG preparations that is well known in the art is the addition of protein-stabilizing excipients to the IG preparation. Known excipients include, but are not limited to, sugars, polyols, amino acids, amines, salts, polymers and surfactants. For example, U.S. Patent 4,499,073 describes stabilization through the selection of pH and ionic strength; JP Patent 54020124 discloses the addition of an amino acid to an intramuscular preparation to render it storage stable and safe; JP 57031623 and JP 57128635 disclose the use of arginine and/or lysine with NaCI in 5 to 15% IG preparations to achieve long-term stability in an intramuscular preparation; JP 4346934 discloses the use of low conductivity (less than mmho), pH 5.3 to 5.7 and optionally one or more stabilizer including PEG, human serum albumin and mannitol; US 4,439,421 teaches the addition of a hydrophilic macromolecule, a polyol and another protein to stabilize against anti-complement generation; U.S. 5,945,098 discloses the stabilization if isotonic solutions by the addition of amino acids (0.1 to 0.3 M glycine) and non-ionic detergents (polysorbate and PEG); US 4,186,192 discloses various additives including amino acids; WO 2005/049078 discloses the stabilization with maltose and additionally glycine to 0.1 M; US 4,362,661 discloses the use of neutral and basic amino acids to impart stability on a 5% IG preparation. Stable liquid formulations also can be prepared, which use carbohydrates in an aqueous medium having a very low ionic strength and a pH of 4.25 (U.S. Patent No. 4,396,608) or a weakly acidic pH of 5-6 (EP 0278422).

[0119] Dimer formation of IG preparations also can be controlled. For example, U.S. Patent No. 5,871,736 discloses IG preparations, particularly liquid preparations, containing one or more amphiphilic stabilizer in order to stabilize against dimer formation. The amphiphilic stabilizers include nicotinic acid and its derivatives, in particular nicotinamide, and mainly in conjunction with the above, amino acids having uncharged lipophilic side chains, e.g., phenylalanine, methionine, leucine, isoleucine, proline and valine.

[0120] The preparations can be prepared by methods known in the art, such as any described herein. Generally, however, the pH of the final preparation is adjusted to a relatively high but acidic pH, namely in the range of about pH 4.2 to 5.4, such as a pH range of 4.6 to 5.1. It has been found that this pH range is particularly useful for improving the storage of IG preparations.

[0121] Generally, final IG preparations have a protein concentration of about 5 to 25% w/v, generally 6 to 15% w/v, 8 to 12% w/v, and typically 10% w/v. Thefinal protein concentration will depend on various factors, such as the administration route the patient to be treated, and the type of condition to be treated.

[0122] It is contemplated herein that any IG preparation used for IV administration can be used in the methods provided herein in combination with a soluble hyaluronidase for subcutaneous administration. Preparations include lyophilized and liquid formulations. Immune globulin (IVIG) is commercially available as Carimune® NF, Flebogamma® 5%, Gam-magard® Liquid, Gammagard® S/D, Gamunex®, Iveegam® EN, Octagam® and Polygam® S/D. Typically, such preparations all use a method of cold alcohol fractionation, but use different methods to isolate and purify the immune globulin and different methods to reduce the potential virus contamination. Further, other preparations presently formulated for intramuscular or subcutaneous administration can be used in the combinations and methods provided herein.

[0123] Exemplary of an IG preparation is Immune Globulin Intravenous (Human), 10% (IVIG, 10%, marketed as Gammagard® liquid, Baxter Healthcare Corporation), which is a liquid unmodified IgG preparation with a distribution of IgG subclasses similar to that of normal plasma. The preparation contains intact fragment crystallizable (Fc) and Fab regions. The preparation contains 100 mg/ml proteins, with at least 98% being IgG; IgA is present at a concentration of 37 μg/ml, and IgM is present only in trace amounts. It has an osmolality that is similar to physiologic osmolality and contains no added sugars, sodium or preservatives. It is formulated with glycine for stabilization at a pH of 4.6 to 5.1. The manufacturing process employs a modified Cohn-Oncley cold alcohol fractionation procedure and further purifications by a continuous process through the use of weak cation exchange chromatography and weak anion exchange chromatography. The manufacturing process also includes 3 independent viral inactivation or removal steps: solvent/de-tergent (S/D) treatment, nanofiltration and incubation at a low pH and elevated temperature. D. Hyaluronidase [0124] Provided herein are combinations containing immunoglobulin and a soluble hyaluronidase, and methods of using such combinations for subcutaneous administration for the treatment of IG-mediated diseases and conditions. Hyaluronidases are a family of enzymes that degrade hyaluronic acid, which is an essential component of the extracellular matrix and a major constituent of the interstitial barrier. By catalyzing the hydrolysis of hyaluronic acid, a major constituent of the interstitial barrier, hyaluronidase lowers the viscosity of hyaluronic acid, thereby increasing tissue permeability. As such, hyaluronidases have been used, for example, as a spreading or dispersing agent in conjunction with other agents, drugs and proteins to enhance their dispersion and delivery. Exemplary of hyaluronidases in the combinations and methods provided herein are soluble hyaluronidases.

[0125] There are three general classes of hyaluronidases; mammalian hyaluronidase, bacterial hyaluronidase and hyaluronidase from leeches, other parasites and crustaceans. Mammalian-type hyaluronidases (EC 3.2.1.35) are endo-/?-A/-acetyl-hexosaminidases that hydrolyze the ^l-^glycosidic bond of hyaluronan into various oligosaccharide lengths such as tetrasaccharides and hexasaccharides. They have both hydrolytic and transglycosidase activities, and can degrade hyaluronan and chondroitin sulfates (CS), generally C4-S and C6-S. Hyaluronidases of this type include, but are not limited to, hyaluronidases from cows (bovine) (SEQ ID NOS:10 and 11), mouse (SEQ ID NOS:17-19, 32), pig (SEQ ID NOS:20-21), rat (SEQ ID NOS:22-24, 31), rabbit (SEQ ID NO:25), sheep (ovine) (SEQ ID NOS:26 and 27), orangutan (SEQ ID NO:28), cynomolgus monkey (SEQ ID NO:29), guinea pig (SEQ ID NO:30), and human hyaluronidases.

[0126] Mammalian hyaluronidases can be further subdivided into those that are neutral active, predominantly found in testes extracts, and acid active, predominantly found in organs such as the liver. Exemplary neutral active hyaluronidases include PH20, including but not limited to, PH20 derived from different species such as ovine (SEQ ID NO:27), bovine (SEQ ID NO: 11) and human (SEQ ID NO: 1). Human PH20 (also known as SPAM1 or sperm surface protein PH20), is generally locked to the plasma membrane via a glycosylphosphatidyl inositol (GPI) anchor. It is naturally involved in sperm-egg adhesion and aids penetration by sperm of the layer of cumulus cells by digesting hyaluronic acid. The PH20 mRNA transcript is normally translated to generate a 509 amino acid precursor polypeptide (SEQ ID NO: 1) containing a 35 amino acid signal sequence at the N-terminus (amino acid residue positions 1-35) and a 19 amino acid GPI anchor at the C-terminus (amino acid residue positions 491-509) The mature PH20 is, therefore, a 474 amino acid polypeptide set forth in SEQ ID NO:2). Bovine PH20 is a 553 amino acid precursor polypeptide (SEQ ID NO: 11). Alignment of bovine PH20 with the human PH20 shows only weak homology, with multiple gaps existing from amino acid 470 through to the respective carboxy termini due to the absence of a GPI anchor in the bovine polypeptide (see e.g., Frost Gl (2007) Expert Opin. Drug. Deliv. 4: 427-440). In fact, no clear GPI anchor is predicted in any other PH20 species besides humans. Thus, PH20 polypeptides produced from ovine and bovine exist as soluble forms. Though bovine PH20 exists very loosely attached to the plasma membrane, it is not anchored via a phospholipase sensitive anchor (Lalancette et al, Biol Reprod. 2001 Aug ;65(2) :628- 36.). This unique feature of bovine hyaluronidase has permitted the use of the soluble bovine testes hyaluronidase enzyme as an extract for clinical use (Wydase™, Hyalase™).

[0127] Besides human PH20 (also termed SPAM1), five hyaluronidase-like genes have been identified in the human genome, HYAL1, HYAL2, HYAL3, HYAL4 and HYALP1. HYALP1 is a pseudogene, and HYAL3 (SEQ ID NO:38) has not been shown to possess enzyme activity toward any known substrates. HYAL4 (precursor polypeptide set forth in SEQ ID NO:39) is a chondroitinase and exhibits little activity towards hyaluronan. HYAL1 (precursor polypeptide set forth in SEQ ID NO:36) is the prototypical acid-active enzyme and PH20 (precursor polypeptide set forth in SEQ ID NO: 1) is the prototypical neutral-active enzyme. Acid-active hyaluronidases, such as H YAL1 and HYAL2 (precursor polypeptide set forth in SEQ ID NO:37) generally lack catalytic activity at neutral pH (i.e. pH 7). For example, HYAL1 has little catalytic activity in vitro over pH 4.5 (Frost et al. (1997) Anal. Biochemistry, 251:263-269). HYAL2 is an acid-active enzyme with a very low specific activity in vitro. The hyaluronidase-like enzymes can also be characterized by those which are generally locked to the plasma membrane via a glycosylphosphatidyl inositol anchor such as human HYAL2 and human PH20 (Danilkovitch-Miagkova, et al. (2003) Proc Natl Acad Sci USA. 100(8):4580-5), and those which are generally soluble such as human HYAL1 (Frost et al, (1997) Biochem Biophys Res Commun. 236(1):10-5).

[0128] Glycosylation, including N- and O-linked glycosylation, of some hyaluronidases can be very important for their catalytic activity and stability. While altering the type of glycan modifying a glycoprotein can have dramatic affects on a protein’s antigenicity, structural folding, solubility, and stability, most enzymes are not thought to require glycosylation for optimal enzyme activity. Such hyaluronidases are unique in this regard, in that removal of N-linked glycosylation can result in near complete inactivation of the hyaluronidase activity. For such hyaluronidases, the presence of N-linked glycans is critical for generating an active enzyme.

[0129] N-linked oligosaccharides fall into several major types (oligomannose, complex, hybrid, sulfated), all of which have (Man) 3-GlcNAc-GlcNAc-cores attached via the amide nitrogen of Asn residues that fall within-Asn-Xaa-Thr/Ser-sequences (where Xaa is not Pro). Glycosylation at an-Asn-Xaa-Cys-site has been reported for coagulation protein C. In some instances, the hyaluronidase can contain both N-glycosidic and O-glycosidic linkages. For example, PH20 has O-linked oligosaccharides as well as N-linked oligosaccharides.

[0130] There are seven potential N-linked glycosylation sites at N82, N166, N235, N254, N368, N393, N490 of human PH20 exemplified in SEQ ID NO: 1. Disulfide bonds form between the cysteine residues C60 and C351 and between C224 and C238 to form the core hyaluronidase domain. However, additional cysteines are required in the carboxy terminus for neutral enzyme catalytic activity such that amino acids 36 to 464 of SEQ ID NO:1 contains the minimally active human PH20 hyaluronidase domain. Thus, N-linked glycosylation site N-490 is not required for proper hyaluronidase activity.

Soluble Hyaluronidase [0131] Provided in the combinations and methods herein are soluble hyaluronidases. Soluble hyaluronidases include any that exist in soluble form, including, but not limited to, Hyall, bovine PH20 and ovine PH20, allelic variants thereof and other variants. Also included among soluble hyaluronidase are any hyaluronidase that has been modified to be soluble. For example, human PH20, which is normally membrane anchored via a GPI anchor, can be made soluble by truncation of and removal of all or a portion of the GPI anchor at the C-terminus. Soluble hyaluronidases also include neutral active and acid active hyaluronidases, however, neutral active hyaluronidases are contemplated for use herein for purposes of subcutaneous administration.

[0132] Thus, exemplary of a soluble hyaluronidase is PH20 from any species, such as any set forth in any of SEQ ID NOS: 1,2, 11,25, 27, 30 and 31, or truncated forms thereof lacking all or a portion of the C-terminal GPI anchor, so long as the hyaluronidase is soluble and retains hyaluronidase activity. Also included among soluble hyaluronidases are allelic variants or other variants of any of SEQ ID NOS: 1,2, 11,25, 27, 30 and 31, or truncated forms thereof. Allelic variants and other variants are known to one of skill in the art, and include polypeptides having 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95% or more sequence identify to any of SEQ ID NOS: 1,2, 11,25, 27, 30 and 31, or truncated forms thereof.

[0133] Typically, for use in the methods herein, a soluble human PH20 is used. Although PH20 from other animals can be utilized, such preparations are potentially immunogenic, since they are animal proteins. For example, a significant proportion of patients demonstrate prior sensitization secondary to ingested foods, and since these are animal proteins, all patients have a risk of subsequent sensitization. Thus, non-human preparations may not be suitable for chronic use. If non-human preparations are desired, it is contemplated herein that such polypeptides can be prepared to have reduced immunogenicity. Such modifications are within the level of one of skill in the art. Hyaluronidases, including PH20, used in the methods herein can be recombinantly produced or can be purified or partially-purified from natural sources, such as, for example, from testes extracts.

Soluble Human PH20 [0134] Exemplary of a soluble hyaluronidase is soluble human PH20. Soluble forms of recombinant human PH20 have been produced and can be used in the methods described herein for co-administration or co-formulation with immunoglobulin for subcutaneous administration to treat IG-treatable diseases and conditions. The production of such soluble forms of PH20 is described in Application Nos. 11/065,716 and 11/238,171, and in Examples 2-6, below. Soluble forms include, but are not limited to, any having C-terminal truncations to generate polypeptides containing amino acid 1 to amino acid 347, 372, 394, 413, 430, 447, 467, 477, 478, 479, 480, 481, 482 and 483 of the sequence of amino acids set forth in SEQ ID NOS 1. When expressed in mammalian cells, the 35 amino acid N-terminal signal sequence is cleaved during processing, and the mature form of the protein is secreted. Thus, the mature soluble polypeptides contain amino acids 36 to 347, 372, 394, 413, 430, 447, 467, 477, 478, 479, 480, 481,482 and 483 of SEQ ID NO:1. Deletion mutants ending at amino acid position 477 to 483 (corresponding to the precursor polypeptide set forth in SEQ ID NO: 1) exhibit higher secreted hyaluronidase activity than the full length GPI-anchored form. Hence, exemplary of soluble hyaluronidases are those that are 442, 443, 444, 445, 446 or 447 amino acids in length, such as set forth in any of SEQ ID NOS:4-9, or allelic or species variants or other variants thereof. Generally soluble forms of PH20 are produced using protein expression systems that facilitate correct N-glycosylation to ensure the polypeptide retains activity, since glycosylation is important for the catalytic activity and stability of hyaluronidases. Such cells include, for example Chinese Hamster Ovary (CHO) cells (e.g. DG44 CHO cells).

Recombinant soluble human PH20 (rHuPH20) [0135] Recombinant soluble forms of human PH20 (soluble rHuPH20) have been generated and can be produced and purified using the methods described herein. The generation of such soluble forms of rHuPH20 are described in U.S. Patent Application Nos. 11/065,716 and 11/238,171 (published as U.S. published patent application Nos. US20050260186 and US 20060104968), and in Examples 2-6, below. Exemplary of such polypeptides are those generated from a nucleic acid molecule encoding amino acids 1-482 set forth in SEQ ID NO:3. Post translational processing removes the 35 amino acid signal sequence, resulting in the secretion of a 447 amino acid soluble rHuPH20 (SEQ ID NO:4). Resulting purified rHuPH20can be heterogenous due to peptidases present in the culture medium upon production and purification. Typically, rHuPH20 is produced in cells that facilitate correct N-glycosylation to retain activity, such as CHO cells (e.g. DG44 CHO cells). E. Methods of Producing Nucleic Acids encoding a soluble Hyaluronidase and Polypeptides Thereof [0136] Polypeptides of a soluble hyaluronidase set forth herein, can be obtained by methods well known in the art for protein purification and recombinant protein expression. Any method known to those of skill in the art for identification of nucleic acids that encode desired genes can be used. Any method available in the art can be used to obtain a full length (/'.e., encompassing the entire coding region) cDNA or genomic DNA clone encoding a hyaluronidase, such as from a cell or tissue source. Modified or variant soluble hyaluronidases, can be engineered from a wildtype polypeptide, such as by site-directed mutagenesis.

[0137] Polypeptides can be cloned or isolated using any available methods known in the art for cloning and isolating nucleic acid molecules. Such methods include PCR amplification of nucleic acids and screening of libraries, including nucleic acid hybridization screening, antibody-based screening and activity-based screening.

[0138] Methods for amplification of nucleic acids can be used to isolate nucleic acid molecules encoding a desired polypeptide, including for example, polymerase chain reaction (PCR) methods. A nucleic acid containing material can be used as a starting material from which a desired polypeptide-encoding nucleic acid molecule can be isolated. For example, DNA and mRNA preparations, cell extracts, tissue extracts, fluid samples (e.g. blood, serum, saliva), samples from healthy and/or diseased subjects can be used in amplification methods. Nucleic acid libraries also can be used as a source of starting material. Primers can be designed to amplify a desired polypeptide. For example, primers can be designed based on expressed sequences from which a desired polypeptide is generated. Primers can be designed based on back-translation of a polypeptide amino acid sequence. Nucleic acid molecules generated by amplification can be sequenced and confirmed to encode a desired polypeptide.

[0139] Additional nucleotide sequences can be joined to a polypeptide-encoding nucleic acid molecule, including linker sequences containing restriction endonuclease sites for the purpose of cloning the synthetic gene into a vector, for example, a protein expression vector or a vector designed for the amplification of the core protein coding DNA sequences. Furthermore, additional nucleotide sequences specifying functional DNA elements can be operatively linked to a polypeptide-encoding nucleic acid molecule. Examples of such sequences include, but are not limited to, promoter sequences designed to facilitate intracellular protein expression, and secretion sequences, for example heterologous signal sequences, designed to facilitate protein secretion. Such sequences are known to those of skill in the art. Additional nucleotide residues sequences such as sequences of bases specifying protein binding regions also can be linked to enzyme-encoding nucleic acid molecules. Such regions include, but are not limited to, sequences of residues that facilitate or encode proteins that facilitate uptake of an enzyme into specific target cells, or otherwise alter pharmacokinetics of a product of a synthetic gene. For example, enzymes can be linked to PEG moieties.

[0140] In addition, tags or other moieties can be added, for example, to aid in detection or affinity purification of the polypeptide. For example, additional nucleotide residues sequences such as sequences of bases specifying an epitope tag or other detectable marker also can be linked to enzyme-encoding nucleic acid molecules. Exemplary of such sequences include nucleic acid sequences encoding a His tag (e.g., 6xHis, HHHHHH; SEQ ID NO:54) or Flag Tag (DYKDDDDK; SEQ ID NO:55).

[0141] The identified and isolated nucleic acids can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art can be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene, La Jolla, CA). Other expression vectors include the HZ24 expression vector exemplified herein. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vectorwhich has complementary cohesive termini. Insertion can be effected usingTOPO cloning vectors (I NVITROGEN, Carlsbad, CA). If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and protein gene can be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via, for example, transformation, transfection, infection, electroporation and sonoporation, so that many copies of the gene sequence are generated.

[0142] In specific examples, transformation of host cells with recombinant DNA molecules that incorporate the isolated protein gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene can be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA. 1. Vectors and cells [0143] For recombinant expression of one or more of the desired proteins, such as any described herein, the nucleic acid containing all or a portion of the nucleotide sequence encoding the protein can be inserted into an appropriate expression vector, i.e.} a vector that contains the necessary elements for the transcription and translation of the inserted protein coding sequence. The necessary transcriptional and translational signals also can be supplied by the native promoter for enzyme genes, and/or their flanking regions.

[0144] Also described are vectors that contain a nucleic acid encoding the enzyme. Cells containing the vectors also are described. The cells include eukaryotic and prokaryotic cells, and the vectors are any suitable for use therein.

[0145] Prokaryotic and eukaryotic cells, including endothelial cells, containing the vectors are described. Such cells include bacterial cells, yeast cells, fungal cells, Archea, plant cells, insect cells and animal cells. The cells are used to produce a protein thereof by growing the above-described cells under conditions whereby the encoded protein is expressed by the cell, and recovering the expressed protein. For purposes herein, for example, the enzyme can be secreted into the medium.

[0146] Described herein are vectors that contain a sequence of nucleotides that encodes the soluble hyaluronidase polypeptide coupled to the native or heterologous signal sequence, as well as multiple copies thereof. The vectors can be selected for expression of the enzyme protein in the cell or such that the enzyme protein is expressed as a secreted protein.

[0147] A variety of host-vector systems can be used to express the protein coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g. vaccinia virus, adenovirus and other viruses); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system used, any one of a number of suitable transcription and translation elements can be used.

[0148] Any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a chimeric gene containing appropriate transcriptional/translational control signals and protein coding sequences. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of nucleic acid sequences encoding protein, or domains, derivatives, fragments or homologs thereof, can be regulated by a second nucleic acid sequence so that the genes or fragments thereof are expressed in a host transformed with the recombinant DNA molecule(s). For example, expression of the proteins can be controlled by any promoter/enhancer known in the art. In a specific example, the promoter is not native to the genes for a desired protein. Promoters which can be used include but are not limited to the SV40 early promoter (Bernoist and Chambon, Nature 290:304-310 (1981)), the promoter contained in the 3’ long terminal repeat of Rous sarcoma virus (Yamamoto et al. Cell 22:787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the β-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)); see also "Useful Proteins from Recombinant Bacteria": in Scientific American 242:79-94 (1980)); plant expression vectors containing the nopaline synthetase promoter (Herrara-Estrella et al., Nature 303:209-213 (1984)) or the cauliflower mosaic virus 35S RNA promoter (Garder et al., Nucleic Acids Res. 9:2871 (1981)), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., Nature 310:115-120 (1984)); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, Hepatology 7:425-515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan et al., Nature 315:115-122 (1985)), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., Cell 38:647-658 (1984); Adams et al., Nature 318:533-538 (1985); Alexander et al., Mol. Cell Biol. 7:1436-1444 (1987)), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 45:485-495 (1986)), albumin gene control region which is active in liver (Pinckert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., Mol. Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science 235:53-58 1987)), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al., Genes and Devel. 1:161-171 (1987)), beta globin gene control region which is active in myeloid cells (Magram et al., Nature 315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al., Cell 48:703-712 (1987)), myosin light chain-2 gene control region which is active in skeletal muscle (Shani, Nature 314:283-286 (1985)), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al., Science 234:1372-1378 (1986)).

[0149] In a specific example, a vector is used that contains a promoter operably linked to nucleic acids encoding a desired protein, or a domain, fragment, derivative or homolog, thereof, one or more origins of replication, and optionally, one or more selectable markers (e.g., an antibiotic resistance gene). Exemplary plasmid vectors for transformation of E.coli cells, include, for example, the pQE expression vectors (available from Qiagen, Valencia, CA; see also literature published by Qiagen describing the system). pQE vectors have a phage T5 promoter (recognized by E. coli RNA polymerase) and a double lac operator repression module to provide tightly regulated, high-level expression of recombinant proteins in E. coli, a synthetic ribosomal binding site (RBS II) for efficient translation, a 6XHis tag coding sequence, to and T1 transcriptional terminators, ColE1 origin of replication, and a beta-lactamase gene for conferring ampicillin resistance. The pQE vectors enable placement of a 6xHis tag at either the N- or C-terminus of the recombinant protein. Such plasmids include pQE 32, pQE 30, and pQE 31 which provide multiple cloning sites for all three reading frames and provide for the expression of N-terminally 6xHis-tagged proteins. Other exemplary plasmid vectors for transformation of E. coli cells, include, for example, the pET expression vectors (see, U.S. patent 4,952,496; available from NOVAGEN, Madison, Wl; see, also literature published by Novagen describing the system). Such plasmids include pET 11a, which contains the T7lac promoter, T7 terminator, the inducible E. coli lac operator, and the lac repressor gene; pET 12a-c, which contains the T7 promoter, T7 terminator, and the E. coli ompT secretion signal; and pET 15b and pET19b (NOVAGEN, Madison, Wl), which contain a His-Tag™ leader sequence for use in purification with a His column and a thrombin cleavage site that permits cleavage following purification over the column, the T7-lac promoter region and the T7 terminator.

[0150] Exemplary of a vector for mammalian cell expression is the HZ24 expression vector. The HZ24 expression vector was derived from the pCI vector backbone (Promega). It contains DNA encoding the Beta-lactamase resistance gene (AmpR), an F1 origin of replication, a Cytomegalovirus immediate-early enhancer/promoter region (CMV), and an SV40 late polyadenylation signal (SV40). The expression vector also has an internal ribosome entry site (IRES) from the ECMV virus (Clontech) and the mouse dihydrofolate reductase (DHFR) gene. 2. Expression [0151] Soluble hyaluronidase polypeptides can be produced by any method known to those of skill in the art including in vivo and in vitro methods. Desired proteins can be expressed in any organism suitable to produce the required amounts and forms of the proteins, such as for example, needed for administration and treatment. Expression hosts include prokaryotic and eukaryotic organisms such as E.coli, yeast, plants, insect cells, mammalian cells, including human cell lines and transgenic animals. Expression hosts can differ in their protein production levels as well as the types of post-translational modifications that are present on the expressed proteins. The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need and methods for purification.

[0152] Many expression vectors are available and known to those of skill in the art and can be used for expression of proteins. The choice of expression vector will be influenced by the choice of host expression system. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector.

[0153] Soluble hyaluronidase polypeptides also can be utilized or expressed as protein fusions. For example, an enzyme fusion can be generated to add additional functionality to an enzyme. Examples of enzyme fusion proteins include, but are not limited to, fusions of a signal sequence, a tag such as for localization, e.g. a his6 tag or a myc tag, or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and/or membrane association. a. Prokaryotic Cells [0154] Prokaryotes, especially E.coli, provide a system for producing large amounts of proteins. Transformation of E.coli is simple and rapid technique well known to those of skill in the art. Expression vectors for E.coli can contain inducible promoters, such promoters are useful for inducing high levels of protein expression and for expressing proteins that exhibit some toxicity to the host cells. Examples of inducible promoters include the lac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters and the temperature regulated λΡί. promoter.

[0155] Proteins, such as any provided herein, can be expressed in the cytoplasmic environment of E.coli. The cytoplasm is a reducing environment and for some molecules, this can result in the formation of insoluble inclusion bodies. Reducing agents such as dithiothreitol and β-mercaptoethanol and denaturants, such as guanidine-HCI and urea can be used to resolubilize the proteins. An alternative approach is the expression of proteins in the periplasmic space of bacteria which provides an oxidizing environment and chaperonin-like and disulfide isomerases and can lead to the production of soluble protein. Typically, a leader sequence is fused to the protein to be expressed which directs the protein to the periplasm. The leader is then removed by signal peptidases inside the periplasm. Examples of periplasmic-targeting leader sequences include the pelB leader from the pectate lyase gene and the leader derived from the alkaline phosphatase gene. In some cases, periplasmic expression allows leakage of the expressed protein into the culture medium. The secretion of proteins allows quick and simple purification from the culture supernatant. Proteins that are not secreted can be obtained from the periplasm by osmotic lysis. Similar to cytoplasmic expression, in some cases proteins can become insoluble and denaturants and reducing agents can be used to facilitate solubilization and refolding. Temperature of induction and growth also can influence expression levels and solubility, typically temperatures between 25°C and 37°C are used. Typically, bacteria produce aglycosylated proteins. Thus, if proteins require glycosylation for function, glycosylation can be added in vitro after purification from host cells. b. Yeast Cells [0156] Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe, Yarrowia lipolytica, Kluyveromyces lactis and Pichia pastoris are well known yeast expression hosts that can be used for production of proteins, such as any described herein. Yeast can be transformed with episomal replicating vectors or by stable chromosomal integration by homologous recombination. Typically, inducible promoters are used to regulate gene expression. Examples of such promoters include GAL1, GAL7 and GAL5 and metallothionein promoters, such asCUPI, AOX1 or other Pichia or other yeast promoter. Expression vectors often include a selectable marker such as LEU2, TRP1, HIS3 and URA3 for selection and maintenance of the transformed DNA. Proteins expressed in yeast are often soluble. Co-expression with chaperonins such as Bipand protein disulfide isomerasecan improve expression levels and solubility. Additionally, proteins expressed in yeast can be directed for secretion using secretion signal peptide fusions such as the yeast mating type alpha-factor secretion signal from Saccharomyces cerevisae and fusions with yeast cell surface proteins such as the Aga2p mating adhesion receptor or the Arxula adeninivorans glucoamylase. A protease cleavage site such as for the Kex-2 protease, can be engineered to remove the fused sequences from the expressed polypeptides as they exit the secretion pathway. Yeast also is capable of glycosylation at Asn-X-Ser/Thr motifs. c. Insect Cells [0157] Insect cells, particularly using baculovirus expression, are useful for expressing polypeptides such as hyaluro- nidase polypeptides. Insect cells express high levels of protein and are capable of most of the post-translational modifications used by higher eukaryotes. Baculovirus have a restrictive host range which improves the safety and reduces regulatory concerns of eukaryotic expression. Typical expression vectors use a promoter for high level expression such as the polyhedrin promoter of baculovirus. Commonly used baculovirus systems include the baculoviruses such as Autographs californica nuclear polyhedrosis virus (AcNPV), and the Bombyx mori nuclear polyhedrosis virus (BmNPV) and an insect cell line such as Sf9 derived from Spodoptera frugiperda, Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1). For high-level expression, the nucleotide sequence of the molecule to be expressed is fused immediately downstream of the polyhedrin initiation codon of the virus. Mammalian secretion signals are accurately processed in insect cells and can be used to secrete the expressed protein into the culture medium. In addition, the cell lines Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1) produce proteins with glycosylation patterns similar to mammalian cell systems.

[0158] An alternative expression system in insect cells is the use of stably transformed cells. Cell lines such as the Schneider 2 (S2) and Kc cells (Drosophila melanogaster) and C7 cells (Aedes albopictus) can be used for expression. The Drosophila metallothionein promoter can be used to induce high levels of expression in the presence of heavy metal induction with cadmium or copper. Expression vectors are typically maintained by the use of selectable markers such as neomycin and hygromycin. d. Mammalian Cells [0159] Mammalian expression systems can be used to express proteins including soluble hyaluronidase polypeptides. Expression constructs can be transferred to mammalian cells by viral infection such as adenovirus or by direct DNA transfer such as liposomes, calcium phosphate, DEAE-dextran and by physical means such as electroporation and microinjection. Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translational initiation sequence (Kozak consensus sequence) and polyadenylation elements. IRES elements also can be added to permit bicistronic expression with another gene, such as a selectable marker. Such vectors often include transcriptional promoter-enhancers for high-level expression, for example the SV40 promoter-enhancer, the human cytomegalovirus (CMV) promoter and the long terminal repeat of Rous sarcoma virus (RSV). These promoter-enhancers are active in many cell types. Tissue and cell-type promoters and enhancer regions also can be used for expression. Exemplary promoter/enhancer regions include, but are not limited to, those from genes such as elastase I, insulin, immunoglobulin, mouse mammary tumor virus, albumin, alpha fetoprotein, alpha 1 antitrypsin, beta globin, myelin basic protein, myosin light chain 2, and gonadotropic releasing hormone gene control. Selectable markers can be used to select for and maintain cells with the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR) and thymidine kinase. For example, expression can be performed in the presence of methotrexate to select for only those cellsexpressing the DHFR gene. Fusion with cell surface signaling molecules such as TCR-ζ and FceRI-y can direct expression of the proteins in an active state on the cell surface.

[0160] Many cell lines are available for mammalian expression including mouse, rat human, monkey, chicken and hamster cells. Exemplary cell lines include but are not limited to CHO, Balb/3T3, HeLa, MT2, mouse NSO (nonsecreting) and other myeloma cell lines, hybridomaand heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293,293S, 2B8, and HKB cells. Cell lines also are available adapted to serum-free media which facilitates purification of secreted proteins from the cell culture media. Examples include CHO-S cells (Invitrogen, Carlsbad, CA, cat # 11619-012) and the serum free EBNA-1 cell line (Pham et al., (2003) Biotechnol. Bioeng. 84:332-42.). Cell lines also are available that are adapted to grow in special mediums optimized for maximal expression. For example, DG44 CHO cells are adapted to grow in suspension culture in a chemically defined, animal product-free medium. e. Plants [0161] Transgenic plant cells and plants can be used to express proteins such as any described herein. Expression constructs are typically transferred to plants using direct DNA transfer such as microprojectile bombardment and PEG-mediated transfer into protoplasts, and with agrobacterium-mediated transformation. Expression vectors can include promoter and enhancer sequences, transcriptional termination elements and translational control elements. Expression vectors and transformation techniques are usually divided between dicot hosts, such as Arabidopsis and tobacco, and monocot hosts, such as corn and rice. Examples of plant promoters used for expression include the cauliflower mosaic virus promoter, the nopaline synthase promoter, the ribose bisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters. Selectable markers such as hygromycin, phosphomannose isomerase and neomycin phosphotransferase are often used to facilitate selection and maintenance of transformed cells. Transformed plant cells can be maintained in culture as cells, aggregates (callus tissue) or regenerated into whole plants. Transgenic plant cells also can include algae engineered to produce hyaluronidase polypeptides. Because plants have different glycosylation pat- terns than mammalian cells, this can influence the choice of protein produced in these hosts. 3. Purification Techniques [0162] Method for purification of polypeptides, including soluble hyaluronidase polypeptides or other proteins, from host cells will depend on the chosen host cells and expression systems. For secreted molecules, proteins are generally purified from the culture media after removing the cells. For intracellular expression, cells can be lysed and the proteins purified from the extract. When transgenic organisms such as transgenic plants and animals are used for expression, tissues or organs can be used as starting material to make a lysed cell extract. Additionally, transgenic animal production can include the production of polypeptides in milk or eggs, which can be collected, and if necessary, the proteins can be extracted and further purified using standard methods in the art.

[0163] Proteins, such as soluble hyaluronidase polypeptides, can be purified using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate precipitation and ionic exchange chromatography, such as anion exchange. Affinity purification techniques also can be utilized to improve the efficiency and purity of the preparations. For example, antibodies, receptors and other molecules that bind hyaluronidase enzymes can be used in affinity purification. Expression constructs also can be engineered to add an affinity tag to a protein such as a myc epitope, GST fusion or His6 and affinity purified with myc antibody, glutathione resin and Ni-resin, respectively. Purity can be assessed by any method known in the art including gel electrophoresis and staining and spectrophotometric techniques. F. Preparation, Formulation and Administration Of Immune Globulins and Soluble Hyaluronidase Polypeptides [0164] Pharmaceutical compositions of immune globulins and soluble hyaluronidases are provided herein for subcutaneous administration. Formulations of pharmaceutical compositions of soluble hyaluronidases, for example, PFI20, are known in the art (see e.g. published U.S. Application Nos. US20040268425, US20050260186 and US20060104968). Soluble hyaluronidases may be co-formulated or co-administered with pharmaceutical formulations of immune globulin to enhance the delivery of immune globulins to desired sites within the body by increasing the bioavailability of immune globulins. For example, co-administration or co-formulation of IG with a hyaluronidase can improve the extent and/or rate of absorption and thus bioavailability of an agent by causing more of it to reach the bloodstream and/or less of it being degraded after administration by more rapid permeation. Increased absorption and bioavailability can be achieved, for example, by accelerating interstitial flow and potentially connective transport following administration by applying hydrostatic pressure associated with the volume injection combined with a reduction in impedance to flow associated with degradation of hyaluronan. Thus, soluble hyaluronidases can be used to achieve elevated and/or more rapidly achieved concentrations of the immune globulin following subcutaneous administration compared to conventional methods of subcutaneous administration, to provide, for example, a more potent and/or more rapid response for a given dose. Alternatively, the soluble hyaluronidase can be used to allow a given response to be achieved with a lower dose of administered IG. The ability of a soluble hyaluronidase to enhance bulk fluid flow at and near a site of injection or infusion also can improve other aspects of associated pharmacologic delivery. For example, the increase in bulk fluid flow can help to allow the volume of fluid injected to be more readily dispersed from the site of injection (reducing potentially painful or other adverse consequences of injection). This is particularly important for subcutaneous infusions to permit higher doses to be administered. In addition to increased bioavailability, co-administration or co-formulation of IG with soluble hyaluronidase provides for a safer or more convenient route of administration compared to conventional intravenous routes of administration.

[0165] Thus, by virtue of the increased bioavailability, immune globulins can be administered subcutaneously at dosages and frequencies for which current intravenous (IVIG) preparations are prepared and administered. The advantages over current subcutaneous formulations of IG is that co-administered or co-formulated hyaluronidase/IG can result in more favorable dosing regimens, for example, less frequent dosing. By less frequent or lower dosing, side effects associated with toxicity can be reduced. Generally, the pharmacokinetic and/or pharmacodynamics of subcutaneous IG therapy is improved. In addition, subcutaneous administrations of IG also has advantages over current intravenous infusions. For example, subcutaneous infusion permits infusion by the patient or family as opposed to a skilled nurse; infusion can be achieved at higher rates such that IG is infused in 1-3 hours compared to 5-10 hours for conventional IVIG therapies; there is no requirement for functional veins; there is no infusion related side effects such as thrombosis, headache, thrombophlebitis, and nausea and less probability of adverse events; and infusion can be performed at home or anywhere.

[0166] The compositions can be formulated in lyophilized or liquid form. Where the compositions are provided in lyophilized form they can be reconstituted just prior to use by an appropriate buffer, for example, a sterile saline solution. The compositions can be provided together or separately. For purposes herein, such compositions typically are provided separately. The soluble hyaluronidase and IG can be packaged as separate compositions for administration together, sequentially or intermittently. The combinations can be packaged as a kit. 1. Formulations [0167] The compounds can be formulated into any suitable pharmaceutical preparations for subcutaneous administration such as solutions, suspensions, powders, or sustained release formulations. Typically, the compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126). Pharmaceutically acceptable compositions are prepared in view of approvals for a regulatory agency or other agency prepared in accordance with generally recognized pharmacopeia for use in animals and in humans. The formulation should suit the mode of administration.

[0168] Pharmaceutical compositions can include carriers such as a diluent, adjuvant, excipient, or vehicle with which a hyaluronidase or IG is administered. Examples of suitable pharmaceutical carriers are described in "Remington’s Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, generally in purified form or partially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions. Compositions can contain along with an active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a bindersuch as starch, natural gums, such as gum acaciagelatin, glucose, molasses, polyvinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. A composition, if desired, also can contain minor amounts of wetting or emulsifying agents, or pH buffering agents, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.

[0169] Pharmaceutically therapeutically active compounds and derivatives thereof are typically formulated and administered in unit dosage forms or multiple dosage forms. Each unit dose contains a predetermined quantity of therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit dose forms can be administered in fractions or multiples thereof. A multiple dose form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dose form. Examples of multiple dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit doses that are not segregated in packaging. Generally, dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier can be prepared.

[0170] Compositions provided herein typically are formulated for administration by subcutaneous route, although other routes of administration are contemplated, such as any route known to those of skill in the art including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, epidural, nasal, oral, vaginal, rectal, topical, local, , otic, inhalational, buccal (e.g., sublingual), and transdermal administration or any route. Formulations suited for such routes are known to one of skill in the art. Administration can be local, topical or systemic depending upon the locus of treatment. Local administration to an area in need of treatment can be achieved by, for example, but not limited to, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant. Compositions also can be administered with other biologically active agents, either sequentially, intermittently or in the same composition. Administration also can include controlled release systems including controlled release formulations and device controlled release, such as by means of a pump.

[0171] The most suitable route in any given case depends on a variety of factors, such as the nature of the disease, the progress of the disease, the severity of the disease the particular composition which is used. For purposes herein, it is desired that hyaluronidases are administered so that they reach the interstitium of skin or tissues, thereby degrading the interstitial space for subsequent delivery of immunoglobulin. Thus, direct administration under the skin, such as by subcutaneous administration methods, is contemplated. Thus, in one example, local administration can be achieved by injection, such as from a syringe or other article of manufacture containing a injection device such as a needle. In another example, local administration can be achieved by infusion, which can be facilitated by the use of a pump or other similar device. Other modes of administration also are contemplated. Pharmaceutical composition can be formulated in dosage forms appropriate for each route of administration.

[0172] Subcutaneous administration, generally characterized by injection or infusion, is contemplated herein. Inject- ables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. The pharmaceutical compositions may contain other minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins. Implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained (see, e. g., U. S. Patent No. 3,710,795) is also contemplated herein. The percentage of active compound contained in such compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject.

[0173] Injectables are designed for local and systemic administration. For purposes herein, local administration is desired for direct administration to the affected interstitium. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous. If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.

[0174] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multiple-dose containers, which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEENs 80). A sequestering or chelating agent of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0175] The concentration of the pharmaceutically active compound is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as is known in the art. The unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. The volume of liquid solution or reconstituted powder preparation, containing the pharmaceutically active compound, is a function of the disease to be treated and the particular article of manufacture chosen for package. All preparations for parenteral administration must be sterile, as is known and practiced in the art.

[0176] In one example, pharmaceutical preparation can be in liquid form, for example, solutions, syrups orsuspensions. If provided in liquid form, the pharmaceutical preparations can be provided as a concentrated preparation to be diluted to a therapeutically effective concentration before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). In another example, pharmaceutical preparations can be presented in lyophilized form for reconstitution with water or other suitable vehicle before use.

[0177] Administration methods can be employed to decrease the exposure of selected compounds to degradative processes, such as proteolytic degradation and immunological intervention via antigenic and immunogenic responses. Examples of such methods include local administration at the site of treatment. Pegylation of therapeutics has been reported to increase resistance to proteolysis, increase plasma half-life, and decrease antigenicity and immunogenicity. Examples of pegylation methodologies are known in the art (see for example, Lu and Felix, Int. J. Peptide Protein Res., 43:127-138,1994; Lu and Felix, Peptide Res., 6:142-6,1993; Felix etal., Int. J. Peptide Res., 46 :253-64,1995; Benhar et al., J. Biol. Chem., 269: 13398-404, 1994; Brumeanu et al., J Immunol., 154: 3088-95, 1995; see also, Caliceti et al. (2003) Adv. Drug Deliv. Rev. 55(10):1261-77 and Molineux (2003) Pharmacotherapy 23 (8 Pt2):3S-8S). Pegylation also can be used in the delivery of nucleic acid molecules in vivo. For example, pegylation of adenovirus can increase stability and gene transfer (see, e.g., Cheng et al. (2003) Pharm. Res. 20(9): 1444-51).

Lyophilized powders [0178] Of interest herein are lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They may also be reconstituted and formulated as solids or gels.

[0179] The sterile, lyophilized powder is prepared by dissolving an active compound in a buffer solution. The buffer solution may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. Briefly, the lyophilized powder is prepared by dissolving an excipient, such as dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent, in a suitable buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art. Then, a selected enzyme is added to the resulting mixture, and stirred until it dissolves. The resulting mixture is sterile filtered or treated to remove particulates and to insure sterility, and apportioned into vials for lyophilization. Each vial will contain a single dosage or multiple dosages of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature. Reconstitution of this lyophilized powder with a buffer solution provides a formulation for use in parenteral administration. 2. Dosage and Administration [0180] The soluble hyaluronidase provided herein can be formulated as pharmaceutical compositions, typically for single dosage administration, in combination with IG. The selected soluble hyaluronidase is included in an amount sufficient to exert a therapeutically useful effect of the IG in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration can be determined empirically by testing the polypeptides in known in vitro and in vivo systems such as by using the assays provided herein or known in the art (see e.g., Taliani et al. (1996) Anal. Biochem., 240: 60-67; Filocamo et al. (1997) J Virology, 71: 1417-1427; Sudo et al. (1996) Antiviral Res. 32: 9-18; Buffard et al. (1995) Virology, 209:52-59; Bianchi et al. (1996) Anal. Biochem., 237: 239-244; Hamatake et al. (1996) Intervirology 39:249-258; Steinkuhler et al. (1998) Biochem., 37:8899-8905; D’Souza et al. (1995) J Gen. Virol., 76:1729-1736; Takeshita et al. (1997) Anal. Biochem., 247:242-246; see also e.g, Shimizu et al. (1994) J. Virol. 68:8406-8408; Mizutani et al. (1996) J. Virol. 70:7219-7223; Mizutani et al. (1996) Biochem. Biophys. Res. Commun., 227:822-826; Lu et al. (1996) Proc. Natl. Acad. Sci (USA), 93:1412-1417; Hahm et al., (1996) Virology, 226:318-326; Ito et al. (1996) J. Gen. Virol., 77:1043-1054; Mizutani et al. (1995) Biochem. Biophys. Res. Commun., 212:906-911; Cho et al. (1997) J. Virol. Meth. 65:201-207 and then extrapolated therefrom for dosages for humans.

[0181] Typically, a therapeutically effective dose is at or about 500 Units to 100,000 Units of a soluble hyaluronidase. For example, soluble hyaluronidase can be administered subcutaneously at or about 500 Units, 1000 Units, 2000 Units, 5000 Units, 10,000 Units, 30,000 Units, 40,000 Units,, 50,000 Units, 60,000 Units, 70,000 Units, 80,000 Units, 90,000 Units, 100,000 Units or more. In some examples, dosages can be provided as a ratio IG administered. For example, hyaluronidase can be administered at 10 U/gram to 500 U/g or more of IG, for example, at or about 10 U/g, 20 U/g, 30U/g, 40 U/g, 50 U/g, 60 U/g, 70 U/g, 80 U/g, 90 U/g, 100 U/g, 150 U/g, 200 U/g, 300 U/g, 400 U/g, 500 U/g or more. Typically, volumes of injections or infusions of hyaluronidase contemplated herein are from at or about 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml or more. The hyaluronidase can be provided as a stock solution at or about 50 U/ml, 100 U/ml, 150 U/ml, 200 U/ml, 400 U/ml or 500 U/ml or can be provided in a more concentrated form, for example at or about 1000 U/ml, 1500 Units/ml, 2000 U/ml, 4000 U/ml or 5000 U/ml for use directly or for dilution to the effective concentration prior to use. The soluble hyaluronidase can be provided as a liquid or lyophilized formulation. Lyophilized formulations are ideal for storage of large Units doses of soluble hyaluronidase.

[0182] The immune globulin preparations provided herein can be formulated as pharmaceutical compositions forsingle or multiple dosage use. Generally, the IG preparations are formulated in pharmaceutical compositions to achieve dosage regimes (doses and frequencies) for which current intravenous (IVIG) preparations are prepared and administered for particular IG-treatable diseases or conditions. One of skill in the art is familiar with dosage regimes for IVIG administration of particular diseases or conditions. For example, Section H below provides exemplary dosage regimes (doses and frequencies) of IG for particular diseases and conditions. Other dosage regimes are well known to those of skill in the art. In some examples, the dosage frequency can be daily over an interval of time given over consecutive or alternate days, for example, 1,2, 3, 4, 5, 6, 7, 8, 9,10 or more days. In other examples, the dosage regime is weekly, for example, once every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks or more. Typically, immune globulin preparations are formulated for single dose administration in an amount sufficient to provide a once monthly dose, but can be provided in lesser amounts for multiple dosage administrations. For example, once monthly doses of IG preparations can be administered daily, weekly, biweekly or once a month. Dosage regimes can be continued for months oryears. The IG preparations can be provided in lyophilized or liquid form as discussed elsewhere herein.

[0183] The immune globulin is provided in a therapeutically effective dose. Therapeutically effective concentration can be determined empirically by testing the compounds in known In vitro and in vivo systems, such as the assays provided herein. The concentration of a selected immune globulin in the composition depends on absorption, inactivation and excretion rates of the complex, the physicochemical characteristics of the complex, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. For example, it is understood that the precise dosage and duration of treatment is a function of the tissue being treated and may be determined empirically using known testing protocols or by extrapolation from In vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the age of the individual treated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope thereof. The amount of a selected immune globulin preparation to be administered for the treatment of a disease or condition, for example an IG-treatable disease or condition, can be determined by standard clinical techniques. In addition, in vitro assays and animal models can be employed to help identify optimal dosage ranges.

[0184] Hence, the precise dosage, which can be determined empirically, can depend on the particular immune globulin preparation, the regime and dosing schedule with the soluble hyaluronidase, the route of administration, the type of disease to be treated and the seriousness of the disease. Generally, the IG preparations have a protein concentration that is or is about 5 to 15% w/v, 6 to 15% w/v, or 8 to 12% w/v of IG composition, such as, for example, 10% w/v. For example, IG is provided in an amount that permits subcutaneous administration of a dose equivalent to a once monthly IV dose for the particular indication being treated. The particular once monthly IV dose is a function of the disease to be treated, and thus can vary. Exemplary dosages ranges for subcutaneous administration of IG are from at or about 1 gram (g), 5g, 10g, 20g, 30g, 40g, 50g, 60g, 70g, 80g, 90g, 100g or 200g. The particular dosage and formulation thereof depends upon the indication and individual. For example, dosages can be administered at 50 mg/kg body weight (BW), 100 mg/kg BW, 200 mg/kg BW, 300 mg/kg BW, 400 mg/kg BW, 500 mg/kg BW, 600 mg/kg BW, or more. If necessary dosage can be empirically determined. To achieve such dosages, volumes of IG preparations administered subcutaneously can be at or about 50 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml, 700 ml or more. For example, a 10% liquid IG formulation (100 mg/ml) for indications described herein can be administered in a volume of 50 ml to 700 ml to achieve a dosage of 0.5 g to 70 g of IG.

[0185] Where large volumes are administered, administration is typically by infusion. Subjects can be dosed at rates of infusion at or about 0.5 ml/kg/BW/h, 1 ml/kg/BW/h, 2 ml/kg/BW/h, 3 ml/kg/BW/h, 4 ml/kg/BW/h, or 5 ml/kg/BW/h. The infusion rate can be empirically determined, and typically is a function of the tolerability of the subject. If an adverse reaction occurs during the infusion, the rate of infusion can be slowed to the rate immediately below that at which the adverse event occurred. If the adverse event resolves in response to the reduction in rate, the infusion rate can be slowly increased at the discretion of the physician. Subcutaneous IG infusion can be facilitated by gravity, pump infusion or injection of a full 20-30 gram dose. Generally, for infusions intravenous infusion pumps can be employed. IG can be infused at rates at or about 5 ml/h, 10 ml/h, 30 ml/h, 60 ml/h, 120 ml/h, 240 ml/h or 300 ml/h. Infusion rates can be increased during the course of treatment so long as the infusion is tolerated by the patient. Generally, time of administration of infusion is at or about 0.5 h, 1 h, 1.5h, 2 h, 2.5h, 3 h, 4 h or more. Due to the high rate of infusion achieved by subcutaneous administration of IG coformulated and/or co-administered with hyaluronidase, the time of infusion is significantly less than for conventional IVIG therapies. Where infusion time exceeds the desired limit, a second infusion site can be started at the physician and subject’s discretion. The second site typically is started at least 10 cm from the initial site.

[0186] Techniques for infusion are known to one of skill in the art, and are within the skill of a treating physician. Generally, the appropriate dose of IG can be pooled into a standard IV bag. For example, a non-vented infusion set can be used that has a Y-port near its terminus. A 24-gauge subcutaneous infusion needle can be inserted at a site of the subject’s preferences, but the abdomen and secondarily the thighs are recommended because of the volume of solution to be infused. The hyaluronidase and IG can be provided in the same Y port apparatus. Other articles of manufacture also can be used herein for purposes of infusion by gravity or a pump, and include, but are not limited to tubes, bottles, syringes or other containers.

[0187] The soluble hyaluronidase can be administered subsequently, intermittently or simultaneously from the IG preparation. Generally, the hyaluronidase is administered prior to administration of the IG preparation to permit the hyaluronidase to degrade the hyaluronic acid in the interstitial space. For example, the soluble hyaluronidase can be administered 1 minute, 2 minute, 3 minute, 4 minute, 5 minute, 6 minute, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes or 30 minutes prior to administration of the IG preparation. In some examples, the hyaluronidase is administered together with the immune globulin preparation. As will be appreciated by those of skill in the art, the desired proximity of co-administration depends in significant part on the effective half lives of the agents in the particular tissue setting, and the particular disease being treated, and can be readily optimized by testing the effects of administering the agents at varying times in suitable models, such as in suitable animal models. In some situations, the optimal timing of administration of the hyaluronidase will exceed 60 minutes.

[0188] Generally, prior to infusion of IG, a soluble hyaluronidase is injected at a rate of at or about 0.2 ml/min, 0.5 ml/min, 1 ml/min. 2 ml/min, 5 ml/min, 10 ml/min or more. For example, the soluble hyaluronidase can be injected through the same Y-port used for subsequent infusion of IG. As noted above, the volume of soluble hyaluronidase administered is a function of the dosage required, but can be varied depending on the concentration of a soluble hyaluronidase stock formulation available. For example, it is contemplated herein that soluble hyaluronidase is not administered in volumes greater than about 50 ml, and typically is administered in a volume of 5-30 ml. A syringe pump can be used for the higher volumes, at the discretion of the physician.

[0189] In the event that an infusion is not tolerated (e.g., it causes moderate to severe local reactions), a second infusion site can be started so that the subject receives the full dosage.

[0190] An IG preparation can be administered at once, or can be divided into a number of smaller doses to be administered at intervals of time. Selected IG preparations can be administered in one or more doses over the course of a treatment time for example over several hours, days, weeks, or months. In some cases, continuous administration is useful. It is understood that the precise dosage and course of administration depends on the indication and patients tolerability.

[0191] Also, it is understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values also can vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or use of compositions and combinations containing them. The compositions can be administered hourly, daily, weekly, monthly, yearly or once. Generally, dosage regimens are chosen to limit toxicity. It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or bone marrow, liver or kidney or other tissue dysfunctions. Conversely, the attending physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects). G. Methods of Assessing Activity, Bioavailability and Pharmacokinetics [0192] Assays can be used to assess the in vitro and in vivo activities of immune globulin alone or in combination with a soluble hyaluronidase. Included among such assays are those that assess the pharmacokinetic properties of subcutaneously-administered immune globulin, including bioavailability, and tolerability. The biological activity of both immune globulin and hyaluronidase also can be assessed using assays well known in the art. Such assays can be used, for example, to determine appropriate dosages of immune globulin and hyaluronidase, and the frequency of dosing, for treatment. 1. Pharmacokinetics and tolerability [0193] Pharmacokinetic and tolerability studies, such as those described in Examples 1, below, can be performed using animal models or can be performed during clinical studies with patients. Animal models include, but are not limited to, mice, rats, rabbits, dogs, guinea pigs and non-human primate models, such as cynomolgus monkeys or rhesus macaques. In some instances, pharmacokinetic and tolerability studies are performed using healthy animals. In other examples, the studies are performed using animal models of a disease for which therapy with immune globulin is considered, such as animal models of any of the diseases and conditions described below.

[0194] The pharmacokinetics of subcutaneously administered immune globulin can be assessed by measuring such parameters as the maximum (peak) plasma immune globulin concentration (Cmax), the peak time (i.e. when maximum plasma immune globulin concentration occurs; Tmax), the minimum plasma immune globulin concentration (i.e. the minimum plasma concentration between doses of immune globulin; Cmin), the elimination half-life (T1/2) and area under the curve (i.e. the area under the curve generated by plotting time versus plasma immune globulin concentration; AUC), following administration. The absolute bioavailability of subcutaneously administered immune globulin is determined by comparing the area under the curve of immune globulin following subcutaneous delivery (AUC^) with the AUC of immune globulin following intravenous delivery (AUCjy). Absolute bioavailability (F), can be calculated using the formula: F = ([AUC]sc X dosesc) / ([AUC]iv x doseiv). The concentration of immune globulin in the plasma following subcutaneous administration can be measured using any method known in the art suitable for assessing concentrations of immune globulin in samples of blood. Exemplary methods include, but are not limited to, ELISA and nephelometry.

[0195] A range of doses and different dosing frequency of dosing can be administered in the pharmacokinetic studies to assess the effect of increasing or decreasing concentrations of immune globulin and/or hyaluronidase in the dose. Pharmacokinetic properties of subcutaneously administered immune globulin, such as bioavailability, also can be assessed with or without co-administration of hyaluronidase. For example, dogs, such as beagles, can be administered immune globulin subcutaneously in combination with hyaluronidase, or alone. Intravenous doses of immune globulin also are given to another group of beagles. Blood samples can then be taken at various time points and the amount of immune globulin in the plasma determine, such as by nephelometry. The AUC can then be measured and the bioavailability of subcutaneously administered immune globulin administered with or without hyaluronidase can be determined. Such studies can be performed to assess the effect of co-administration with hyaluronidase on pharmacokinetic properties, such as bioavailability, of subcutaneously administered immune globulin.

[0196] Studies to assess safety and tolerability also are known in the art and can be used herein. Following subcutaneous administration of immune globulin, with or without co-administration of hyaluronidase, the development of any adverse reactions can be monitored. Adverse reactions can include, but are not limited to, injection site reactions, such as edema or swelling, headache, fever, fatigue, chills, flushing, dizziness, urticaria, wheezing or chest tightness, nausea, vomiting, rigors, back pain, chest pain, muscle cramps, seizures or convulsions, changes in blood pressure and anaphylactic or severe hypersensitivity responses. Typically, a range of doses and different dosing frequencies are be administered in the safety and tolerability studies to assess the effect of increasing or decreasing concentrations of immune globulin and/or hyaluronidase in the dose. 2. Biological activity a. Immune globulin [0197] The ability of immune globulin to act as a therapeutic agent can be assessed in vitro or in vivo. For example, in vitro assays can be performed to assess the ability of immune globulin to neutralize viral or bacterial infectivity (Hiemstra et al., (1994) J Lab Clin Med 123:241-6). Other in vitro assays can be utilized to assess other biological activities of immune globulin. For example, the ability of immune globulin preparations to interact with and modulate complement activation products, bind idiotypic antibody, bindFc receptors on macrophages, and suppress various inflammatory mediators including cytokines, chemokines, and metalloproteinases, can be assessed using any method known in the art, including, but not limited to, ELISA, Western blot, Northern blot, and flow cytometry to assess marker expression. For example, the effect of immune globulin on the expression of chemokine receptors on peripheral blood mononuclear cells can be assessed using flow cytomtery (Trebst et al., (2006) Eur J Neurology). In another example, the effect of immune globulin on metalloproteinase expression in macrophages can be assessed using Northern blot analysis (Shapiro et al., (2002) Cancer 95:2032-2037).

[0198] In vivo studies using animal models also can be performed to assess the therapeutic activity of immune globulin. Immune globulin can be administered to animal models infected with one or more microorganisms and the effect on progression of infection can be assessed, such as by measuring the number of microorganisms or measuring weight as a marker of morbidity. The therapeutic effect of immune globulin also can be assessed using animal models of the diseases and conditions for which therapy using immune globulin is considered. Such animal models are known in the art, and include, but are not limited to, small animal models for X-linked agammaglobulinemia (XLA), SCID, Wiskott-Aldrich syndrome, Kawasaki disease, Guillain-Barre syndrome, ITP, polymyositis, Lambert-Eaton myasthenic syndrome, Myasthenia gravis and Moersch-Woltmann syndrome (Czitrom et al (1985) J Immunol 134:2276-2280, Ellmeier et al., (2000) J Exp Med. 192: 1611-1624, Ohno (2006) Drug Discovery Today: Disease Models 3:83-89, Oyaizu et al (1988) J Exp Med 2017-2022, Hansen etal., (2002) Blood 100:2087-2093, Strongwateretal., (1984) Arthritis Rheum. 27:433-42, Kim et al. (1998) Annals NY Acad Sci 841:670-676, Christadoss et al. (2000) 94:75-87, Sommer et al., (2005) Lancet 365:1406-1411, U.S. Patent No.7309810) b. Hyaluronidase [0199] Hyaluronidase activity can be assessed using methods well known in the art. In one example, activity is measured using a microturbidity assay. This is based on the formation of an insoluble precipitate when hyaluronic acid binds with serum albumin. The activity is measured by incubating hyaluronidase with sodium hyaluronate (hyaluronic acid) for a set period of time (e.g. 10 minutes) and then precipitating the undigested sodium hyaluronate with the addition of acidified serum albumin. The turbidity of the resulting sample is measured at 640 nm after an additional development period. The decrease in turbidity resulting from hyaluronidase activity on the sodium hyaluronate substrate is a measure of hyaluronidase enzymatic activity. In another example, hyaluronidase activity is measured using a microtiter assay in which residual biotinylated hyaluronic acid is measured following incubation with hyaluronidase (see e.g. Frost and Stern (1997) Anal. Biochem. 251:263-269, U.S. Patent Publication No. 20050260186). The free carboxyl groups on the glucuronic acid residues of hyaluronic acid are biotinylated, and the biotinylated hyaluronic acid substrate is covalently couple to a microtiter plate. Following incubation with hyaluronidase, the residual biotinylated hyaluronic acid substrate is detected using an avidin-peroxidase reaction, and compared to that obtained following reaction with hyaluronidase standards of known activity. Other assays to measure hyaluronidase activity also are known in the art and can be used in the methods herein (see e.g. Delpech etal., (1995) Anal. Biochem. 229:35-41; Takahashi etal., (2003) Anal. Biochem. 322:257-263).

[0200] The ability of hyaluronidase to act as a spreading or diffusing agent also can be assessed. For example, trypan blue dye can be injected subcutaneously with or without hyaluronidase into the lateral skin on each side of nude mice. The dye area is then measured, such as with a microcaliper, to determine the ability of hyaluronidase to act as aspreading agent (U.S. Patent No. 20060104968). H. Therapeutic uses [0201] The methods described herein can be used for treatment of any condition for which immune globulin is employed. Immune globulin (IG) can be administered subcutaneously, in combination with hyaluronidase, to treat any condition that is amendable to treatment with immune globulin. This section provides exemplary therapeutic uses of IG. The therapeutic uses described below are exemplary and do not limit the applications of the methods described herein. Therapeutic uses include, but are not limited to, immunoglobulin replacement therapy and immunomodulation therapy for various immunological, hematological, neurological, inflammatory, dermatological and/or infectious diseases and conditions. In some examples, immune globulin is administered to augment the immune response in healthy patients, such as following possible exposure to infectious disease (e.g. accidental needle stick injury). It is within the skill of a treating physician to identify such diseases or conditions.

[0202] Immune globulin can be co-administered with hyaluronidase subcutaneously, in combination with other agents used in the treatment of these diseases and conditions. For example, other agents that can be administered include, but are not limited to, antibiotics, chemotherapeutics, steroidal anti-inflammatories, non-steroidal anti-inflammatories, and other immunomodulatory agents such as cytokines, chemokines and growth factors.

[0203] If necessary, a particular dosage and duration and treatment protocol can be empirically determined or extrapolated. For example, exemplary doses of intravenously administered immune globulin can be used as a starting point to determine appropriate dosages. Dosage levels can be determined based on a variety of factors, such as body weight of the individual, general health, age, the activity of the specific compound employed, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, and the patient’s disposition to the disease and the judgment of the treating physician. Generally, dosages of immune globulin are from or about 100 mg per kg body weight (i.e. 100 mg/kg BW) to 2 g/kg BW, and dosages of hyaluronidase are from or about 10 U/gram to 500 U/g or more of immune globulin, for example, at or about 10 U/g, 20 U/g, 30U/g, 40 U/g, 50 U/g, 60 U/g, 70 U/g, 80 U/g, 90 U/g, 100 U/g, 150 U/g, 200 U/g, 300 U/g, 400 U/g, 500 U/g or more. It is understood that the amount to administer will be a function of the indication treated, and possibly side effects that will be tolerated. Dosages can be empirically determined using recognized models for each disorder.

[0204] Upon improvement of a patient’s condition, a maintenance dose of immune globulin can be administered subcutaneously in combination with hyaluronidase, if necessary, and the dosage, the dosage form, or frequency of administration, or a combination thereof can be modified. In some cases, a subject can require intermittent treatment on a long-term basis upon any recurrence of disease symptoms. I. Primary immune deficiency with antibody deficiency [0205] Immune globulin can be used to treat primary immune deficiency with antibody deficiency. Primary immune deficiency encompasses many disorders that are characterized by a deficiency of one or more proteins of the immune system. Typically, primary immune deficiencies are inherited disorders, and many are manifest by failure of protective antibody production. Thus, immune globulin can be administered as immunoglobulin replacement therapy to patients presenting with such diseases. Exemplary of primary immune deficiencies include, but are not limited to, common variable immunodeficiency (CVID), congenital agammaglobulinemia, Wiskott-Aldrich syndrome, severe combined immunodeficiency (SCID), primary hypogammaglobulinemia, primary immunodeficiency diseases with antibody deficiency, X-linked agammaglobulinaemia (XLA), hypogammaglobulinaemia of infancy, and paraneoplastic cerebellar degeneration with no antibodies. Immune globulin can be administered subcutaneously, in combination with hyaluronidase, to patients with primary im mune deficiency with antibody deficiency at doses sim ilar to the doses used for intravenous adm inistration of immune globulin. Exemplary doses include, for example, between 100 mg/kg BW and 800 mg/kg BW immune globulin, at four week intervals. The dose can be increased or decreased, as can the frequency of the doses, depending on the clinical response. 2. Acquired hypogammaglobulinemia secondary to hematological malignancies [0206] Patients with acquired hypogammaglobulinemia secondary to hematological malignancies, such as Chronic Lymphocytic Leukemia (CLL), multiple myeloma (MM), non-Hodgkin’s lymphoma (NHL) &amp; other relevant malignancies and post-hematopoietic stem cell transplantation, are susceptible to bacterial infections due to. Hypogammaglobulinemia is caused by a lack of B-lymphocytes and a resulting low level of antibodies in the blood, and can occur in patients with CLL, MM, NHL and as a result of both leukemia-related immune dysfunction and therapy-related immunosuppression. The deficiency in humoral immunity is largely responsible for the increased risk of infection-related morbidity and mortality in these patients, especially by encapsulated microorganisms. For example, Streptococcus pneumoniae, Haemophilus influenzae, and Staphyloccus aureus, as well as Legionella and Nocardia spp. are frequent bacterial pathogens that cause pneumonia in patients with CLL. Opportunistic infections such as Pneumocystis carinii, fungi, viruses, and mycobacteria also have been observed. The number and severity of infections in these patients can be significantly reduced by administration of immune globulin (Griffiths et al., (1989) Blood 73:366-368, Chapel et al. (1994) Lancet 343:1059-1063). Such patients, therefore, can be administered immune globulin subcutaneously in combination with hyaluronidase using the methods described herein to prevent recurrent infections. Exemplary dosages include those used for intravenous administration of immune globulin to patients with acquired hypogammaglobulinemia secondary to hematological malignancies. For example, about 400 mg/kg BW immune globulin, in combination with hyaluronidase, can be administered subcutaneously every 3 to 4 weeks. In a further example, an additional dose of 400 mg/kg BW can be administered in the first month of therapy where the patient’s serum IgG is less than 4 g/L. The amount of immune globulin administered, and the frequency of the doses, can be increased or decreased as appropriate. 3. Kawasaki’s disease [0207] Kawasaki disease is an acute, febrile, multi-system disease of children and young infants often involving the coronary arteries. It also is known as lymph node syndrome, mucocutaneous node disease, infantile polyarteritis and Kawasaki syndrome, and is a poorly understood self-limited vasculitis that affects many organs, including the skin and mucous membranes, lymph nodes, blood vessel walls, and the heart. Coronary artery aneurysms can occur from the second week of illness during the convalescent stage. Although the cause of the condition is unknown, there is evidence that the characteristic vasculitis results from an immune reaction characterized by T-cell and macrophage activation to an unknown antigen, secretion of cytokines, polyclonal B-cell hyperactivity, and the formation of autoantibodies to endothelial cells and smooth muscle cells. In genetically susceptible individuals, one or more uncharacterized common infectious agents, possibly with super-antigen activity, may trigger the disease. Immune globulin administered early in Kawasaki disease can prevent coronary artery pathology. Subcutaneous administration of immune globulin in combination with hyaluronidase to patients with ongoing inflammation associated with Kawasaki disease can ameliorate symptoms. Exemplary dosages include those used for intravenous administration of immune globulin to patients with Kawasaki disease. For example, a patient with Kawasaki disease can be administered about 1-2 g per kg patient body weight of immune globulin. This can be administered, for example, in four doses of 400 mg/kg BW for four consecutive days. In another example, 1 g/kg BW immune globulin is administered as a single dose over a 10 hour period. The amount of immune globulin administered can be increased or decreased as appropriate. 4. Chronic inflammatory demyelinating polyneuropathy [0208] Chronic inflammatory demyelinating polyneuropathy (CIDP) is a neurological disorder characterized by progressive weakness and impaired sensory function in the legs and arms. The disorder, which is sometimes called chronic relapsing polyneuropathy, is caused by damage to the myelin sheath of the peripheral nerves. Although it can occur at any age and in both genders, CIDP is more common in young adults and in men more so than women. It often presents with symptoms that include tingling or numbness (beginning in the toes and fingers), weakness of the arms and legs, loss of deep tendon reflexes (areflexia), fatigue, and abnormal sensations. CIDP is closely related to Guillain-Barre syndrome and it is considered the chronic counterpart of that acute disease. There is no specific diagnostic test but characteristic clinical and laboratory findings help distinguish this disorder from other immune mediated neuropathic syndromes. Studies indicate that treatment with immune globulin reduces symptoms (van Schaik et al., (2002) Lancet Neurol. 1:497-498). Thus, immune globulin can be co-administered with hyaluronidase subcutaneously to patients presenting with CIDP using the methods described herein. Exemplary dosages include those used for intravenous administration of immune globulin to patients with CIDP. In one example, a patient with CIDP is administered about 2 g/kg BW of immune globulin subcutaneously, in combination with hyaluronidase. This can be administered, for example, in five doses of 400 mg/kg BW for five consecutive days. The amount of immune globulin administered can be increased or decreased as appropriate. 5. Guillain-Barre Syndrome [0209] Guillain-Barre syndrome is a neurologic autoimmune disorder involving inflammatory demyelination of peripheral nerves. The first symptoms include varying degrees of weakness or tingling sensations in the legs, which can spread to the arms and upper body. These symptoms can increase in intensity until the muscles cannot be used at all and the patient is almost totally paralyzed, resulting in a life-threatening condition. Although recovery is generally good or complete in the majority of patients, persistent disability has been reported to occur in about 20% and death in 4 to 15% of patients. Guillain-Barre syndrome can occur a few days or weeks after symptoms of a respiratory or gastrointestinal viral infection. In some instances, surgery or vaccinations can trigger the syndrome. The disorder can develop over the course of hours or days, or it may take up to 3 to 4 weeks. A nerve conduction velocity (NCV) test can give a doctor clues to aid the diagnosis. In some instances, a spinal tap can be used in diagnosis as the cerebrospinal fluid in Guillain-Barre syndrome patients typically contains more protein than normal subjects.

[0210] Although there is no known cure for Guillain-Barre syndrome, treatment with immune globulin can lessen the severity of the illness and accelerate recovery. Immune globulin can be administered subcutaneously to patients in combination with hyaluronidase at an appropriate dose, such as, for example, a dose similar to the dose use to administer immune globulin intravenously to patients with Guillain-Barre syndrome. For example, a patient with Guillain-Barre syndrome can administered about 2 g/kg BW of immune globulin, in combination with hyaluronidase, subcutaneously. This can be administered, for example, in five doses of 400 mg/kg BW for five consecutive days. The amount of immune globulin administered can be increased or decrease depending on, for example, the severity of the disease and the clinical response to therapy, which can be readily evaluated by one of skill in the art. 6. Idiopathic thrombocytopenic purpura [0211] Idiopathic thrombocytopenic purpura (ITP), also known as primary immune thrombocytopenic purpura and autoimmune thrombocytopenic purpura, is a reduction in platelet count (thrombocytopenia) resulting from shortened platelet survival due to anti-platelet antibodies. When platelet counts are very low (e.g. <30 X 109/L), bleeding into the skin (purpura) and mucous membranes can occur. Bone marrow platelet production (megakaryopoiesis) in patients with ITP is morphologically normal. In some instances, there is additional impairment of platelet function related to antibody binding to glycoproteins on the platelet surface. ITP can present as chronic and acute forms. Approximately 80% of adults with ITP have the chronic form of disease. The highest incidence of chronic ITP is in women aged 15-50 years, although some reports suggest increasing incidence with age. ITP is relatively common in patients with HIV. While ITP can be found at any stage of the infection, its prevalence increases as HIV disease advances.

[0212] Studies have demonstrated that immune globulin can be used to treat patients with ITP (Godeau et al. (1993) Blood 82(5):1415-21, Godeau et al. (1999) Br J Haematol 1999;107(4):716-9). Immune globulin can be administered subcutaneously to patients in combination with hyaluronidase at a dose similar to the dose use to administer immune globulin intravenously, to treat patients with ITP. For example, a patient with ITP can administered about 1 to 2 g /kg of immune globulin, in combination with hyaluronidase, subcutaneously. This can be administered over several days, or can be administered in one dose. In some examples, five doses of 400 mg/kg BW immune globulin on consecutive days is administered. In another example, 1 g/kg BW is administered for 1-2 consecutive days, depending on platelet count and clinical response. The amount of immune globulin administered, and the frequency of the doses, can be increased or decrease depending on, for example, platelet count and the clinical response to therapy, which can be readily evaluated by one of skill in the art. 7. Inflammatory myopathies: polymyositis, dermatomyositis and inclusion body myositis [0213] Inflammatory myopathies are a group of muscle diseases involving the inflammation and degeneration of skeletal muscle tissues. These disorders are acquired and all present with significant muscle weakness and the presence of an inflammatory response within the muscle. Dermatomyositis (DM) is the most easily recognized of the inflammatory myopathies due to its distinctive rash, which occurs as a patchy, dusky, reddish or lilac rash on the eyelids, cheeks, and bridge of the nose, and on the back or upper chest, elbows, knees and knuckles. In some patients, calcified nodules or hardened bumps develop under the skin. The rash often precedes muscle weakness, which typically develops over a period of weeks but may develop over months or even days. Dermatomyositis can occur at any age from childhood to adulthood and is more common in females than males. Approximately one third of DM patients report difficulty swallowing. Muscle pain and tenderness generally occurs in less than 25% of adults with DM, but more than 50% of children with DM complain of muscle pain and tenderness.

[0214] Polymyositis (PM) does not have the characteristic rash of dermatomyositis, and the onset of muscle weakness usually progresses slower than DM. Many PM patients present have difficulty in swallowing. In some instances, the patients also have difficulty breathing due to muscle failure. As many as one third of PM patients have muscle pain. PM. The disease affects more women than men, and rarely affects people under the age of 20, although cases of childhood and infant polymyositis have been reported.

[0215] Inclusion body myositis (IBM) is very similar to polymyositis. Onset of muscle weakness in IBM is usually very gradual, taking place over months or years. It is different from PM in that both proximal and distal muscles are affected, while generally only the proximal muscles are affected in PM. Typical findings include weakness of the wrist flexors and finger flexors. Atrophy of the forearms is characteristic of the disease, and atrophy of the quadriceps muscle is common with varying degrees of weakness in other muscles. Approximately half of the patients afFlicted with IBM have difficulty swallowing. Symptoms of IBM usually begin after age 50, although no age group is excluded. IBM occurs more frequently in men than women. About one in ten cases of IBM may be hereditary.

[0216] Studies indicate that administration of immune globulin can benefit patients with these inflammatory myopathies. Immune globulin can improve muscle strength, reduce inflammation and reduce disease progression and severity (Dala-kas et al. (1993) N Engl J Med 329(27):1993-2000; Dalakas et al. (2001) Neurology 56(3):323-7, Dalakas (2004) Pharmacol Ther 102(3):177-93, Walter et al. (2000) J Neurol 247(1):22-8). Immune globulin can be administered subcutaneously to patients with DM, PM or IBM in combination with hyaluronidase at a dose similar to the dose used to administer immune globulin intravenously. For example, 2 g/kg BW of immune globulin can be administered, typically over several days, such as, for example, five doses of 400 mg/kg BW on consecutive days. 8. Lambert-Eaton myasthenic syndrome [0217] Lambert-Eaton myasthenic syndrome (LEMS) is a rare autoimmune disorder of neuromuscular transmission first recognized clinically in association with lung cancer and subsequently in cases in which no neoplasm was detected. Patients with LEMS have a presynaptic neuromuscular junction defect. The disease is characterized clinically by proximal muscle weakness with augmentation of strength after exercise, mild oculomotor signs, depressed deep tendon reflexes and autonomic dysfunction (dry mouth, constipation, erectile failure). Subcutaneous administration of immune globulin in combination with hyaluronidase to patients with LEMS can ameliorate symptoms. Exemplary dosages include those used for intravenous administration of immune globulin to patients with LEMS. For example, a patient with LEMS can be administered 2 g per kg patient body weight of immune globulin over several doses. For example, five doses of 400 mg/kg BW immune globulin can be administered on five consecutive days. The amount of immune globulin administered can be increased or decreased as appropriate. 9. Multifocal motor neuropathy [0218] Multifocal motor neuropathy (MMN) with conduction block is an acquired immune-mediated demyelinating neuropathy with slowly progressive weakness, fasciculations, and cramping, without significant sensory involvement. The duration of disease prior to diagnosis ranges from several months to more than 15 years. The precise cause of MMN is unknown. Histopathologic and electrodiagnostic studies demonstrate the presence of both demyelinating and axonal injury. Motor nerves are primarily affected, although mild demyelination has been demonstrated in sensory nerves as well. Efficacy of immunomodulatory and immunosuppressive treatment further supports the immune nature of MMN. Titers of anti-GM1 antibodies are elevated in over half of the patients with MMN. Although the role of the anti-GM1 antibodies in the disease in unknown, their presence can be used as a diagnostic marker for MMN.

[0219] Subcutaneous administration of immune globulin in combination with hyaluronidase to patients with MMN can ameliorate symptoms. Exemplary dosages include those used for intravenous administration of immune globulin to patients with MMN. For example, a patient with MMN can be administered 2 g per kg patient body weight of immune globulin over several doses. For example, five doses of 400 mg/kg BW immune globulin can be administered on five consecutive days. In another example, 1 g/kg BW can be administered on 2 consecutive days. Some patients can be given maintenance therapy, which can include, for example, doses of 400 mg/kg BW to 2 g/kg BW, given every 2-6 weeks. The amount of immune globulin administered can be increased or decreased as appropriate, taking into account the patients response. 10. Myasthenia Gravis [0220] Myasthenia gravis (MG) is a chronic autoimmune neuromuscular disease characterized by varying degrees of weakness of the skeletal muscles of the body. It is associated with the presence of antibodies to acetylcholine receptors (AChR) or to muscle-specific tyrosine kinase (MuSK) at the neuromuscular junction, although some patients are antibody negative. The clinical features of MG include fluctuating weakness and fatigability of voluntary muscles, particularly levator palpebrae, extraocular, bulbar, limb and respiratory muscles. Patients usually present with unilateral or bilateral drooping of eyelid (ptosis), double vision (diplopia), difficulty in swallowing (dysphagia) and proximal muscle weakness. Weakness of respiratory muscles can result in respiratory failure in severe cases or in acute severe exacerbations (myasthenic crisis). Myasthenia gravis occurs in all ethnic groups and both genders. It most commonly affects young adult women under 40 and older men over 60, but it can occur at any age. In some instances, thymectomy is performed to reduce symptoms.

[0221] Immune globulin can be used, for example, as maintenance therapy for patients with moderate to severe MG, typically when other treatments have been ineffective or caused severe side efFects, and also can be administered prior to thymectomy orduring an acute exacerbation of the disease (myasthemic crisis). Immune globulin can be administered subcutaneously, in combination with hyaluronidase, to patients with Myasthenia gravis using the methods described herein. Exemplary dosages include those used for intravenous administration of immune globulin to patients with MG. For exam pie, a patient with MG can be administered doses of400 mg/kg BWto2g/kg BW every 4-6 weeks for maintenance therapy. Prior to thymectomy or during myasthemic crisis, 1-2 g/kg BW can be administered over several doses, such as,for example,fivedoses of400 mg/kg BWonfive consecutive days. In another example, 1 g/kg BWcan be administered on 2 consecutive days. 11. Moersch-Woltmann syndrome [0222] Moersch-Woltmann syndrome, also known as stiff person syndrome or stiffman syndrome, is a rare neurological disorder with features of an autoimmune disease. Patients present with symptoms related to muscular rigidity and superimposed episodic spasms. Muscle rigidity spreads to involve axial muscles, primarily abdominal and thoracolumbar, as well as proximal limb muscles. Typically, co-contraction of truncal agonist and antagonistic muscles leads to a boardlike appearance with hyperlordosis. Less frequently, respiratory muscle involvement leads to breathing difficulty and facial muscle involvement to a mask-like face. Treatment with immune globulin can effect decreased stiffness and heightened sensitivity scores in patients with Moersch-Woltmann syndrome (Dalakas et al. (2001) N Engl J Med 345(26):1870-6). Immune globulin can be administered subcutaneously, in combination with hyaluronidase, to patients with Moersch-Woltmann syndrome using the methods described herein. Exemplary dosages include those used for intravenous administration of immune globulin to patients with Moersch-Woltmann syndrome. For example, immune globulin can be administered at doses of 400 mg/kg BW on five consecutive days. Some patients can be given maintenance therapy, which can include, for example, 1-2 g/kg BW immune globulin every 4-6 weeks. The amount of immune globulin administered can be increased or decreased as appropriate. 12. Alzheimer’s Disease [0223] Treatment for Alzheimer’s disease includes treatment with intravenous immunoglobulin (see e.g. Dodel et al. (2004) J Neurol Neurosurg. Psychiatry, 75:1472-4; Solomon et al. (2007) Curr. Opin. Mol. Ther., 9:79-85; Relkin et al. (2008) Neurobiol Aging). IG contains antibodies that bind to beta amyloid (AB), which is a central component of the plaque in the brains of Alzheimer’s patients. Thus, IG can help to promote the clearance of AB from the brain and block AB’s toxic effects on brain cells. Hence, immune globulin can be administered subcutaneously, in combination with hyaluronidase, to patients with Alzheimer’s disease using the methods described herein. Subjects to be treated include patients having mild, moderate or advanced Alzheimer’s disease. It is within the level of skill of a treating physician to identify patients for treatment. Immune globulin in combination with hyaluronidase can be administered every week, every two weeks or once a month. Treatment can continue over the course of months or years. IG can be administered at doses at or between 200 mg/kg BW to 2 g/kg BW every week or every two weeks, and generally at least 200 mg/kg to 2 g/kg BW at least once a month. Treatment with immune globulin can effect an increase in patients’ anti-amyloid beta antibody levels compared to levels before treatment. 13. Other diseases and conditions [0224] Clinical data indicate that immune globulin can be used in the treatment of many conditions. In some instances, immune globulin can be used as the primary treatment, while in other cases, it is administered as second-line therapy when standard therapies have proven ineffective, have become intolerable, or are contraindicated. It is within the skill of a treating physician to identify such diseases or conditions. Exemplary of these include, but are not limited to, secondary hypogammaglobulinaemia (including iatrogenic immunodeficiency); specific antibody deficiency; Acute disseminated encephalomyelitis; ANCA-positive systemic necrotizing vasculitis; Autoimmune haemolytic anaemia; Bullous pemphigoid; Cicatricial pemphigoid; Evans syndrome (including autoimmune haemolytic anaemia with immune thrombocytopenia); Foeto-maternal/neonatal alloimmune thrombocytopenia (FMAIT/NAIT); Alzheimer’s Disease, Haemophagocytic syndrome; High-risk allogeneic haemopoietic stem cell transplantation; IgM paraproteinaemic neuropathy; kidney transplantation; multiple sclerosis; Opsoclonus myoclonus ataxia; Pemphigusfoliaceus; Pemphigus vulgaris; Post-transfusion purpura; Toxic epidermal necrolysis/Steven Johnson syndrome (TEN/SJS); Toxic shock syndrome; Systemic lupus erythematosus; multiple myeloma; sepsis; bone marrow transplantation, B cell tumors; and trauma.

[0225] Immune globulin also has been shown to have antimicrobial activity against a number of bacterial, viral and fungal infections, including, but not limited to, Haemophilus influenzae type B, Psuedomonas aeruginosa types A and B, Staphylococcus aureus, Group B Streptococcus, Streptococcus pneumoniae types 1,3, 4, 6, 7, 8, 9, 12, 14, 18, 19, and 23, Adenovirus types 2 and 5, Cytomegalovirus, Epstein Barr virus VCA, Hepatitis A virus, Hepatitis B virus, Herpes simplex virus-1, Herpes simplex virus-2, Influenza A, Measles, Parainfluenza types 1,2 and 3, Polio, Varicella zoster virus, Apergillus and Candida albicans. Thus, immune globulin can be administered subcutaneously in combination with hyaluronidase to patients with bacterial, viral and fungal infections to augment the patient’s immune system and treat the disease. In some examples, antibiotics or other antimicrobials also are administered. I. Articles of manufacture and kits [0226] Pharmaceutical compositions of immune globulin and a soluble hyaluronidase, provided together or separately, can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition which is effective for treating a IG-treatable disease or condition, and a label that indicates that the composition and combinations are to be used for treating a IG-treatable diseases and conditions. Exemplary of articles of manufacture are containers including single chamber and dual chamber containers. The containers include, but are not limited to, tubes, bottles and syringes. The containers can further include a needle for subcutaneous administration.

[0227] The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, for example, U.S. Patent Nos. 5,323,907, 5,033,252 and 5,052,558. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. Awide array of formulations of the compounds and compositions provided herein are contemplated as are a variety of treatments for any IG-treatable disease or condition.

[0228] Compositions of immune globulin and a soluble hyaluronidase, provided together or separately, also can be provided as kits. Kits can include a pharmaceutical composition described herein and an item for administration. For example compositions can be supplied with a device for administration, such as a syringe, an inhaler, a dosage cup, a dropper, or an applicator. The kit can, optionally, include instructions for application including dosages, dosing regimens and instructions for modes of administration. Kits also can include a pharmaceutical composition described herein and an item for diagnosis. For example, such kits can include an item for measuring the concentration, amount or activity of IG.

J. EXAMPLES

[0229] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1.

Soluble Recombinant Human PH20 (rHuPH20) Facilitates Subcutaneous Administration of Immune Globulin (IG) and Bioavailability [0230] Subcutaneous (SQ) administration of immune globulin (IG) results in reduced bioavailability compared to intravenous (IV) administration. One study reported 63% bioavailability compared to IV administration, thereby requiring an SQ dose of 137% of the IV does to be used to achieve an equivalent bioavailability, i.e. area under the time-concentration curve (AUC) (Ochs et al. (2006) J Clin. Immunol., 26:265-273). Thus, experiments were performed to determine if subcutaneous administration of IG (GAMMAGARD LIQUID (GGL), Baxter Biosciences) in the presence of soluble recombinant human PH20 (rHuPH20) increased the bioavailability of IG upon SQ administration, obviating the need for increased doses. The experimental study was designed to assess 1) the ability of subjects to tolerate a monthly dose of GGL in a single site via subcutaneous (SQ) route; 2) the dose of rHuPH20 per gram of GGL required to tolerate a monthly dose of GGL with no more than mild local adverse drug reactions; 3) the time required for SC administration; and 3) a comparison of the bioavailability measured by area under the curve (AUC) of GGL IV versus SQ.

[0231] Briefly, eleven adult immunodeficient patients who were on stable doses of intravenous gammaglobulin (IVIG) were enrolled in the study. All patients remained on the same monthly doses of IVIG as they received prior to the study. For subcutaneous administration, patients that were receiving up to 600 mg/kg body weight of GGL every four weeks IV, received SQ infusions in a single site beginning at a dose that was % of the 4-week IV dose, i.e. a 1-week dose, to determine the dose of rHuPH20 required to tolerate the 1-week dose. Subsequently, dosages of rHuPH20 were reduced and patients received the 2, 3, and finally a full four-week dose of GGL to determine the minimum rHuPH20 required to tolerate a once monthly infusion of GGL SQ.

[0232] Initial infusions were conducted using 150 U of rHuPH20 per gram of GGL. The rHuPH20 was administered through a 24-gauge SQ needle at a concentration of 150 U/ml or 1500 U/ml at a rate of 1-2 ml/min., prior to the infusion of the GGL. If 1500 U/ml rHuPH20 was used, it was diluted as follows: a) if the volume of the concentrated (1500 U/ml) rHuPH20 solution needed was 1.5 ml of below, it was diluted 1:10 using normal saline for injection; b) if the volume of the concentrated (1500 U/ml) rHuPH20 needed was above 1.5 ml but below 15 ml, it was diluted to 15 1 with normal saline for injection; c) if the volume of the concentrated (1500 U/ml) rHuPH20 solution needed was 15 ml or above, it was used undiluted.

[0233] Immediately after the infusion of rHuPH20 (and within 5 minutes), the GGL was infused through the same 24-gauge SQ needle beginning at a dose that was 1Λ of the 4-week dose (i.e. 1 week dose) to determine the amount of rHuPH20 needed to give one fourth of the 4-week dose in a single site. Each patient was assessed to determine if the infusion was tolerated; the infusion was deemed not to be tolerated if there were moderate or severe local reactions requiring more than one site of administration or an inability to complete the infusion in less than 3 hours. If the weekly dose of GGL was tolerated, the one-half dose (two week dose) was administered using rHuPH20 at 100 U/g GGL, and the dose of rHuPH20 was further reduced to 66 U/g GGL, then 50 U/g GGL. The dose of rHuPH20 was repeated, on a per-gram GGL basis, with increased amounts of GGL until a full 4-week dose of GGL in a single site was tolerated. If the amount of rHuPH20 was not tolerated at any point, then that weekly dose of GGL was repeated at the next interval and the amount of rHuPH20 increased until the dose was tolerated. If a subject fails to tolerate a dose for 2 successive increases in rHuPH20 dose (i.e. 3 attempts at a distinct dose of GGL), then the previously tolerated dose was determined to be the maximum tolerated dose.

[0234] The first 4 patients were evaluated for tolerability only; the last 7 patients had an IV infusion, followed by a pharmacokinetic (PK) study to compare the IV infusion to the subsequent SQ infusions. For the SQ infusions, after a monthly dose administration of GGL was achieved, the same monthly dose was repeated and a PK study performed to evaluate the T1/2, Tmax, and AUC. If AUC(SQ) was not within 90% of the AUC(IV), the dose of rHuPH20 was increased 4-fold at the next infusion, and the PK assessment was repeated.

[0235] Ten of the 11 patients achieved monthly doses of 25.5 to 61.2 grams of GGL (255 to 612 ml) in a single SQ site, at rates of 120 to 300 ml/hour. The eleventh patient withdrew following the 1-week infusion citing local discomfort. For the first patient (39001), the initial infusions were done by gravity, however, the rates were not acceptable despite increasing the dose of rHuPH20 from 150 to 300 U/g GGL. Therefore, all subsequent infusions were done using an IV peristaltic pump. The remaining 9 patients achieved the GGL monthly dose without the need to repeat doses or increase the concentration of rHuPH20. Thus, all 9 patients in whom an attempt was made to reduce the dose of rHuPH20 completed the study and were able to tolerate the infusions using 50 U/g GGL. To determine if 50 U/g GGL was the minimum rHuPH20 that could be administered, a dose of 25 U/g GGL was attempted in two patients without success: one had discomfort and the other had reduced tolerability and required administration at two sites. Thus, the minimum amount of rHuPH20 that permitted a monthly dose administration of GGL SQ was 50 U/g GGL. The results are summarized in Table 3 below. The results show that all but the first two patients were infused at rates up to 300 ml/hr with infusion times of 1.64 h (270 ml) to 3.55 h (537 ml). The rate of administration was limited primarily by the type of pump used. The IV pump frequently alarmed at rapid infusion rates. One infusion was slowed and one was interrupted due to mild infusion-site pain. Both infusions resumed and were completed.

1. Tolerability Assessment [0236] The subjects were assessed for their tolerability to the SQ infusions. Most infusions were associated with only mild infusion-related reactions (Table 4). Most common mild reactions were infusion site erythema, pain, swelling, warmth and pruritus. Moderate infusion site reactions included three cases of pain, and one case each of pruritus, swelling and warmth. No severe reactions were reported. There were complaints of transient burning during the infusion of the rHuPH20 in 10 of the infusions, with five occurring in one patient.

[0237] Systemic adverse events considered to be possibly or probably related to the infusions are listed in Table 5. Three were moderate and none were severe. Only the episode of mild chest pain was associated with interruption of the infusion, but the infusion was completed and subsequent infusions were well tolerated. Table 6 depicts the proportion of subcutaneous infusions that were completed without interruption for an adverse event.

[0238] One serious adverse event, an anaphylactic reaction, occurred in one patient, unrelated to study therapy. The patient, who had a history of previous allergic reactions to antibiotics, received an antibiotic on the day following her infusion with GGL/rHuPH20, and subsequently developed anaphylaxis. This patient recovered completely and was able to successfully complete the study.

[0239] There were no documented bacterial infections during this trial of PID patients. There were 9 reported cases of viral infections, all deemed not related to study therapy: 2 cases of viral gastroenteritis, 1 case of herpetic keratitis, 2 cases of sinusitis, 1 case of conjunctivitis, and 3 cases (in one patient) of influenza-like illness.

2. Pharmacokinetic Assessment [0240] Pharmacokinetic (PK) analysis was performed on serum IgG levels. The 7 patients enrolled in the PK assessment phase of this study achieved monthly doses of 27 to 61 grams of GGL in a single site using 50 U of rHuPH20 per gram GGL (see Table 3). The PK study was performed after receiving a second monthly dose infusion of GGL. Serum samples were collected pre-infusion, 1 h post-infusion and on days 1,2, 3, 4, 5, 7,14,21 and 28 (if on 28 day schedule) post-infusion. The pharmacokinetic parameters of the 7 patients are depicted in Table 7. The ratio of AUC(SQ) to AUC(IV) for the 7 patients is shown in Table 8. The results show that five of the 7 patients had AUC(SQ) within 90% of AUC (IV). Increasing the dose or rHuPH20 four-fold in the 2 subjects with a ratio less than 90% did notfurther improve bioavailability.

3. Summary of Results [0241] The use of rHuPH20 by injection prior to infusion with GGL made it possible to infuse as much as 600 ml of GGL in a single subcutaneous site at infusion rates up to 300 ml per hour in this study. The rate was limited primarily by the IV pumps that were used, which are designed to alarm and shut off when the IV infiltrates and pressure increases. Although pump shut off did not occur until rates approached the 300 ml/h, the need to restart the pump did increase the time of infusion. Since there is no need to pressure alarms for subcutaneous administration, the problem of shutting off should be eliminated by switching to pumps capable of generating more pressure without alarming.

[0242] Although most of the infusions were associated with some swelling, redness, or occasionally pain or itching, all but a few were mild. Two of the six moderate reactions occurred in infusions where the rHuPH20 was reduced in an effort to find the minimum effective dose; the rHuPH20 dose of 50 U/g GGL was tolerated by 10 of the 11 subjects. One subject, who had not previously experienced subcutaneous infusions, withdrew after the first one week dose citing discomfort at the site of the lower abdomen. All other infusions were completed despite the mild reactions, with 97% of the infusions being completed without interruption. Most reactions were treated with cool packs and only a few required acetaminophen or diphenhydramine.

[0243] The mean bioavailability of GGL upon subcutaneously administering in combination with rHuPH20 was 92% of the bioavailability of GGL following IV administration. This study suggests that the increased diffusion afforded by the rHuPH20 improved absorption of GGL. Further, the trough levels achieved by monthly subcutaneous dosing of GGL in this study are identical to those achieved by IV administration. Thus, there is no need to increase the frequency of administration of GGL by subcutaneous dosing versus IV dosing; subcutaneous administration of GGL in combination with rHuPH20 requires only a single SQ site and can be achieved at rates of infusion up to 300 ml/h.

[0244] In conclusion, rHuPH20 facilitated administration of a full monthly dose of GGL in a single site at rates up to 300 ml/h in a group of adult immunodeficient subjects. The bioavailability of the combination was 92% of the IV bioavailability, based on AUC of the time versus IgG concentration curve. This suggests that rHuPH20 improves absorption of subcutaneous administered GGL. Most of the local side effects were mild and did not result in slowing or interrupting the infusions.

Example 2.

Generation of a soluble rHuPH20 -expressing cell line [0245] The HZ24 plasmid (set forth in SEQ ID NO:52) was used to transfect Chinese Hamster Ovary (CHO cells) (see e.g. application Nos. 10,795,095, 11/065,716 and 11/238,171). The HZ24 plasmid vector for expression of soluble rHuPH20 contains a pCI vector backbone (Promega), DNA encoding amino acids 1-482 of human PH20 hyaluronidase (SEQ ID NO:49), an internal ribosomal entry site (IRES) from the ECMV virus (Clontech), and the mouse dihydrofolate reductase (DHFR) gene. The pCI vector backbone also includes DNA encoding the Beta-lactamase resistance gene (AmpR), an f1 origin of replication, a Cytomegalovirus immediate-early enhancer/promoter region (CMV), a chimeric intron, and an SV40 late polyadenylation signal (SV40). The DNA encoding the soluble rHuPH20 construct contains an Nhel site and a Kozak consensus sequence prior to the DNA encoding the methionine at amino acid position 1 of the native 35 amino acid signal sequence of human PH20, and a stop codon following the DNA encoding the tyrosine corresponding to amino acid position 482 of the human PH20 hyaluronidase set forth in SEQ ID NO:1), followed by a BamHlrestrictionsite.TheconstructpCI-PH20-IRES-DHFR-SV40pa(HZ24), therefore, results in a single mRNAspecies driven by the CMV promoter that encodes amino acids 1-482 of human PH20 (set forth in SEQ ID NO:3) and amino acids 1-186 of mouse dihydrofolate reductase (set forth in SEQ ID NO:53), separated by the internal ribosomal entry site (IRES).

[0246] Non-transfected DG44 CHO cells growing in GIBCO Modified CD-CHO media for DHFR(-) cells, supplemented with 4 mM Glutamine and 18 ml/L Plurionic F68/L (Gibco), were seeded at 0.5 x 106 cells/ml in ashakerflaskin preparation for transfection. Cells were grown at 37° C in 5% C02 in a humidified incubator, shaking at 120 rpm. Exponentially growing non-transfected DG44 CHO cells were tested for viability prior to transfection.

[0247] Sixty million viable cells of the non-transfected DG44 CHO cell culture were pelleted and resuspended to a density of 2 X 107 cells in 0.7 mL of 2x transfection buffer (2x HeBS: 40 mM Hepes, pH 7.0, 274 mM NaCI, 10 mM KC1, 1.4 mM Na2HP04,12 mM dextrose). To each aliquot of resuspended cells, 0.09 mL (250 μg) of the linear HZ24 plasmid (linearized by overnight digestion with Cla I (New England Biolabs) was added, and the cell/DNA solutions were transferred into 0.4 cm gap BTX (Gentronics) electroporation cuvettes at room temperature. A negative control electroporation was performed with no plasmid DNA mixed with the cells. The cell/plasmid mixes were electroporated with a capacitor discharge of 330 V and 960 μΡ or at 350 V and 960 μΡ.

[0248] The cells were removed from the cuvettes after electroporation and transferred into 5 mL of Modified CD-CHO media for DHFR(-) cells, supplemented with 4 mM Glutamine and 18 ml/L Plurionic F68/L (Gibco), and allowed to grow in a well of a 6-well tissue culture plate without selection for 2 days at 37° C in 5% C02 in a humidified incubator.

[0249] Two days post-electroporation, 0.5 mL of tissue culture media was removed from each well and tested for the presence of hyaluronidase activity, using the microturbidity assay described in Example 3.

[0250] Cells from Transfection 2 (350V) were collected from the tissue culture well, counted and diluted to 1 X 104 to 2 X 104 viable cells per mL. A 0.1 mL aliquot of the cell suspension was transferred to each well of five, 96 well round bottom tissue culture plates. One hundred microliters of CD-CHO media (GIBCO) containing 4 mM GlutaMAX™-1 supplement (GIBCO™, Invitrogen Corporation) and without hypoxanthine and thymidine supplements were added to the wells containing cells (final volume 0.2 mL).

[0251] Ten clones were identified from the 5 plates grown without methotrexate.

Table 10. Hyaluronidase activity of identified clones

[0252] Six HZ24 clones were expanded in culture and transferred into shaker flasks as single cell suspensions. Clones 3D3,3E5,2G8,2D9,1E11, and 4D10 were plated into 96-well round bottom tissue culture plates using a two-dimensional infinite dilution strategy in which cells were diluted 1:2 down the plate, and 1:3 across the plate, starting at 5000 cells in the top left hand well. Diluted clones were grown in a background of 500 non-transfected DG44 CHO cells per well, to provide necessary growth factors for the initial days in culture. Ten plates were made per subclone, with 5 plates containing 50 nM methotrexate and 5 plates without methotrexate.

[0253] Clone 3D3 produced 24 visual subclones (13 from the no methotrexate treatment, and 11 from the 50 nM methotrexate treatment. Significant hyaluronidase activity was measured in the supernatants from 8 of the 24 subclones (>50 Units/mL), and these 8 subclones were expanded into T-25 tissue culture flasks. Clones isolated from the methotrexate treatment protocol were expanded in the presence of 50 nM methotrexate. Clone 3D35M was further expanded in 500 nM methotrexate giving rise to clones producing in excess of 1,000 Units/ml in shaker flasks (clone 3D35M; or Gen1 3D35M). A master cell bank (MCB) of the 3D35M cells was then prepared.

Example 3.

Determination of hyaluronidase activity of soluble rHuPH20 [0254] Hyaluronidase activity of soluble rHuPH20 in samples such as cell cultures, purification fractions and purified solutions was determined using a tubidometric assay, which based on the formation of an insoluble precipitate when hyaluronic acid binds with serum albumin. The activity is measured by incubating soluble rHuPH20 with sodium hyalur-onate (hyaluronic acid) for a set period of time (10 minutes) and then precipitating the undigested sodium hyaluronate with the addition of acidified serum albumin. The turbidity of the resulting sample is measured at 640 nm after a 30 minute development period. The decrease in turbidity resulting from enzyme activity on the sodium hyaluronate substrate is a measure of the soluble rHuPH20 hyaluronidase activity. The method is performed using a calibration curve generated with dilutions of a soluble rHuPH20 assay working reference standard, and sample activity measurements are made relative to this calibration curve.

[0255] Dilutions of the sample were prepared in Enzyme Diluent Solutions. The Enzyme Diluent Solution was prepared by dissolving 33.0 ± 0.05 mg of hydrolyzed gelatin in 25.0 mL of the 50 mM PIPES Reaction Buffer (140 mM NaCI, 50 mM PIPES, pH 5.5) and 25.0 mL of SWFI, and diluting 0.2 mL of 25% Buminate solution into the mixture and vortexing for 30 seconds. This was performed within 2 hours of use and stored on ice until needed. The samples were diluted to an estimated 1-2 U/mL. Generally, the maximum dilution per step did not exceed 1:100 and the initial sample size for the first dilution was not be less than 20 μί. The minimum sample volumes needed to perform the assay were: In-process Samples, FPLC Fractions: 80 μί; Tissue Culture Supernatants:1 mL; Concentrated Material 80 μί; Purified or Final Step Material: 80 μί. The dilutions were made in in triplicate in a Low Protein Binding 96-well plate, and 30 μί of each dilution was transferred to Optilux black/clear bottom plates (BD BioSciences).

[0256] Dilutions of known soluble rHuPH20 with a concentration of 2.5 U/mL were prepared in Enzyme Diluent Solution to generate a standard curve and added to the Optilux plate in triplicate. The dilutions included 0 U/mL, 0.25 U/mL, 0.5 U/mL, 1.0 U/mL, 1.5 U/mL, 2.0 U/mL, and 2.5 U/mL. "Reagent blank" wells that contained 60 μί of Enzyme Diluent Solution were included in the plate as a negative control. The plate was then covered and warmed on a heat block for 5 minutes at 37°C. The cover was removed and the plate was shaken for 10 seconds. After shaking, the plate was returned to the plate to the heat block and the MULTIDROP 384 Liquid Handling Device was primed with the warm 0.25mg/mL sodium hyaluronate solution (prepared by dissolving 100 mg of sodium hyaluronate (LifeCore Biomedical) in 20.0 mL of SWFI. This was mixed by gently rotating and/or rocking at 2-8°C for 2-4 hours, or until completely dissolved). The reaction plate was transferred to the MULTIDROP 384 and the reaction was initiated by pressing the start key to dispense 30 μί sodium hyaluronate into each well. The plate was then removed from the MULTIDROP 384 and shaken for 10 seconds before being transferred to a heat block with the plate cover replaced. The plate was incubated at 37°C for 10 minutes [0257] The MULTIDROP 384 was prepared to stop the reaction by priming the machine with Serum Working Solution and changing the volume setting to 240 μί. (25 mL of Serum Stock Solution [1 volume of Horse Serum (Sigma) was diluted with 9 volumes of 500 mM Acetate Buffer Solution and the pH was adjusted to 3.1 with hydrochloric acid] in 75 mL of 500 mM Acetate Buffer Solution). The plate was removed from the heat block and placed onto the MULTIDROP 384 and 240 μί of serum Working Solutions was dispensed into the wells. The plate was removed and shaken on a plate reader for 10 seconds. After a further 15 minutes, the turbidity of the samples was measured at 640 nm and the hyaluronidase activity (in U/mL) of each sample was determined by fitting to the standard curve.

[0258] Specific activity (Units/mg) was calculated by dividing the hyaluronidase activity (U/ml) by the protein concentration (mg/mL).

Example 4

Production and Purification of Gen1 Human sPH20 A. 5 L Bioreactor Process [0259] A vial of 3D35M was thawed and expanded from shaker flasks through 1 L spinner flasks in CD-CHO media (Invitrogen, Carlsbad Calif.) supplemented with 100 nM Methotrexate and GlutaMAX™-1 (Invitrogen). Cells were transferred from spinner flasks to a 5 L bioreactor (Braun) at an inoculation density of 4 x 105 viable cells per ml. Parameters were temperature Setpoint 37°C, pH 7.2 (starting Setpoint), with Dissolved Oxygen Setpoint 25% and an air overlay of 0-100 cc/min. At 168 hrs, 250 ml of Feed #1 Medium (CD CHO with 50 g/L Glucose) was added. At 216 hours, 250 ml of Feed #2 Medium (CD CHO with 50 g/L Glucose and 10 mM Sodium Butyrate) was added, and at 264 hours 250 ml of Feed #2 Medium was added. This process resulted in a final productivity of 1600 Units per ml with a maximal cell density of 6 x 106 cells/ml. The addition of sodium butyrate was to dramatically enhance the production of soluble rHuPH20 in the final stages of production.

[0260] Conditioned media from the 3D35M clone was clarified by depth filtration and tangential flow diafiltration into 10 mM Hepes pH 7.0. Soluble rHuPH20 was then purified by sequential chromatography on Q Sepharose (Pharmacia) ion exchange, Phenyl Sepharose (Pharmacia) hydrophobic interaction chromatography, phenyl boronate (Prometics) and Hydroxapatite Chromatography (Biorad, Richmond, CA).

[0261] Soluble rHuPH20 bound to Q Sepharose and eluted at 400 mM NaCI in the same buffer. The eluate was diluted with 2M ammonium sulfate to a final concentration of500 mM ammonium sulfate and passed through a Phenyl Sepharose (low sub) column, followed by binding under the same conditions to a phenyl boronate resin. The soluble rHuPH20 was eluted from the phenyl sepharose resin in Hepes pH 6.9 after washing at pH 9.0 in 50 mM bicine without ammonium sulfate. The eluate was loaded onto a ceramic hydroxyapatite resin at pH 6.9 in 5 mM potassium phosphate and 1 mM CaCI2 and eluted with 80 mM potassium phosphate, pH 7.4 with 0.1 mM CaCI2.

[0262] The resultant purified soluble rHuPH20 possessed a specific activity in excess of 65,000 USP Units/mg protein by way of the microturbidity assay (Example 16) using the USP reference standard. Purified sPH20 eluted as a single peak from 24 to 26 minutes from a Pharmacia 5RPC styrene divinylbenzene column with a gradient between 0.1% TFA/H20 and 0.1 % TFA/90% acetonitrile/10% H20 and resolved as a single broad 61 kDa band by SDS electrophoresis that reduced to a sharp 51 kDa band upon treatment with PNGASE-F. N-terminal amino acid sequencing revealed that the leader peptide had been efficiently removed. B. Upstream Cell Culture Expansion Process into 100 L Bioreactor Cell Culture [0263] A scaled-up process was used to separately purify soluble rHuPH20 from four different vials of 3D35M cell to produce 4 separate batches of sHuPH20; HUA0406C, HUA0410C, HUA0415CandHUA0420C. Each vial was separately expanded and cultured through a 125 L bioreactor, then purified using column chromatography. Samples were taken throughout the process to assess such parameters as enzyme yield. The description of the process provided below sets forth representative specifications for such things as bioreactor starting and feed media volumes, transfer cell densities, and wash and elution volumes. The exact numbers vary slightly with each batch, and are detailed in Tables 11 to 18.

[0264] Four vials of 3D35M cells were thawed in a 37°C water bath, CD CHO containing 100 nM methotrexate and 40 mL/L GlutaMAX was added and the cells were centrifuged. The cells were re-suspended in a 125 mL shake flask with 20 mL of fresh media and placed in a 37°C, 7% C02 incubator. The cells were expanded up to 40 mL in the 125 mL shake flask. When the cell density reached 1.5 - 2.5 x 106 cells/mL, the culture was expanded into a 125 mL spinner flask in a 100 mL culture volume. The flask was incubated at 37°C, 7% C02. When the cell density reached 1.5- 2.5 x 106 cells/mL, the culture was expanded into a 250 mL spinner flask in 200 mL culture volume, and the flask was incubated at 37°C, 7% C02. When the cell density reached 1.5 - 2.5 x 106 cells/mL, the culture was expanded into a 1 L spinner flask in 800 mL culture volume and incubated at 37°C, 7% C02. When the cell density reached 1.5 - 2.5 x 106 cells/mL, the culture was expanded into a 6 L spinner flask in 5 L culture volume and incubated at 37°C, 7% C02 When the cell density reached 1.5 - 2.5 x 106 cells/mL, the culture was expanded into a 36 L spinner flask in 20 L culture volume and incubated at 37°C, 7% C02.

[0265] A 125 L reactor was sterilized with steam at 121°C, 20 PSI and 65 L of CD CHO media was added. Before use, the reactor was checked for contamination. When the cell density in the 36 L spinner flasks reached 1.8 -2.5 x 106 cells/mL, 20 L cell culture were transferred from the 36L spinner flasks to the 125 L bioreactor (Braun), resulting a final volume of 85 L and a seeding density of approximately 4 x 105 cells/mL. Parameters were temperature setpoint, 37°C; pH: 7.2; Dissolved oxygen: 25% ± 10%; Impeller Speed 50 rpm; Vessel Pressure 3 psi; Air Sparge 1 L/ min.; Air Overlay: 1 L/min. The reactor was sampled daily for cell counts, pH verification, media analysis, protein production and retention. Nutrient feeds were added during the run. At Day 6, 3.4 L of Feed #1 Medium (CD CHO + 50 g/L Glucose + 40 mL/L

GlutaMAX™-1) was added, and culture temperature was changed to 36.5°C. At day 9 , 3.5 L of Feed #2 (CD CHO + 50 g/L Glucose + 40 mL/L GlutaMAX™-1 + 1.1 g/L Sodium Butyrate) was added, and culture temperature was changed to 36°C. At day 11,3.7 L of Feed #3 (CD CHO + 50 g/L Glucose + 40 mL/L GlutaMAX™-1 + 1.1 g/L Sodium Butyrate) was added, and the culture temperature was changed to 35.5°C. The reactor was harvested at 14 days or when the viability of the cells dropped below 50%. The process resulted in production of soluble rHuPH20 with an enzymatic activity of 1600 Units/ml with a maximal cell density of 8 million cells/mL. At harvest, the culture was sampled for mycoplasma, bioburden, endotoxin, and virus in vitro and in vivo, transmission electron microscopy (TEM) for viral particles, and enzyme activity.

[0266] The one hundred liter bioreactor cell culture harvest was filtered through a series of disposable capsule filters having a polyethersulfone medium (Sartorius): first through a 8.0 μίτι depth capsule, a 0.65 μιτι depth capsule, a 0.22 μίτι capsule, and finally through a 0.22 μιτι Sartopore 2000 cm2 filter and into a 100 L sterile storage bag. The culture was concentrated 10X using two TFF with Spiral Polyethersulfone 30 kDa MWCO filters (Millipore), followed by a 6x buffer exchange with 10 mM HEPES, 25 mM Na2S04, pH 7.0 into a 0.22 μιτι final filter into a 20 L sterile storage bag. Table 11 provides monitoring data related to the cell culture, harvest, concentration and buffer exchange steps.

Table 11. Monitoring data for cell culture, harvest, concentration and buffer exchange steps.

[0267] AQ Sepharose (Pharmacia) ion exchange column (3 L resin, Height = 20 cm, Diameter = 14 cm) was prepared. Wash samples were collected for a determination of pH, conductivity and endotoxin (LAL) assay. The column was equilibrated with 5 column volumes of 10 mM Tris, 20 mM Na2S04, pH 7.5. The concentrated, diafiltered harvest was loaded onto the Q column at a flow rate of 100 cm/hr. The column was washed with 5 column volumes of 10 mM Tris, 20 mM Na2S04, pH 7.5 and 10 mM Hepes, 50 mM NaCI, pH 7.0. The protein was eluted with 10 mM Hepes, 400 mM NaCI, pH 7.0 and filtered through a 0.22 μιτι final filter into a sterile bag.

[0268] Phenyl-Sepharose (Pharmacia) hydrophobic interaction chromatography was next performed. A Phenyl-Sepha-rose (PS) column (9.1 L resin, Height = 29 cm, Diameter = 20cm) was prepared. The column was equilibrated with 5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate, 0.1 mM CaCI2, pH 7.0. The protein eluate from above was supplemented with 2M ammonium sulfate, 1 M potassium phosphate and 1 M CaCI2 stock solutions to final concentrations of 5 mM, 0.5 M and 0.1 mM, respectively. The protein was loaded onto the PS column at a flow rate of 100 cm/hr. 5 mM potassium phosphate, 0.5 M ammonium sulfate and 0.1 mM CaCI2 pH 7.0 was added at 100 cm/hr. The flow through was passed through a 0.22 μιτι final filter into a sterile bag.

[0269] The PS-purified protein was the loaded onto an aminophenyl boronate column (ProMedics) (6.3 L resin, Height = 20 cm, Diameter = 20cm) that had been equilibrated with 5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate. The protein was passed through the column at a flow rate of 100 cm/hr, and the column was washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate, pH 7.0. The column was then washed with 20 mM bicine, 100 mM NaCI, pH 9.0 and the protein eluted with 50 mM Hepes, 100 mM NaCI pH 6.9 through a sterile filter and into a 20 L sterile bag. The eluate was tested for bioburden, protein concentration and enzyme activity.

[0270] A hydroxyapatite (HAP) column (BioRad) (1.6 L resin, Height = 10 cm, Diameter = 14 cm) was equilibrated with 5 mM potassium phosphate, 100 mM NaCI, 0.1 mM CaCI2 pH 7.0. Wash samples were collected and tested for pH, conductivity and endotoxin (LAL assay). The aminophenyl boronate purified protein was supplemented with potassium phosphate and CaCI2 to yield final concentrations of 5 mM potassium phosphate and 0.1 mM CaCI2 and loaded onto the HAP column at a flow rate of 100 cm/hr. The column was washed with 5 mM potassium phosphate pH 7.0, 100 mM NaCI, 0.1 mM CaCI2, then 10 mM potassium phosphate pH 7.0, 100 mM NaCI, 0.1 mM CaCI2 pH. The protein was eluted with 70 mM potassium phosphate pH 7.0 and filtered through a 0.22 μίτι filter into a 5 L sterile storage bag. The eluate was tested for bioburden, protein concentration and enzyme activity.

[0271] The HAP-purified protein was then pumped through a 20 nM viral removal filter via a pressure tank. The protein was added to the DV20 pressure tank and filter (Pall Corporation), passing through an Ultipor DV20 Filter with 20 nm pores (Pall Corporation) into a sterile 20 L storage bag. The filtrate was tested for protein concentration, enzyme activity, oligosaccharide, monosaccharide and sialic acid profiling, and process-related impurities. The protein in the filtrate was then concentrated to 1 mg/mL using a 10 kD molecular weight cut off (MWCO) Sartocon Slice tangential flow filtration (TFF) system (Sartorius). The filter was first prepared by washing with a Hepes/saline solution (10 mM Hepes, 130 mM NaCI, pH 7.0) and the permeate was sampled for pH and conductivity. Following concentration, the concentrated protein was sampled and tested for protein concentration and enzyme activity. A 6X buffer exchange was performed on the concentrated protein into the final buffer: 10 mM Hepes, 130 mM NaCI, pH 7.0. The concentrated protein was passed though a 0.22 μίτι filter into a 20 L sterile storage bag. The protein was sampled and tested for protein concentration, enzyme activity, free sulfhydryl groups, oligosaccharide profiling and osmolarity.

[0272] Tables 12 to 18 provide monitoring data related to each of the purification steps described above, for each 3D35M cell lot.

Table 12. Q sepharose column data

Table 13. Phenyl Sepharose column data

Table 14. Amino Phenyl Boronate column data

Table 15. Hydroxyapatite column data

Tahle -IR nV90 filtration data

Table 17. Final concentration data

(continued)

Table 18. Buffer Exchange into Final Formulation data

[0273] The purified and concentrated soluble rHuPH20 protein was aseptically filled into sterile vials with 5 mL and 1 mL fill volumes. The protein was passed though a 0.22 μη filter to an operator controlled pump that was used to fill the vials using a gravimetric readout. The vials were closed with stoppers and secured with crimped caps. The closed vials were visually inspected forforeign particles and then labeled. Following labeling, the vials were flash-frozen by submersion in liquid nitrogen for no longer than 1 minute and stored at <-15°C (-20 ± 5 °C).

Example 5

Production Gen2 Cells Containing Soluble human PH20 (rHuPH20) [0274] The Gen1 3D35M cell line described in Example 2 was adapted to higher methotrexate levels to produce generation 2 (Gen2) clones. 3D35M cells were seeded from established methotrexate-containing cultures into CD CHO medium containing 4mM GlutaMAX-1™ and 1.0 μΜ methotrexate. The cells were adapted to a higher methotrexate level by growing and passaging them 9 times over a period of 46 days in a 37°C, 7% C02 humidified incubator. The amplified population of cells was cloned out by limiting dilution in 96-well tissue culture plates containing medium with 2.0 μΜ methotrexate. After approximately 4 weeks, clones were identified and clone 3E10B was selected for expansion. 3E10B cells were grown in CD CHO medium containing 4 mM GlutaMAX-1™ and 2.0 μΜ methotrexate for 20 passages. A master cell bank (MCB) of the 3E10B cell line was created and frozen and used for subsequent studies.

[0275] Amplification of the cell line continued by culturing 3E10B cells in CD CHO medium containing 4 mM GlutaMAX-1 ™ and 4.0 μΜ methotrexate. After the 12th passage, cells were frozen in vials as a research cell bank (RCB). One vial of the RCB was thawed and cultured in medium containing 8.0 μΜ methotrexate. After 5 days, the methotrexate concentration in the medium was increased to 16.0 μΜ, then 20.0 μΜ 18 days later. Cells from the 8th passage in medium containing 20.0 μΜ methotrexate were cloned out by limiting dilution in 96-well tissue culture plates containing CD CHO medium containing 4 mM GlutaMAX-1 ™ and 20.0 μΜ methotrexate. Clones were identified 5-6 weeks later and clone 2B2 was selected for expansion in medium containing 20.0 μΜ methotrexate. After the 11th passage, 2B2 cells were frozen in vials as a research cell bank (RCB).

[0276] The resultant 2B2 cells are dihydrofolate reductase deficient (dhfr-) DG44 CHO cells that express soluble recombinant human PH20 (rHuPH20). The soluble PH20 is present in 2B2 cells at a copy number of approximately 206 copies/cell. Southern blot analysis of Spe l-,Xba I-and BamH l/Hind Ill-digested genomic2B2 cell DIMA using a rHuPH20-specific probe revealed the following restriction digest profile: one major hybridizing band of ~7.7 kb and four minor hybridizing bands (-13.9, ~6.6, ~5.7 and ~4.6 kb) with DNA digested with Spe I; one major hybridizing band of ~5.0 kb and two minor hybridizing bands (-13.9 and ~6.5 kb) with DNA digested with Xba I; and one single hybridizing band of ~1.4 kb observed using 2B2 DNA digested with BamH l/Hind III. Sequence analysis of the mRNA transcript indicated that the derived cDNA (SEQ ID NO:56) was identical to the reference sequence (SEQ ID NO:49) except for one base pair difference at position 1131, which was observed to be a thymidine (T) instead of the expected cytosine (C). This is a silent mutation, with no effect on the amino acid sequence.

Example 6 A. Production of Gen2 soluble rHuPH20 in 300 L Bioreactor Cell Culture [0277] A vial of HZ24-2B2 was thawed and expanded from shaker flasks through 36L spinner flasks in CD-CHO media (Invitrogen, Carlsbad, CA) supplemented with 20 μΜ methotrexate and GlutaMAX-1 ™ (Invitrogen). Briefly, the a vial of cells was thawed in a 37°C water bath, media was added and the cells were centrifuged. The cells were re-suspended in a 125 mL shake flask with 20 mL of fresh media and placed in a 37°C, 7% C02 incubator. The cells were expanded up to 40 mL in the 125 mL shake flask. When the cell density reached greater than 1.5 x 106 cells/mL, the culture was expanded into a 125 mL spinner flask in a 100 mL culture volume. The flask was incubated at 37°C, 7% C02. When the cell density reached greater than 1.5 x 106 cells/mL, the culture was expanded into a 250 mL spinner flask in 200 mL culture volume, and the flask was incubated at 37°C, 7% C02. When the cell density reached greater than 1.5 x 106 cells/mL, the culture was expanded into a 1 L spinner flask in 800 mL culture volume and incubated at 37°C, 7% C02. When the cell density reached greater than 1.5 x 106 cells/mL the culture was expanded into a 6 L spinner flask in 5000 mL culture volume and incubated at 37°C, 7% C02. When the cell density reached greater than 1.5 x 106 cells/mL the culture was expanded into a 36 L spinner flask in 32 L culture volume and incubated at 37°C, 7% C02.

[0278] A 400 L reactor was sterilized and 230 mL of CD-CHO media was added. Before use, the reactor was checked for contamination. Approximately 30 L cells were transferred from the 36L spinner flasks to the 400 L bioreactor (Braun) at an inoculation density of 4.0 X 105 viable cells per ml and a total volume of 260L. Parameters were temperature setpoint, 37°C; Impeller Speed 40-55 RPM; Vessel Pressure: 3 psi; Air Sparge 0.5- 1.5 L/Min.; Air Overlay: 3 L/ min.. The reactor was sampled daily for cell counts, pH verification, media analysis, protein production and retention. Also, during the run nutrient feeds were added. At 120 hrs (day 5), 10.4L of Feed #1 Medium (4x CD-CHO + 33 g/L Glucose + 160 mL/L Glutamax-1™ + 83 mL/L Yeastolate + 33 mg/L rHuInsulin) was added. At 168 hours (day 7), 10.8 L of Feed #2 (2x CD-CHO + 33 g/L Glucose + 80 mL/L Glutamax-1 ™ + 167 mL/L Yeastolate + 0.92 g/L Sodium Butyrate) was added, and culture temperature was changed to 36.5°C. At 216 hours (day 9), 10.8 L of Feed #3 (1 x CD-CHO + 50 g/L Glucose + 50 mL/L Glutamax-1 ™ +250 mL/L Yeastolate +1.80 g/L Sodium Butyrate) was added, and culture temperature was changed to 36° C. At 264 hours (day 11), 10.8 L of Feed #4 (1 x CD-CHO + 33 g/L Glucose + 33 mL/L Glutamax-1 ™ + 250 mL/L Yeastolate + 0.92 g/L Sodium Butyrate) was added, and culture temperature was changed to 35.5° C. The addition of the feed media was observed to dramatically enhance the production of soluble rHuPH20 in the final stages of production. The reactor was harvested at 14 or 15 days or when the viability of the cells dropped below 40%. The process resulted in a final productivity of 17,000 Units per ml with a maximal cell density of 12 million cells/mL. At harvest, the culture was sampled for mycoplasma, bioburden, endotoxin and viral in vitro and in vivo, Transmission Electron Microscopy (TEM) and enzyme activity.

[0279] The culture was pumped by a peristaltic pump through four Millistak filtration system modules (Millipore) in parallel, each containing a layer of diatomaceous earth graded to 4-8 μ(η and a layer of diatomaceous earth graded to 1.4-1.1 μηι, followed by a cellulose membrane, then through a second single Millistak filtration system (Millipore) containing a layer of diatomaceous earth graded to 0.4-0.11 μηι and a layer of diatomaceous earth graded to <0.1 μηι, followed by a cellulose membrane, and then through a 0.22 μ(η final filter into a sterile single use flexible bag with a 350 L capacity. The harvested cell culture fluid was supplemented with 10 mM EDTA and 10 mM Tris to a pH of 7.5. The culture was concentrated 10x with a tangential flow filtration (TFF) apparatus using four SartosliceTFF 30kDa molecular weight cut-off (MWCO) polyether sulfone (PES) filter (Sartorious), followed by a 10x buffer exchange with 10 mM Tris, 20mM Na2S04, pH 7.5 into a 0.22 μηι final filter into a 50 L sterile storage bag.

[0280] The concentrated, diafiltered harvest was inactivated for virus. Prior to viral inactivation, a solution of 10% Triton X-100, 3% tri (n-butyl) phosphate (TNBP) was prepared. The concentrated, diafiltered harvest was exposed to 1 % Triton X-100, 0.3% TNBP for 1 hour in a 36 L glass reaction vessel immediately prior to purification on the Q column. B. Purification of Gen2 soluble rHuPH20 [0281] A Q Sepharose (Pharmacia) ion exchange column (9 L resin, H= 29 cm, D= 20 cm) was prepared. Wash samples were collected for a determination of pH, conductivity and endotoxin (LAL) assay. The column was equilibrated with 5 column volumes of 10 mM Tris, 20 mM Na2S04, pH 7.5. Following viral inactivation, the concentrated, diafiltered harvest was loaded onto the Q column at a flow rate of 100 cm/hr. The column was washed with 5 column volumes of 10 mM Tris, 20 mM Na2S04, pH 7.5 and 10 mM Hepes, 50 mM NaCI, pH7.0. The protein was eluted with 10 mM Hepes, 400 mM NaCI, pH 7.0 into a 0.22 μηι final filter into sterile bag. The eluate sample was tested for bioburden, protein concentration and hyaluronidase activity. A280 absorbance reading were taken at the beginning and end of the exchange..

[0282] Phenyl-Sepharose (Pharmacia) hydrophobic interaction chromatography was next performed. A Phenyl-Spe-harose (PS) column (19-21 L resin, H=29 cm, D= 30 cm) was prepared. The wash was collected and sampled for pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 column volumes of 5 mM potassium phos phate, 0.5 M ammonium sulfate, 0.1 mM CaCI2, pH 7.0. The protein eluate from the Q sepharose column was supplemented with 2M ammonium sulfate, 1 M potassium phosphate and 1 M CaCI2 stock solutions to yield final concentrations of 5 mM, 0.5 M and 0.1 mM, respectively. The protein was loaded onto the PS column at a flow rate of 100 cm/hr and the column flow thru collected. The column was washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate and 0.1 mM CaCI2 pH 7.0 at 100 cm/hr and the wash was added to the collected flow thru. Combined with the column wash, the flow through was passed through a 0.22 μηι final filter into a sterile bag. The flow through was sampled for bioburden, protein concentration and enzyme activity.

[0283] An aminophenyl boronate column (Promtics) was prepared. The wash was collected and sampled for pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate. The PS flow through containing purified protein was loaded onto the aminophenyl boronate column at a flow rate of 100 cm/hr. The column was washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate, pH 7.0. The column was washed with 20 mM bicine, 0.5 M ammonium sulfate, pH 9.0. The column was washed with 20 mM bicine, 100 mM sodium chloride, pH 9.0. The protein was eluted with 50 mM Hepes, 100 mM NaCI, pH 6.9 and passed through a sterile filter into a sterile bag. The eluted sample was tested for bioburden, protein concentration and enzyme activity.

[0284] The hydroxyapatite (HAP) column (Biorad) was prepared. The wash was collected and test for pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 mM potassium phosphate, 100 mM NaCI, 0.1 mM CaCI2, pH 7.0. The aminophenyl boronate purified protein was supplemented to final concentrations of 5 mM potassium phosphate and 0.1 mM CaCI2 and loaded onto the HAP column at a flow rate of 100 cm/hr. The column was washed with 5 mM potassium phosphate, pH 7, 100 mM NaCI, 0.1 mM CaCI2. The column was next washed with 10 mM potassium phosphate, pH 7, 100 mM NaCI, 0.1 mM CaCI2. The protein was eluted with 70 mM potassium phosphate, pH 7.0 and passed through a 0.22μΐτι sterile filter into a sterile bag. The eluted sample was tested for bioburden, protein concentration and enzyme activity.

[0285] The HAP purified protein was then passed through a viral removal filter. The sterilized Virosart filter (Sartorius) was first prepared by washing with 2 L of 70 mM potassium phosphate, pH 7.0. Before use, the filtered buffer was sampled for pH and conductivity. The HAP purified protein was pumped via a peristaltic pump through the 20 nM viral removal filter. The filtered protein in 70 mM potassium phosphate, pH 7.0 was passed through a 0.22 μηι final filter into a sterile bag. The viral filtered sample was tested for protein concentration, enzyme activity, oligosaccharide, monosaccharide and sialic acid profiling. The sample also was tested for process related impurities.

[0286] The protein in the filtrate was then concentrated to 10 mg/mL using a 10 kD molecular weight cut ofF(MWCO) Sartocon Slice tangential flow filtration (TFF) system (Sartorius). The filter was first prepared by washing with 10 mM histidine, 130 mM NaCI, pH 6.0 and the permeate was sampled for pH and conductivity. Following concentration, the concentrated protein was sampled and tested for protein concentration and enzyme activity. A 6x buffer exchange was performed on the concentrated protein into the final buffer: 10 mM histidine, 130 mM NaCI, pH 6.0. Following buffer exchange, the concentrated protein was passed though a 0.22 μ(η filter into a 20 L sterile storage bag. The protein was sampled and tested for protein concentration, enzyme activity, free sulfhydryl groups, oligosaccharide profiling and osmolarity.

[0287] The sterile filtered bulk protein was then aseptically dispensed at 20 mL into 30 mL sterile Teflon vials (Nalgene). The vials were then flash frozen and stored at-20 ± 5°C. C. Comparison of production and purification of Gen1 soluble rHuPH20 and Gen2 soluble rHuPH20 [0288] The production and purification of Gen2 soluble rHuPH20 in a 300L bioreactor cell culture contained some changes in the protocols compared to the production and purification Gen1 soluble rHuPH20 in a 100L bioreactor cell culture (described in Example 4.B). Table 19 sets forth exemplary differences, in addition to simple scale up changes, between the methods.

(continued)

(continued)

[0289] Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

SEQUENCE LISTING

[0290] <110> Baxter Healthcare, S.A.

Baxter International, Inc.

Halozyme, Inc.

Schiff, Richard Leibl, Heinz Frost, Gregory <120> Combinations and Methods for

Subcutaneous Administration of Immune Globulin and Hyaluronidase

<130> 0119374-00095/3058PC <140> Not yet assigned <141 > Herewith <150> US 61/069,841 <151 > 2008-03-17 <160> 56 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 509 <212> PRT <213> Homo sapiens <220> <223> precursor human PH20 <400> 1

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Arg Ser Phe Val Lys 15 10 15

Ser Ser Gly Val Ser Gin lie Val Phe Thr Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60

Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg 65 70 75 80 lie Asn Ala Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly 100 105 110

Gly lie Pro Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys 115 120 125

Lys Asp lie Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140

He Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160

Lys Asp Val Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn 165 170 175

Val Gin Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr He Lys Leu Gly Lys 195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240

Val Glu lie Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255

Thr Ala Leu Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val 260 265 270

Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val 275 280 285

Ser Lys lie Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300

Arg lie Val Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu 305 310 315 320

Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie 325 330 335

Val lie Trp Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu 340 345 350

Leu Leu Asp Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin 370 375 380

Gly Val Cys lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400

Asn Pro Asp Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415

Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys 420 425 430

Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys 450 455 460 lie Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin lie 465 470 475 480

Phe Tyr Asn Ala Ser Pro Ser Thr Leu Ser Ala Thr Met Phe lie Val 485 490 495

Ser lie Leu Phe Leu lie lie Ser Ser Val Ala Ser Leu 500 505 <210> 2 <211 >474

<212> PRT <213> Homo sapiens <220> <223> Mature PH20 <400>2

Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro Phe Leu Trp 15 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro 20 25 30

Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg lie Asn Ala 35 40 45

Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu Gly Tyr Tyr 50 55 60

Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly Gly lie Pro 65 70 75 80

Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys Lys Asp lie 85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val lie Asp Trp 100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val 115 120 125

Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn Val Gin Leu 130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe Glu Lys Ala 145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys Leu Leu Arg 165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His 180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu lie 195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu 210 215 220

Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val Ala Ala Thr 225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val Ser Lys lie 245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg lie Val 260 265 270

Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu Leu Val Tyr 275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie Val lie Trp 290 295 300

Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp 305 310 315 320

Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn Val Thr Leu 325 330 335

Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin Gly Val Cys 340 345 350 lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp 355 360 365

Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly 370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys Phe Tyr Cys 385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp 405 410 415

Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys lie Asp Ala 420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin lie Phe Tyr Asn 435 440 445

Ala Ser Pro Ser Thr Leu Ser Ala Thr Met Phe lie Val Ser lie Leu 450 455 460

Phe Leu lie lie Ser Ser Val Ala Ser Leu 465 470 <210> 3 <211 >482

<212> PRT <213> Homo sapiens <220> <223> precursor soluble rHuPH20 <400>3

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Arg Ser Phe Val Lys 15 10 15

Ser Ser Gly Val Ser Gin lie Val Phe Thr Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60

Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg 65 70 75 80 lie Asn Ala Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly 100 105 110

Gly lie Pro Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys 115 120 125

Lys Asp lie Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140 lie Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160

Lys Asp Val Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn 165 170 175

Val Gin Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys 195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240

Val Glu lie Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255

Thr Ala Leu Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val 260 265 270

Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val 275 280 285

Ser Lys lie Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300

Arg lie Val Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu 305 310 315 320

Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie 325 330 335

Val lie Trp Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu 340 345 350

Leu Leu Asp Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin 370 375 380

Gly Val Cys lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400

Asn Pro Asp Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415

Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys 420 425 430

Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys 450 455 460 lie Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin lie 465 470 475 480

Phe Tyr <210> 4 <211 >447

<212> PRT <213> Homo sapiens <220> <223> soluble rHuPH20 1-447 <400>4

Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro Phe Leu Trp 15 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro 20 25 30

Leu Asp Met Ser Leu Phe Ser Phe He Gly Ser Pro Arg lie Asn Ala 35 40 45

Thr Gly Gin Gly Val Thr He Phe Tyr Val Asp Arg Leu Gly Tyr Tyr 50 55 60

Pro Tyr He Asp Ser He Thr Gly Val Thr Val Asn Gly Gly He Pro 65 70 75 80

Gin Lys He Ser Leu Gin Asp His Leu Asp Lys Ala Lys Lys Asp He 85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val He Asp Trp 100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val 115 120 125

Tyr Lys Asn Arg Ser He Glu Leu Val Gin Gin Gin Asn Val Gin Leu 130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe Glu Lys Ala 145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr He Lys Leu Gly Lys Leu Leu Arg 165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His 180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu He 195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu 210 215 220

Tyr Pro Ser He Tyr Leu Asn Thr Gin Gin Ser Pro Val Ala Ala Thr 225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala He Arg Val Ser Lys He 245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg He Val 260 265 270

Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu Leu Val Tyr 275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly He Val He Trp 290 295 300

Gly Thr Leu Ser He Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp 305 310 315 320

Asn Tyr Met Glu Thr He Leu Asn Pro Tyr He He Asn Val Thr Leu 325 330 335

Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin Gly Val Cys 340 345 350

He Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp 355 360 365

Asn Phe Ala He Gin Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly 370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys Phe Tyr Cys 385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp 405 410 415

Thr Asp Ala Val Asp Val Cys He Ala Asp Gly Val Cys He Asp Ala 420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin He Phe Tyr 435 440 445 <210 5 <211> 446 <212> PRT <213> Homo sapiens <220 <223> soluble rHuPH20 1-446 <400> 5

Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro Phe Leu Trp 15 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro 20 25 30

Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg lie Asn Ala 35 40 45

Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu Gly Tyr Tyr 50 55 60

Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly Gly lie Pro 65 70 75 80

Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys Lys Asp lie 85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val lie Asp Trp 100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val 115 120 125

Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn Val Gin Leu 130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe Glu Lys Ala 145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys Leu Leu Arg 165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His 180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu lie 195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu 210 215 220

Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val Ala Ala Thr 225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val Ser Lys lie 245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg lie Val 260 265 270

Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu Leu Val Tyr 275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie Val lie Trp 290 295 300

Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp 305 310 315 320

Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn Val Thr Leu 325 330 335

Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin Gly Val Cys 340 345 350 lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp 355 360 365

Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly 370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys Phe Tyr Cys 385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp 405 410 415

Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys lie Asp Ala 420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin lie Phe 435 440 445 <210> 6 <211 >445

<212> PRT <213> Homo sapiens <220 <223> soluble rHuPH20 1-445 <400 6

Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro Phe Leu Trp 15 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro 20 25 30

Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg lie Asn Ala 35 40 45

Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu Gly Tyr Tyr 50 55 60

Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly Gly lie Pro 65 70 75 80

Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys Lys Asp lie 85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val lie Asp Trp 100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val 115 120 125

Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn Val Gin Leu 130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe Glu Lys Ala 145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys Leu Leu Arg 165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His 180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu lie 195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu 210 215 220

Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val Ala Ala Thr 225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val Ser Lys lie 245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg lie Val 260 265 270

Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu Leu Val Tyr 275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie Val lie Trp 290 295 300

Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp 305 310 315 320

Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn Val Thr Leu 325 330 335

Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin Gly Val Cys 340 345 350 lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp 355 360 365

Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly 370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys Phe Tyr Cys 385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp 405 410 415

Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys lie Asp Ala 420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin lie 435 440 445 <210>7 <211> 444 <212> PRT <213> Homo sapiens <220> <223> soluble rHuPH20 1-444 <400>7

Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro Phe Leu Trp 15 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro 20 25 30

Leu Asp Met Ser Leu Phe Ser Phe He Gly Ser Pro Arg lie Asn Ala 35 40 45

Thr Gly Gin Gly Val Thr He Phe Tyr Val Asp Arg Leu Gly Tyr Tyr 50 55 60

Pro Tyr He Asp Ser He Thr Gly Val Thr Val Asn Gly Gly He Pro 65 70 75 80

Gin Lys He Ser Leu Gin Asp His Leu Asp Lys Ala Lys Lys Asp He 85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val He Asp Trp 100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val 115 120 125

Tyr Lys Asn Arg Ser He Glu Leu Val Gin Gin Gin Asn Val Gin Leu 130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe Glu Lys Ala 145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr He Lys Leu Gly Lys Leu Leu Arg 165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His 180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu He 195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu 210 215 220

Tyr Pro Ser He Tyr Leu Asn Thr Gin Gin Ser Pro Val Ala Ala Thr 225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala He Arg Val Ser Lys He 245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg He Val 260 265 270

Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu Leu Val Tyr 275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly He Val He Trp 290 295 300

Gly Thr Leu Ser He Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp 305 310 315 320

Asn Tyr Met Glu Thr He Leu Asn Pro Tyr He He Asn Val Thr Leu 325 330 335

Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin Gly Val Cys 340 345 350

He Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp 355 360 365

Asn Phe Ala He Gin Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly 370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys Phe Tyr Cys 385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp 405 410 415

Thr Asp Ala Val Asp Val Cys He Ala Asp Gly Val Cys He Asp Ala 420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin 435 440

<210> 8 <211 >443 <212> PRT <213> Homo sapiens <220 <223> soluble rHuPH20 1-443 <400 8

Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro Phe Leu Trp 15 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro 20 25 30

Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg lie Asn Ala 35 40 45

Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu Gly Tyr Tyr 50 55 60

Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly Gly lie Pro 65 70 75 80

Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys Lys Asp lie 85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val lie Asp Trp 100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val 115 120 125

Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn Val Gin Leu 130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe Glu Lys Ala 145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys Leu Leu Arg 165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His 180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu lie 195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu 210 215 220

Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val Ala Ala Thr 225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val Ser Lys lie 245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg lie Val 260 265 270

Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu Leu Val Tyr 275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie Val lie Trp 290 295 300

Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp 305 310 315 320

Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn Val Thr Leu 325 330 335

Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin Gly Val Cys 340 345 350 lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp 355 360 365

Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly 370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys Phe Tyr Cys 385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp 405 410 415

Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys lie Asp Ala 420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro 435 440 <210>9 <211> 442 <212> PRT <213> Homo sapiens <220> <223> soluble rHuPH20 1-442 <400>9

Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro Phe Leu Trp 15 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro 20 25 30

Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg lie Asn Ala 35 40 45

Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu Gly Tyr Tyr 50 55 60

Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly Gly lie Pro 65 70 75 80

Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys Lys Asp lie 85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val lie Asp Trp 100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val 115 120 125

Tyr Lys Asn Arg Ser He Glu Leu Val Gin Gin Gin Asn Val Gin Leu 130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe Glu Lys Ala 145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys Leu Leu Arg 165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His 180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu lie 195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu 210 215 220

Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val Ala Ala Thr 225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val Ser Lys lie 245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg lie Val 260 265 270

Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu Leu Val Tyr 275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie Val lie Trp 290 295 300

Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp 305 310 315 320

Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn Val Thr Leu 325 330 335

Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin Gly Val Cys 340 345 350 lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp 355 360 365

Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly 370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys Phe Tyr Cys 385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp 405 410 415

Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys lie Asp Ala 420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu 435 440

<210> 10 <211 >450 <212> PRT <213> Bos taurus <220> <223> hyaluronidase <400> 10

Met Arg Pro Phe Ser Leu Glu Val Ser Leu His Leu Pro Trp Ala Met 15 10 15

Ala Ala His Leu Leu Pro Val Cys Thr Leu Phe Leu Asn Leu Leu Ser 20 25 30

Met Thr Gin Gly Ser Arg Asp Pro Val Val Pro Asn Gin Pro Phe Thr 35 40 45

Thr lie Trp Asn Ala Asn Thr Glu Trp Cys Met Lys Lys His Gly Val 50 55 60

Asp Val Asp lie Ser lie Phe Asp Val Val Thr Asn Pro Gly Gin Thr 65 70 75 80

Phe Arg Gly Pro Asn Met Thr lie Phe Tyr Ser Ser Gin Leu Gly Thr 85 90 95

Tyr Pro Tyr Tyr Thr Ser Ala Gly Glu Pro Val Phe Gly Gly Leu Pro 100 105 110

Gin Asn Ala Ser Leu Asn Ala His Leu Ala Arg Thr Phe Gin Asp lie 115 120 125

Leu Ala Ala Met Pro Glu Pro Arg Phe Ser Gly Leu Ala Val lie Asp 130 135 140

Trp Glu Ala Trp Arg Pro Arg Trp Ala Phe Asn Trp Asp Thr Lys Asp 145 150 155 160 lie Tyr Arg Gin Arg Ser Arg Ala Leu Val Gin Lys Gin His Pro Asp 165 170 175

Trp Leu Ala Pro Arg Val Glu Ala Ala Ala Gin Asp Gin Phe Glu Gly 180 185 190

Ala Ala Glu Glu Trp Met Ala Gly Thr Leu Lys Leu Gly Gin Ala Leu 195 200 205

Arg Pro Gin Gly Leu Trp Gly Phe Tyr Asn Phe Pro Glu Cys Tyr Asn 210 215 220

Tyr Asp Phe Lys Ser Pro Asn Tyr Thr Gly Arg Cys Pro Leu Asn lie 225 230 235 240

Cys Ala Gin Asn Asp Gin Leu Gly Trp Leu Trp Gly Gin Ser Arg Ala 245 250 255

Leu Tyr Pro Ser lie Tyr Leu Pro Ala Ala Leu Glu Gly Thr Lys Lys 260 265 270

Thr Gin Met Phe Val Gin His Arg Val Ala Glu Ala Phe Arg Val Ala 275 280 285

Ala Gly Ala Gly Asp Pro Lys Leu Pro Val Leu Pro Tyr Met Gin Leu 290 295 300

Phe Tyr Asp Met Thr Asn His Phe Leu Pro Ala Glu Glu Leu Glu His 305 310 315 320

Ser Leu Gly Glu Ser Ala Ala Gin Gly Ala Ala Gly Val Val Leu Trp 325 330 335

Val Ser Trp Leu Ser Thr Ser Thr Lys Glu Ser Cys Gin Ala lie Lys 340 345 350

Glu Tyr Val Asp Thr Thr Leu Gly Pro Ser lie Leu Asn Val Thr Ser 355 360 365

Gly Ala Arg Leu Cys Ser Gin Val Leu Cys Ser Gly His Gly Arg Cys 370 375 380

Ala Arg Arg Pro Ser Tyr Pro Lys Ala Arg Leu lie Leu Asn Ser Thr 385 390 395 400

Ser Phe Ser lie Lys Pro Thr Pro Gly Gly Gly Pro Leu Thr Leu Gin 405 410 415

Gly Ala Leu Ser Leu Glu Asp Arg Leu Arg Met Ala Val Glu Phe Glu 420 425 430

Cys Arg Cys Tyr Arg Gly Trp Arg Gly Thr Arg Cys Glu Gin Trp Gly 435 440 445

Met Trp 450 <210> 11 <211> 553 <212> PRT <213> Bos taurus <220 <223> PH20 <400> 11

Met Arg Met Leu Arg Arg His His lie Ser Phe Arg Ser Phe Ala Gly 15 10 15

Ser Ser Gly Thr Pro Gin Ala Val Phe Thr Phe Leu Leu Leu Pro Cys 20 25 30

Cys Leu Ala Leu Asp Phe Arg Ala Pro Pro Leu lie Ser Asn Thr Ser 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Val Glu Arg Cys Val Asn Arg Arg 50 55 60

Phe Gin Leu Pro Pro Asp Leu Arg Leu Phe Ser Val Lys Gly Ser Pro 65 70 75 80

Gin Lys Ser Ala Thr Gly Gin Phe lie Thr Leu Phe Tyr Ala Asp Arg 85 90 95

Leu Gly Tyr Tyr Pro His lie Asp Glu Lys Thr Gly Lys Thr Val Phe 100 105 110

Gly Gly lie Pro Gin Leu Gly Asn Leu Lys Ser His Met Glu Lys Ala 115 120 125

Lys Asn Asp lie Ala Tyr Tyr lie Pro Asn Asp Ser Val Gly Leu Ala 130 135 140

Val lie Asp Trp Glu Asn Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys 145 150 155 160

Pro Lys Asp Val Tyr Arg Asp Glu Ser Val Glu Leu Val Leu Gin Lys 165 170 175

Asn Pro Gin Leu Ser Phe Pro Glu Ala Ser Lys lie Ala Lys Val Asp 180 185 190

Phe Glu Thr Ala Gly Lys Ser Phe Met Gin Glu Thr Leu Lys Leu Gly 195 200 205

Lys Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp 210 215 220

Cys Tyr Asn His Asn His Asn Gin Pro Thr Tyr Asn Gly Asn Cys Pro 225 230 235 240

Asp Val Glu Lys Arg Arg Asn Asp Asp Leu Glu Trp Leu Trp Lys Glu 245 250 255

Ser Thr Ala Leu Phe Pro Ser Val Tyr Leu Asn lie Arg Leu Lys Ser 260 265 270

Thr Gin Asn Ala Ala Leu Tyr Val Arg Asn Arg Val Gin Glu Ala lie 275 280 285

Arg Leu Ser Lys lie Ala Ser Val Glu Ser Pro Leu Pro Val Phe Val 290 295 300

Tyr Ala Arg Pro Val Phe Thr Asp Gly Ser Ser Thr Tyr Leu Ser Gin 305 310 315 320

Gly Asp Leu Val Asn Ser Val Gly Glu lie Val Ser Leu Gly Ala Ser 325 330 335

Gly lie lie Met Trp Gly Ser Leu Asn Leu Ser Leu Ser Met Gin Ser 340 345 350

Cys Met Asn Leu Gly Thr Tyr Leu Asn Thr Thr Leu Asn Pro Tyr lie 355 360 365 lie Asn Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys His 370 375 380

Asn Glu Gly Val Cys Thr Arg Lys His Trp Asn Ser Ser Asp Tyr Leu 385 390 395 400

His Leu Asn Pro Met Asn Phe Ala lie Gin Thr Gly Glu Gly Gly Lys 405 410 415

Tyr Thr Val Pro Gly Thr Val Thr Leu Glu Asp Leu Gin Lys Phe Ser 420 425 430

Asp Thr Phe Tyr Cys Ser Cys Tyr Ala Asn lie His Cys Lys Lys Arg 435 440 445

Val Asp lie Lys Asn Val His Ser Val Asn Val Cys Met Ala Glu Asp 450 455 460 lie Cys lie Asp Ser Pro Val Lys Leu Gin Pro Ser Asp His Ser Ser 465 470 475 480

Ser Gin Glu Ala Ser Thr Thr Thr Phe Ser Ser lie Ser Pro Ser Thr 485 490 495

Thr Thr Ala Thr Val Ser Pro Cys Thr Pro Glu Lys His Ser Pro Glu 500 505 510

Cys Leu Lys Val Arg Cys Ser Glu Val lie Pro Asn Val Thr Gin Lys 515 520 525

Ala Cys Gin Ser Val Lys Leu Lys Asn lie Ser Tyr Gin Ser Pro lie 530 535 540

Gin Asn lie Lys Asn Gin Thr Thr Tyr 545 550

<210> 12 <211> 331 <212> PRT <213> Vespula vulgaris <220> <223> hyaluronidase A <400 12

Ser Glu Arg Pro Lys Arg Val Phe Asn lie Tyr Trp Asn Val Pro Thr 15 10 15

Phe Met Cys His Gin Tyr Asp Leu Tyr Phe Asp Glu Val Thr Asn Phe 20 25 30

Asn lie Lys Arg Asn Ser Lys Asp Asp Phe Gin Gly Asp Lys lie Ala 35 40 45 lie Phe Tyr Asp Pro Gly Glu Phe Pro Ala Leu Leu Ser Leu Lys Asp 50 55 60

Gly Lys Tyr Lys Lys Arg Asn Gly Gly Val Pro Gin Glu Gly Asn lie 65 70 75 80

Thr lie His Leu Gin Lys Phe lie Glu Asn Leu Asp Lys lie Tyr Pro 85 90 95

Asn Arg Asn Phe Ser Gly lie Gly Val lie Asp Phe Glu Arg Trp Arg 100 105 110

Pro lie Phe Arg Gin Asn Trp Gly Asn Met Lys lie His Lys Asn Phe 115 120 125

Ser lie Asp Leu Val Arg Asn Glu His Pro Thr Trp Asn Lys Lys Met 130 135 140 lie Glu Leu Glu Ala Ser Lys Arg Phe Glu Lys Tyr Ala Arg Phe Phe 145 150 155 160

Met Glu Glu Thr Leu Lys Leu Ala Lys Lys Thr Arg Lys Gin Ala Asp 165 170 175

Trp Gly Tyr Tyr Gly Tyr Pro Tyr Cys Phe Asn Met Ser Pro Asn Asn 180 185 190

Leu Val Pro Glu Cys Asp Val Thr Ala Met His Glu Asn Asp Lys Met 195 200 205

Ser Trp Leu Phe Asn Asn Gin Asn Val Leu Leu Pro Ser Val Tyr Val 210 215 220

Arg Gin Glu Leu Thr Pro Asp Gin Arg lie Gly Leu Val Gin Gly Arg 225 230 235 240

Val Lys Glu Ala Val Arg lie Ser Asn Asn Leu Lys His Ser Pro Lys 245 250 255

Val Leu Ser Tyr Trp Trp Tyr Val Tyr Gin Asp Glu Thr Asn Thr Phe 260 265 270

Leu Thr Glu Thr Asp Val Lys Lys Thr Phe Gin Glu lie Val lie Asn 275 280 285

Gly Gly Asp Gly lie lie lie Trp Gly Ser Ser Ser Asp Val Asn Ser 290 295 300

Leu Ser Lys Cys Lys Arg Leu Gin Asp Tyr Leu Leu Thr Val Leu Gly 305 310 315 320

Pro lie Ala lie Asn Val Thr Glu Ala Val Asn 325 330

<210> 13 <211> 340 <212> PRT <213> Vespula vulgaris <220> <223> hyaluronidase B <400> 13

Asp Arg Thr lie Trp Pro Lys Lys Gly Phe Ser lie Tyr Trp Asn lie 15 10 15

Pro Thr His Phe Cys His Asn Phe Gly Val Tyr Phe Lys Glu Leu Lys 20 25 30

Gin Phe Asn lie Lys Tyr Asn Ser Met Asn Asn Phe Arg Gly Glu Thr 35 40 45 lie Ser Leu Phe Tyr Asp Pro Gly Asn Phe Pro Ser Met Val Leu Leu 50 55 60

Lys Asn Gly Thr Tyr Glu lie Arg Asn Glu Gly Val Pro Gin Lys Gly 65 70 75 80

Asn Leu Thr lie His Leu Glu Gin Phe Thr Lys Glu Leu Asp Glu lie 85 90 95

Tyr Pro Lys Lys lie Ala Gly Gly lie Gly Val lie His Phe His Asn 100 105 110

Trp Arg Pro lie Phe Arg Arg Asn Val Asp Asn Leu Lys lie Asn Lys 115 120 125

Asp lie Ser lie Asp Leu Val Arg Lys Glu His Pro Lys Trp Asp Lys 130 135 140

Ser Met lie Glu Lys Glu Ala Ser Asn Arg Phe Glu Thr Ser Ala Lys 145 150 155 160 lie Phe Met Glu Lys Thr Leu Lys Leu Ala Lys Glu lie Arg Lys Lys 165 170 175

Thr Glu Trp Gly Tyr His Gly Tyr Pro His Cys Leu Ser Gly Ser Thr 180 185 190

Asp Lys Pro Ser Phe Asp Cys Asp Ala Leu Ser Met Ser Glu Asn Asp 195 200 205

Lys Met Ser Trp Leu Phe Asn Asn Gin Asn Val Leu Leu Pro Ser lie 210 215 220

Tyr Leu Lys Asn Val Leu Lys Pro Asp Glu Lys lie His Leu Val Gin 225 230 235 240

Glu Arg Leu Lys Glu Ala lie Arg lie Ser Lys Asn Phe Lys His Leu 245 250 255

Pro Lys Val Leu Pro Tyr Trp Trp Tyr Thr Tyr Gin Asp Lys Glu Ser 260 265 270 lie Phe Leu Thr Glu Ala Asp Val Lys Asn Thr Phe Lys Glu lie Leu 275 280 285

Thr Asn Gly Ala Asp Gly lie lie lie Trp Gly Val Ser Tyr Glu Leu 290 295 300

Thr Asp Arg Lys Arg Cys Glu Lys Leu Lys Glu Tyr Leu Met Lys lie 305 310 315 320

Leu Gly Pro lie Ala Phe Lys Val Thr Lys Ala Val Lys Glu Asn Thr 325 330 335

Pro Leu Asn Phe 340 <210> 14 <211> 382

<212> PRT <213> Apis mellifera <220> <223> hyaluronidase <400> 14

Met Ser Arg Pro Leu Val lie Thr Glu Gly Met Met lie Gly Val Leu 15 10 15

Leu Met Leu Ala Pro lie Asn Ala Leu Leu Leu Gly Phe Val Gin Ser 20 25 30

Thr Pro Asp Asn Asn Lys Thr Val Arg Glu Phe Asn Val Tyr Trp Asn 35 40 45

Val Pro Thr Phe Met Cys His Lys Tyr Gly Leu Arg Phe Glu Glu Val 50 55 60

Ser Glu Lys Tyr Gly lie Leu Gin Asn Trp Met Asp Lys Phe Arg Gly 65 70 75 80

Glu Glu lie Ala lie Leu Tyr Asp Pro Gly Met Phe Pro Ala Leu Leu 85 90 95

Lys Asp Pro Asn Gly Asn Val Val Ala Arg Asn Gly Gly Val Pro Gin 100 105 110

Leu Gly Asn Leu Thr Lys His Leu Gin Val Phe Arg Asp His Leu lie 115 120 125

Asn Gin lie Pro Asp Lys Ser Phe Pro Gly Val Gly Val lie Asp Phe 130 135 140

Glu Ser Trp Arg Pro lie Phe Arg Gin Asn Trp Ala Ser Leu Gin Pro 145 150 155 160

Tyr Lys Lys Leu Ser Val Glu Val Val Arg Arg Glu His Pro Phe Trp 165 170 175

Asp Asp Gin Arg Val Glu Gin Glu Ala Lys Arg Arg Phe Glu Lys Tyr 180 185 190

Gly Gin Leu Phe Met Glu Glu Thr Leu Lys Ala Ala Lys Arg Met Arg 195 200 205

Pro Ala Ala Asn Trp Gly Tyr Tyr Ala Tyr Pro Tyr Cys Tyr Asn Leu 210 215 220

Thr Pro Asn Gin Pro Ser Ala Gin Cys Glu Ala Thr Thr Met Gin Glu 225 230 235 240

Asn Asp Lys Met Ser Trp Leu Phe Glu Ser Glu Asp Val Leu Leu Pro 245 250 255

Ser Val Tyr Leu Arg Trp Asn Leu Thr Ser Gly Glu Arg Val Gly Leu 260 265 270

Val Gly Gly Arg Val Lys Glu Ala Leu Arg lie Ala Arg Gin Met Thr 275 280 285

Thr Ser Arg Lys Lys Val Leu Pro Tyr Tyr Trp Tyr Lys Tyr Gin Asp 290 295 300

Arg Arg Asp Thr Asp Leu Ser Arg Ala Asp Leu Glu Ala Thr Leu Arg 305 310 315 320

Lys lie Thr Asp Leu Gly Ala Asp Gly Phe lie lie Trp Gly Ser Ser 325 330 335

Asp Asp lie Asn Thr Lys Ala Lys Cys Leu Gin Phe Arg Glu Tyr Leu 340 345 350

Asn Asn Glu Leu Gly Pro Ala Val Lys Arg lie Ala Leu Asn Asn Asn 355 360 365

Ala Asn Asp Arg Leu Thr Val Asp Val Ser Val Asp Gin Val 370 375 380

<210> 15 <211> 331 <212> PRT <213> Dolichovespula maculata <220> <223> hyaluronidase <400> 15

Ser Glu Arg Pro Lys Arg Val Phe Asn lie Tyr Trp Asn Val Pro Thr 15 10 15

Phe Met Cys His Gin Tyr Gly Leu Tyr Phe Asp Glu Val Thr Asn Phe 20 25 30

Asn lie Lys His Asn Ser Lys Asp Asp Phe Gin Gly Asp Lys lie Ser 35 40 45 lie Phe Tyr Asp Pro Gly Glu Phe Pro Ala Leu Leu Pro Leu Lys Glu 50 55 60

Gly Asn Tyr Lys lie Arg Asn Gly Gly Val Pro Gin Glu Gly Asn lie 65 70 75 80

Thr lie His Leu Gin Arg Phe lie Glu Asn Leu Asp Lys Thr Tyr Pro 85 90 95

Asn Arg Asn Phe Asn Gly lie Gly Val lie Asp Phe Glu Arg Trp Arg 100 105 110

Pro lie Phe Arg Gin Asn Trp Gly Asn Met Met lie His Lys Lys Phe 115 120 125

Ser lie Asp Leu Val Arg Asn Glu His Pro Phe Trp Asp Lys Lys Met 130 135 140 lie Glu Leu Glu Ala Ser Lys Arg Phe Glu Lys Tyr Ala Arg Leu Phe 145 150 155 160

Met Glu Glu Thr Leu Lys Leu Ala Lys Lys Thr Arg Lys Gin Ala Asp 165 170 175

Trp Gly Tyr Tyr Gly Tyr Pro Tyr Cys Phe Asn Met Ser Pro Asn Asn 180 185 190

Leu Val Pro Asp Cys Asp Ala Thr Ala Met Leu Glu Asn Asp Lys Met 195 200 205

Ser Trp Leu Phe Asn Asn Gin Asn Val Leu Leu Pro Ser Val Tyr lie 210 215 220

Arg His Glu Leu Thr Pro Asp Gin Arg Val Gly Leu Val Gin Gly Arg 225 230 235 240

Val Lys Glu Ala Val Arg lie Ser Asn Asn Leu Lys His Ser Pro Lys 245 250 255

Val Leu Ser Tyr Trp Trp Tyr Val Tyr Gin Asp Asp Thr Asn Thr Phe 260 265 270

Leu Thr Glu Thr Asp Val Lys Lys Thr Phe Gin Glu lie Ala lie Asn 275 280 285

Gly Gly Asp Gly lie lie lie Trp Gly Ser Ser Ser Asp Val Asn Ser 290 295 300

Leu Ser Lys Cys Lys Arg Leu Arg Glu Tyr Leu Leu Thr Val Leu Gly 305 310 315 320

Pro lie Thr Val Asn Val Thr Glu Thr Val Asn 325 330

<210> 16 <211> 367 <212> PRT <213> Polistes annularis <220> <223> hyaluronidase <400> 16

Tyr Val Ser Leu Ser Pro Asp Ser Val Phe Asn lie lie Thr Asp Asp 15 10 15 lie Ser His Gin lie Leu Ser Arg Ser Asn Cys Glu Arg Ser Lys Arg 20 25 30

Pro Lys Arg Val Phe Ser lie Tyr Trp Asn Val Pro Thr Phe Met Cys 35 40 45

His Gin Tyr Gly Met Asn Phe Asp Glu Val Thr Asp Phe Asn lie Lys 50 55 60

His Asn Ser Lys Asp Asn Phe Arg Gly Glu Thr lie Ser lie Tyr Tyr 65 70 75 80

Asp Pro Gly Lys Phe Pro Ala Leu Met Pro Leu Lys Asn Gly Asn Tyr 85 90 95

Glu Glu Arg Asn Gly Gly Val Pro Gin Arg Gly Asn lie Thr lie His 100 105 110

Leu Gin Gin Phe Asn Glu Asp Leu Asp Lys Met Thr Pro Asp Lys Asn 115 120 125

Phe Gly Gly lie Gly Val lie Asp Phe Glu Arg Trp Lys Pro lie Phe 130 135 140

Arg Gin Asn Trp Gly Asn Thr Glu lie His Lys Lys Tyr Ser lie Glu 145 150 155 160

Leu Val Arg Lys Glu His Pro Lys Trp Ser Glu Ser Met lie Glu Ala 165 170 175

Glu Ala Thr Lys Lys Phe Glu Lys Tyr Ala Arg Tyr Phe Met Glu Glu 180 185 190

Thr Leu Lys Leu Ala Lys Lys Thr Arg Lys Arg Ala Lys Trp Gly Tyr 195 200 205

Tyr Gly Phe Pro Tyr Cys Tyr Asn Val Thr Pro Asn Asn Pro Gly Pro 210 215 220

Asp Cys Asp Ala Lys Ala Thr lie Glu Asn Asp Arg Leu Ser Trp Met 225 230 235 240

Tyr Asn Asn Gin Glu lie Leu Phe Pro Ser Val Tyr Val Arg His Glu 245 250 255

Gin Lys Pro Glu Glu Arg Val Tyr Leu Val Gin Gly Arg lie Lys Glu 260 265 270

Ala Val Arg lie Ser Asn Asn Leu Glu His Ser Pro Ser Val Leu Ala 275 280 285

Tyr Trp Trp Tyr Val Tyr Gin Asp Lys Met Asp lie Tyr Leu Ser Glu 290 295 300

Thr Asp Val Glu Lys Thr Phe Gin Glu lie Val Thr Asn Gly Gly Asp 305 310 315 320

Gly lie lie lie Trp Gly Ser Ser Ser Asp Val Asn Ser Leu Ser Lys 325 330 335

Cys Lys Arg Leu Arg Glu Tyr Leu Leu Asn Thr Leu Gly Pro Phe Ala 340 345 350

Val Asn Val Thr Glu Thr Val Asn Gly Arg Ser Ser Leu Asn Phe 355 360 365 <210> 17 <211> 462 <212> PRT <213> Mus musculus <220> <223> hyaluronidase <400> 17

Met Leu Gly Leu Thr Gin His Ala Gin Lys Val Trp Arg Met Lys Pro 15 10 15

Phe Ser Pro Glu Val Ser Pro Gly Ser Ser Pro Ala Thr Ala Gly His 20 25 30

Leu Leu Arg lie Ser Thr Leu Phe Leu Thr Leu Leu Glu Leu Ala Gin 35 40 45

Val Cys Arg Gly Ser Val Val Ser Asn Arg Pro Phe lie Thr Val Trp 50 55 60

Asn Gly Asp Thr His Trp Cys Leu Thr Glu Tyr Gly Val Asp Val Asp 65 70 75 80

Val Ser Val Phe Asp Val Val Ala Asn Lys Glu Gin Ser Phe Gin Gly 85 90 95

Ser Asn Met Thr lie Phe Tyr Arg Glu Glu Leu Gly Thr Tyr Pro Tyr 100 105 110

Tyr Thr Pro Thr Gly Glu Pro Val Phe Gly Gly Leu Pro Gin Asn Ala 115 120 125

Ser Leu Val Thr His Leu Ala His Thr Phe Gin Asp lie Lys Ala Ala 130 135 140

Met Pro Glu Pro Asp Phe Ser Gly Leu Ala Val lie Asp Trp Glu Ala 145 150 155 160

Trp Arg Pro Arg Trp Ala Phe Asn Trp Asp Ser Lys Asp lie Tyr Arg 165 170 175

Gin Arg Ser Met Glu Leu Val Gin Ala Glu His Pro Asp Trp Pro Glu 180 185 190

Thr Leu Val Glu Ala Ala Ala Lys Asn Gin Phe Gin Glu Ala Ala Glu 195 200 205

Ala Trp Met Ala Gly Thr Leu Gin Leu Gly Gin Val Leu Arg Pro Arg 210 215 220

Gly Leu Trp Gly Tyr Tyr Gly Phe Pro Asp Cys Tyr Asn Asn Asp Phe 225 230 235 240

Leu Ser Leu Asn Tyr Thr Gly Gin Cys Pro Val Phe Val Arg Asp Gin 245 250 255

Asn Asp Gin Leu Gly Trp Leu Trp Asn Gin Ser Tyr Ala Leu Tyr Pro 260 265 270

Ser lie Tyr Leu Pro Ala Ala Leu Met Gly Thr Gly Lys Ser Gin Met 275 280 285

Tyr Val Arg His Arg Val Gin Glu Ala Leu Arg Val Ala lie Val Ser 290 295 300

Arg Asp Pro His Val Pro Val Met Pro Tyr Val Gin lie Phe Tyr Glu 305 310 315 320

Met Thr Asp Tyr Leu Leu Pro Leu Glu Glu Leu Glu His Ser Leu Gly 325 330 335

Glu Ser Ala Ala Gin Gly Val Ala Gly Ala Val Leu Trp Leu Ser Ser 340 345 350

Asp Lys Thr Ser Thr Lys Glu Ser Cys Gin Ala lie Lys Ala Tyr Met 355 360 365

Asp Ser Thr Leu Gly Pro Phe lie Val Asn Val Thr Ser Ala Ala Leu 370 375 380

Leu Cys Ser Glu Ala Leu Cys Ser Gly His Gly Arg Cys Val Arg His 385 390 395 400

Pro Ser Tyr Pro Glu Ala Leu Leu Thr Leu Asn Pro Ala Ser Phe Ser 405 410 415 lie Glu Leu Thr His Asp Gly Arg Pro Pro Ser Leu Lys Gly Thr Leu 420 425 430

Ser Leu Lys Asp Arg Ala Gin Met Ala Met Lys Phe Arg Cys Arg Cys 435 440 445

Tyr Arg Gly Trp Arg Gly Lys Trp Cys Asp Lys Arg Gly Met 450 455 460 <210> 18 <211> 473 <212> PRT <213> Mus musculus <220> <223> Hyaluronidase 2 <400> 18

Met Arg Ala Gly Leu Gly Pro lie lie Thr Leu Ala Leu Val Leu Glu 15 10 15

Val Ala Trp Ala Gly Glu Leu Lys Pro Thr Ala Pro Pro lie Phe Thr 20 25 30

Gly Arg Pro Phe Val Val Ala Trp Asn Val Pro Thr Gin Glu Cys Ala 35 40 45

Pro Arg His Lys Val Pro Leu Asp Leu Arg Ala Phe Asp Val Lys Ala 50 55 60

Thr Pro Asn Glu Gly Phe Phe Asn Gin Asn lie Thr Thr Phe Tyr Tyr 65 70 75 80

Asp Arg Leu Gly Leu Tyr Pro Arg Phe Asp Ala Ala Gly Thr Ser Val 85 90 95

His Gly Gly Val Pro Gin Asn Gly Ser Leu Cys Ala His Leu Pro Met 100 105 110

Leu Lys Glu Ser Val Glu Arg Tyr lie Gin Thr Gin Glu Pro Gly Gly 115 120 125

Leu Ala Val lie Asp Trp Glu Glu Trp Arg Pro Val Trp Val Arg Asn 130 135 140

Trp Gin Glu Lys Asp Val Tyr Arg Gin Ser Ser Arg Gin Leu Val Ala 145 150 155 160

Ser Arg His Pro Asp Trp Pro Ser Asp Arg Val Met Lys Gin Ala Gin 165 170 175

Tyr Glu Phe Glu Phe Ala Ala Arg Gin Phe Met Leu Asn Thr Leu Arg 180 185 190

Tyr Val Lys Ala Val Arg Pro Gin His Leu Trp Gly Phe Tyr Leu Phe 195 200 205

Pro Asp Cys Tyr Asn His Asp Tyr Val Gin Asn Trp Glu Ser Tyr Thr 210 215 220

Gly Arg Cys Pro Asp Val Glu Val Ala Arg Asn Asp Gin Leu Ala Trp 225 230 235 240

Leu Trp Ala Glu Ser Thr Ala Leu Phe Pro Ser Val Tyr Leu Asp Glu 245 250 255

Thr Leu Ala Ser Ser Val His Ser Arg Asn Phe Val Ser Phe Arg Val 260 265 270

Arg Glu Ala Leu Arg Val Ala His Thr His His Ala Asn His Ala Leu 275 280 285

Pro Val Tyr Val Phe Thr Arg Pro Thr Tyr Thr Arg Gly Leu Thr Gly 290 295 300

Leu Ser Gin Val Asp Leu lie Ser Thr lie Gly Glu Ser Ala Ala Leu 305 310 315 320

Gly Ser Ala Gly Val lie Phe Trp Gly Asp Ser Glu Asp Ala Ser Ser 325 330 335

Met Glu Thr Cys Gin Tyr Leu Lys Asn Tyr Leu Thr Gin Leu Leu Val 340 345 350

Pro Tyr lie Val Asn Val Ser Trp Ala Thr Gin Tyr Cys Ser Trp Thr 355 360 365

Gin Cys His Gly His Gly Arg Cys Val Arg Arg Asn Pro Ser Ala Asn 370 375 380

Thr Phe Leu His Leu Asn Ala Ser Ser Phe Arg Leu Val Pro Gly His 385 390 395 400

Thr Pro Ser Glu Pro Gin Leu Arg Pro Glu Gly Gin Leu Ser Glu Ala 405 410 415

Asp Leu Asn Tyr Leu Gin Lys His Phe Arg Cys Gin Cys Tyr Leu Gly 420 425 430

Trp Gly Gly Glu Gin Cys Gin Arg Asn Tyr Lys Gly Ala Ala Gly Asn 435 440 445

Ala Ser Arg Ala Trp Ala Gly Ser His Leu Thr Ser Leu Leu Gly Leu 450 455 460

Val Ala Val Ala Leu Thr Trp Thr Leu 465 470 <210 19 <211> 412 <212> PRT <213> Mus musculus <220> <223> hyalurinidase 3 <400> 19

Met lie Met His Leu Gly Leu Met Met Val Val Gly Leu Thr Leu Cys 15 10 15

Leu Met His Gly Gin Ala Leu Leu Gin Val Pro Glu His Pro Phe Ser 20 25 30

Val Val Trp Asn Val Pro Ser Ala Arg Cys Lys Ala His Phe Gly Val 35 40 45

His Leu Pro Leu Asp Ala Leu Gly lie Val Ala Asn His Gly Gin His 50 55 60

Phe His Gly Gin Asn lie Ser lie Phe Tyr Lys Asn Gin Phe Gly Leu 65 70 75 80

Tyr Pro Tyr Phe Gly Pro Arg Gly Thr Ala His Asn Gly Gly lie Pro 85 90 95

Gin Ala Val Ser Leu Asp His His Leu Ala Arg Ala Ala His Gin lie 100 105 110

Leu His Ser Leu Gly Ser Ser Phe Ala Gly Leu Ala Val Leu Asp Trp 115 120 125

Glu Glu Trp Tyr Pro Leu Trp Ala Gly Asn Trp Gly Pro His Arg Gin 130 135 140

Val Tyr Leu Ala Ala Ser Trp Val Trp Thr Gin Gin Met Phe Pro Gly 145 150 155 160

Leu Asp Pro Gin Glu Gin Leu His Lys Ala His Thr Ser Phe Glu Gin 165 170 175

Ala Ala Arg Ala Leu Met Glu Tyr Thr Leu Gin Leu Gly Arg Thr Leu 180 185 190

Arg Pro Ser Gly Leu Trp Gly Phe Tyr Arg Tyr Pro Ala Cys Gly Asn 195 200 205

Gly Trp His Lys Met Ala Ser Asn Tyr Thr Gly His Cys His Ala Ala 210 215 220 lie Thr Thr Gin Asn Thr Gin Leu Arg Trp Leu Trp Ala Ala Ser Ser 225 230 235 240

Ala Leu Phe Pro Ser lie Tyr Leu Pro Pro Arg Leu Pro Leu Ala Tyr 245 250 255

Arg Gin Ala Phe Val Arg His Arg Leu Glu Glu Ala Phe Arg Val Ala 260 265 270

Leu Leu Glu His Ser His Pro Leu Pro Val Leu Ala Tyr Ser Arg Leu 275 280 285

Thr His Arg Ser Ser Gly Arg Phe Leu Ser Leu Asp Asp Leu Met Gin 290 295 300

Thr lie Gly Val Ser Ala Ala Leu Gly Thr Ala Gly Val Val Leu Trp 305 310 315 320

Gly Asp Leu Ser Phe Ser Ser Ser Glu Glu Lys Cys Trp Arg Leu His 325 330 335

Asp Tyr Leu Val Gly Thr Leu Gly Pro Tyr Val lie Asn Val Thr Lys 340 345 350

Ala Asp Met Ala Cys Ser His Gin Arg Cys His Gly His Gly Arg Cys 355 360 365

Ala Arg Lys Asp Pro Gly Gin Met Glu Ala Phe Leu His Leu Gin Pro 370 375 380

Asp Asp Ser Leu Gly Ala Trp Asn Ser Phe Arg Cys His Cys Tyr Ser 385 390 395 400

Gly Trp Ala Gly Pro Thr Cys Leu Glu Pro Lys Pro 405 410 <210> 20 <211> 435 <212> PRT <213> Sus scrofa <220> <223> hyalauronidase <400> 20

Met Ala Ala His Leu Leu Pro lie Cys Thr Leu Phe Leu Asn Leu Leu 15 10 15

Ser Val Ala Gin Gly Ser Arg Asp Pro Val Val Leu Asn Arg Pro Phe 20 25 30

Thr Thr lie Trp Asn Ala Asn Thr Gin Trp Cys Leu Lys Arg His Gly 35 40 45

Val Asp Val Asp Val Ser Val Phe Glu Val Val Val Asn Pro Gly Gin 50 55 60

Thr Phe Arg Gly Pro Asn Met Thr lie Phe Tyr Ser Ser Gin Leu Gly 65 70 75 80

Thr Tyr Pro Tyr Tyr Thr Ser Ala Gly Glu Pro Val Phe Gly Gly Leu 85 90 95

Pro Gin Asn Ala Ser Leu Asp Val His Leu Asn Arg Thr Phe Lys Asp 100 105 110 lie Leu Ala Ala Met Pro Glu Ser Asn Phe Ser Gly Leu Ala Val lie 115 120 125

Asp Trp Glu Ala Trp Arg Pro Arg Trp Ala Phe Asn Trp Asp Ala Lys 130 135 140

Asp lie Tyr Arg Gin Arg Ser Arg Ala Leu Val Gin Lys Gin His Pro 145 150 155 160

Asp Trp Pro Ala Pro Trp Val Glu Ala Ala Ala Gin Asp Gin Phe Gin 165 170 175

Glu Ala Ala Gin Thr Trp Met Ala Gly Thr Leu Lys Leu Gly Gin Thr 180 185 190

Leu Arg Pro His Gly Leu Trp Gly Phe Tyr Gly Phe Pro Asp Cys Tyr 195 200 205

Asn Tyr Asp Phe Gin Ser Ser Asn Tyr Thr Gly Gin Cys Pro Pro Gly 210 215 220

Val Ser Ala Gin Asn Asp Gin Leu Gly Trp Leu Trp Gly Gin Ser Arg 225 230 235 240

Ala Leu Tyr Pro Ser He Tyr Leu Pro Ser Ala Leu Glu Gly Thr Asn 245 250 255

Lys Thr Gin Leu Tyr Val Gin His Arg Val Asn Glu Ala Phe Arg Val 260 265 270

Ala Ala Ala Ala Gly Asp Pro Asn Leu Pro Val Leu Pro Tyr Ala Gin 275 280 285 lie Phe His Asp Met Thr Asn Arg Leu Leu Ser Arg Glu Glu Leu Glu 290 295 300

His Ser Leu Gly Glu Ser Ala Ala Gin Gly Ala Ala Gly Val Val Leu 305 310 315 320

Trp Val Ser Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gin Ser He 325 330 335

Lys Glu Tyr Val Asp Thr Thr Leu Gly Pro Phe He Leu Asn Val Thr 340 345 350

Ser Gly Ala Leu Leu Cys Ser Gin Ala Val Cys Ser Gly His Gly Arg 355 360 365

Cys Val Arg Arg Pro Ser His Thr Glu Ala Leu Pro He Leu Asn Pro 370 375 380

Ser Ser Phe Ser He Lys Pro Thr Pro Gly Gly Gly Pro Leu Thr Leu 385 390 395 400

Gin Gly Ala Leu Ser Leu Lys Asp Arg Val Gin Met Ala Glu Glu Phe 405 410 415

Gin Cys Arg Cys Tyr Pro Gly Trp Arg Gly Thr Trp Cys Glu Gin Gin 420 425 430

Gly Thr Arg 435 <210> 21 <211> 419 <212> PRT <213> Sus scrofa <220> <223> hyaluronidase 3 <400> 21 ' Met Thr Met Gin Leu Gly Leu Ala Leu Val Leu Gly Val Ala Met Cys 15 10 15

Leu Gly Cys Gly Gin Pro Leu Leu Arg Ala Pro Glu Arg Pro Phe Cys 20 25 30

Val Leu Trp Asn Val Pro Ser Ala Arg Cys Lys Ala Arg Phe Gly Val 35 40 45

His Leu Pro Leu Glu Ala Leu Gly lie Thr Ala Asn His Gly Gin Arg 50 55 60

Phe His Gly Gin Asn lie Thr lie Phe Tyr Lys Ser Gin Leu Gly Leu 65 70 75 80

Tyr Pro Tyr Phe Gly Pro Arg Gly Thr Ala His Asn Gly Gly lie Pro 85 90 95

Gin Ala Val Ser Leu Asp His His Leu Ala Arg Ala Ala Tyr Gin lie 100 105 110

His Arg Ser Leu Arg Pro Gly Phe Thr Gly Leu Ala Val Leu Asp Trp 115 120 125

Glu Glu Trp Cys Pro Leu Trp Ala Gly Asn Trp Gly Arg Arg Gin Ala 130 135 140

Tyr Gin Ala Ala Ser Cys Ala Trp Ala Gin Arg Val Tyr Pro Asn Leu 145 150 155 160

Asp Pro Gin Glu Gin Leu Cys Lys Ala Arg Ala Gly Phe Glu Glu Ala 165 170 175

Ala Arg Ala Leu Met Glu Asp Thr Leu Arg Leu Gly Arg Met Leu Arg 180 185 190

Pro His Gly Leu Trp Gly Phe Tyr His Tyr Pro Ala Cys Gly Asn Gly 195 200 205

Trp His Gly Thr Ala Ser Asn Tyr Thr Gly His Cys His Ala Ala Ala 210 215 220

Leu Ala Arg Asn Thr Gin Leu Tyr Trp Leu Trp Ala Ala Ser Ser Ala 225 230 235 240

Leu Phe Pro Ser lie Tyr Leu Pro Pro Gly Leu Pro Pro Ala Tyr His 245 250 255

Gin Ala Phe Val Arg Tyr Arg Leu Glu Glu Ala Phe Arg Val Ala Leu 260 265 270

Val Gly His Pro His Pro Leu Pro Val Leu Ala Tyr Ala Arg Leu Thr 275 280 285

His Arg Asn Ser Gly Arg Phe Leu Ser Gin Asp Glu Leu Val Gin Thr 290 295 300 lie Gly Val Ser Ala Ala Leu Gly Ala Ser Gly Val Val Leu Trp Gly 305 310 315 320

Asp Leu Ser Phe Ser Ser Ser Glu Glu Glu Cys Trp His Leu Arg Gly 325 330 335

Tyr Leu Val Gly Thr Leu Gly Pro Tyr Val lie Asn Val Thr Arg Ala 340 345 350

Ala Met Ala Cys Ser His Gin Arg Cys His Gly His Gly Arg Cys Ala 355 360 365

Trp Gin Asp Pro Gly Gin Leu Lys Val Phe Leu His Leu His Pro Gly 370 375 380

Gly Ser Pro Gly Ala Trp Glu Ser Phe Ser Cys Arg Cys Tyr Trp Gly 385 390 395 400

Trp Ala Gly Pro Thr Cys Gin Glu Pro Arg Pro Glu Leu Gly Pro Glu 405 410 415

Glu Ala Thr <210> 22 <211> 449

<212> PRT <213> Rattus norvegicus <220> <223> hyaluronidase 1 <400> 22

Met Lys Pro Phe Ser Pro Glu Val Ser Pro Asp Pro Cys Pro Ala Thr 15 10 15

Ala Ala His Leu Leu Arg Thr Tyr Thr Leu Phe Leu Thr Leu Leu Glu 20 25 30

Leu Ala Gin Gly Cys Arg Gly Ser Met Val Ser Asn Arg Pro Phe lie 35 40 45

Thr Val Trp Asn Ala Asp Thr His Trp Cys Leu Lys Asp His Gly Val 50 55 60

Asp Val Asp Val Ser Val Phe Asp Val Val Ala Asn Lys Glu Gin Asn 65 70 75 80

Phe Gin Gly Pro Asn Met Thr lie Phe Tyr Arg Glu Glu Leu Gly Thr 85 90 95

Tyr Pro Tyr Tyr Thr Pro Thr Gly Glu Pro Val Phe Gly Gly Leu Pro 100 105 110

Gin Asn Ala Ser Leu Val Thr His Leu Ala His Ala Phe Gin Asp lie 115 120 125

Lys Ala Ala Met Pro Glu Pro Asp Phe Ser Gly Leu Ala Val lie Asp 130 135 140

Trp Glu Ala Trp Arg Pro Arg Trp Ala Phe Asn Trp Asp Ser Lys Asp 145 150 155 160 lie Tyr Gin Gin Arg Ser Met Glu Leu Val Arg Ala Glu His Pro Asp 165 170 175

Trp Pro Glu Thr Leu Val Glu Ala Glu Ala Gin Gly Gin Phe Gin Glu 180 185 190

Ala Ala Glu Ala Trp Met Ala Gly Thr Leu Gin Leu Gly Gin Val Leu 195 200 205

Arg Pro Arg Gly Leu Trp Gly Tyr Tyr Gly Phe Pro Asp Cys Tyr Asn 210 215 220

Tyr Asp Phe Leu Ser Pro Asn Tyr Thr Gly Gin Cys Ser Leu Ser lie 225 230 235 240

His Asp Gin Asn Asp Gin Leu Gly Trp Leu Trp Asn Gin Ser Tyr Ala 245 250 255

Leu Tyr Pro Ser lie Tyr Leu Pro Ala Ala Leu Met Gly Thr Gly Lys 260 265 270

Ser Gin Met Tyr Val Arg Tyr Arg Val Gin Glu Ala Phe Arg Leu Ala 275 280 285

Leu Val Ser Arg Asp Pro His Val Pro lie Met Pro Tyr Val Gin lie 290 295 300

Phe Tyr Glu Lys Thr Asp Tyr Leu Leu Pro Leu Glu Glu Leu Glu His 305 310 315 320

Ser Leu Gly Glu Ser Ala Ala Gin Gly Ala Ala Gly Ala Val Leu Trp 325 330 335 lie Ser Ser Glu Lys Thr Ser Thr Lys Glu Ser Cys Gin Ala lie Lys 340 345 350

Ala Tyr Met Asp Ser Thr Leu Gly Pro Phe lie Leu Asn Val Thr Ser 355 360 365

Ala Ala Leu Leu Cys Ser Glu Ala Leu Cys Ser Gly Arg Gly Arg Cys 370 375 380

Val Arg His Pro Ser Tyr Pro Glu Ala Leu Leu Thr Leu Ser Pro Ala 385 390 395 400

Ser Phe Ser lie Glu Pro Thr His Asp Gly Arg Pro Leu Ser Leu Lys 405 410 415

Gly Thr Leu Ser Leu Lys Asp Arg Ala Gin Met Ala Met Lys Phe Lys 420 425 430

Cys Arg Cys Tyr Arg Gly Trp Ser Gly Glu Trp Cys Lys Lys Gin Asp 435 440 445

Met <210> 23

<211> 473 <212> PRT <213> Rattus norvegicus <220> <223> hyaluronidase 2 <400> 23

Met Arg Ala Gly Leu Gly Pro lie lie Thr Leu Ala Leu Val Leu Glu 15 10 15

Val Ala Trp Ala Ser Glu Leu Lys Pro Thr Ala Pro Pro lie Phe Thr 20 25 30

Gly Arg Pro Phe Val Val Ala Trp Asn Val Pro Thr Gin Glu Cys Ala 35 40 45

Pro Arg His Lys Val Pro Leu Asp Leu Arg Ala Phe Asp Val Glu Ala 50 55 60

Thr Pro Asn Glu Gly Phe Phe Asn Gin Asn lie Thr Thr Phe Tyr Tyr 65 70 75 80

Asp Arg Leu Gly Leu Tyr Pro Arg Phe Asp Ala Ala Gly Met Ser Val 85 90 95

His Gly Gly Val Pro Gin Asn Gly Ser Leu Cys Ala His Leu Pro Met 100 105 110

Leu Lys Glu Ala Val Glu Arg Tyr lie Gin Thr Gin Glu Pro Ala Gly 115 120 125

Leu Ala Val lie Asp Trp Glu Glu Trp Arg Pro Val Trp Val Arg Asn 130 135 140

Trp Gin Glu Lys Asp Val Tyr Arg Gin Ser Ser Arg Gin Leu Val Ala 145 150 155 160

Ser Arg His Pro Asp Trp Pro Ser Asp Arg lie Val Lys Gin Ala Gin 165 170 175

Tyr Glu Phe Glu Phe Ala Ala Arg Gin Phe Met Leu Asn Thr Leu Arg 180 185 190

Tyr Val Lys Ala Val Arg Pro Gin His Leu Trp Gly Phe Tyr Leu Phe 195 200 205

Pro Asp Cys Tyr Asn His Asp Tyr Val Gin Asn Trp Asp Ser Tyr Thr 210 215 220

Gly Arg Cys Pro Asp Val Glu Val Ala Gin Asn Asp Gin Leu Ala Trp 225 230 235 240

Leu Trp Ala Glu Asn Thr Ala Leu Phe Pro Ser Val Tyr Leu Asp Lys 245 250 255

Thr Leu Ala Ser Ser Lys His Ser Arg Asn Phe Val Ser Phe Arg Val 260 265 270

Gin Glu Ala Leu Arg Val Ala His Thr His His Ala Asn His Ala Leu 275 280 285

Pro Val Tyr Val Phe Thr Arg Pro Thr Tyr Thr Arg Arg Leu Thr Glu 290 295 300

Leu Asn Gin Met Asp Leu lie Ser Thr lie Gly Glu Ser Ala Ala Leu 305 310 315 320

Gly Ser Ala Gly Val lie Phe Trp Gly Asp Ser Val Tyr Ala Ser Ser 325 330 335

Met Glu Asn Cys Gin Asn Leu Lys Lys Tyr Leu Thr Gin Thr Leu Val 340 345 350

Pro Tyr lie Val Asn Val Ser Trp Ala Thr Gin Tyr Cys Ser Trp Thr 355 360 365

Gin Cys His Gly His Gly Arg Cys Val Arg Arg Asn Pro Ser Ala Ser 370 375 380

Thr Phe Leu His Leu Ser Pro Ser Ser Phe Arg Leu Val Pro Gly Arg 385 390 395 400

Thr Pro Ser Glu Pro Gin Leu Arg Pro Glu Gly Glu Leu Ser Glu Asp 405 410 415

Asp Leu Ser Tyr Leu Gin Met His Phe Arg Cys His Cys Tyr Leu Gly 420 425 430

Trp Gly Gly Glu Gin Cys Gin Trp Asn His Lys Arg Ala Ala Gly Asp 435 440 445

Ala Ser Arg Ala Trp Ala Gly Ala His Leu Ala Ser Leu Leu Gly Leu 450 455 460

Val Ala Met Thr Leu Thr Trp Thr Leu 465 470

<210> 24 <211> 412 <212> PRT <213> Rattus norvegicus <220> <223> hyaluronidase 3 <400> 24

Met lie Thr Gin Leu Gly Leu Thr Leu Val Val Gly Leu Thr Leu Cys 15 10 15

Leu Val His Val Gin Ala Leu Leu Gin Val Pro Glu Phe Pro Phe Ser 20 25 30

Val Leu Trp Asn Val Pro Ser Ala Arg Cys Lys Thr Arg Phe Gly Val 35 40 45

His Leu Pro Leu Asp Ala Leu Gly lie lie Ala Asn His Gly Gin Arg 50 55 60

Phe His Gly Gin Asn lie Thr lie Phe Tyr Lys Asn Gin Phe Gly Leu 65 70 75 80

Tyr Pro Tyr Phe Gly Pro Arg Gly Thr Ala His Asn Gly Gly lie Pro 85 90 95

Gin Ala Val Ser Leu Asp His His Leu Ala Gin Ala Ala His Gin lie 100 105 110

Leu His Asn Leu Gly Ser Ser Phe Ala Gly Leu Ala Val Leu Asp Trp 115 120 125

Glu Glu Trp Tyr Pro Leu Trp Ala Gly Asn Trp Gly Thr His Arg Gin 130 135 140

Val Tyr Gin Ala Ala Ser Trp Ala Trp Ala Gin Gin Met Phe Pro Asp 145 150 155 160

Leu Asn Pro Gin Glu Gin Leu His Lys Ala Gin Thr Gly Phe Glu Gin 165 170 175

Ala Ala Arg Ala Leu Met Glu His Thr Leu Arg Leu Gly Gin Met Leu 180 185 190

Arg Pro His Gly Leu Trp Gly Phe Tyr Arg Tyr Pro Val Cys Gly Asn 195 200 205

Gly Trp His Asn Met Ala Ser Asn Tyr Thr Gly His Cys His Pro Ala 210 215 220

He lie Thr Arg Asn Thr Gin Leu Arg Trp Leu Trp Ala Ala Ser Ser 225 230 235 240

Ala Leu Phe Pro Ser lie Tyr Leu Pro Pro Arg Leu Pro Pro Ala Tyr 245 250 255

His Gin Thr Phe Val Arg His Arg Leu Glu Glu Ala Phe Arg Val Ala 260 265 270

Leu Thr Gly His Ala His Pro Leu Pro Val Leu Ala Tyr Val Arg Leu 275 280 285

Thr His Arg Ser Ser Gly Arg Phe Leu Ser Leu Asp Asp Leu Met Gin 290 295 300

Thr He Gly Val Ser Ala Ala Leu Gly Ala Ala Gly Val Val Leu Trp 305 310 315 320

Gly Asp Leu Ser Val Ser Ser Ser Glu Glu Glu Cys Trp Arg Leu His 325 330 335

Asp Tyr Leu Val Gly Thr Leu Gly Pro Tyr Val He Asn Val Thr Lys 340 345 350

Ala Ala Thr Ala Cys Ser His Gin Arg Cys His Gly His Gly Arg Cys 355 360 365

Ser Trp Lys Asp Pro Gly Gin Met Glu Ala Phe Leu His Leu Gin Pro 370 375 380

Asp Asp Asn Leu Gly Ala Trp Lys Ser Phe Arg Cys Arg Cys Tyr Leu 385 390 395 400

Gly Trp Ser Gly Pro Thr Cys Leu Glu Pro Lys Pro 405 410

<210> 25 <211> 545 <212> PRT <213> Oryctolagus cuniculus <220 <223> PH20 <400> 25

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Gly Ser Ala Val Glu 15 10 15

Leu Ser Gly Val Phe Gin lie Val Phe lie Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Ala Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Thr Glu Phe Cys Leu Gly Lys Ser 50 55 60

Gly Glu Pro Leu Asp Met Ser Leu Phe Ser Leu Phe Gly Ser Pro Arg 65 70 75 80

Lys Asn Lys Thr Gly Gin Gly lie Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Pro His Thr Gly Ala lie Val His Gly 100 105 110

Arg lie Pro Gin Leu Gly Pro Leu Gin Gin His Leu Thr Lys Leu Arg 115 120 125

Gin Glu lie Leu Tyr Tyr Met Pro Lys Asp Asn Val Gly Leu Ala Val 130 135 140 lie Asp Trp Glu Glu Trp Leu Pro Thr Trp Leu Arg Asn Trp Lys Pro 145 150 155 160

Lys Asp lie Tyr Arg lie Lys Ser lie Glu Leu Val Lys Ser Gin His 165 170 175

Pro Gin Tyr Asn His Ser Tyr Ala Thr Glu Lys Ala Lys Arg Asp Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Met Glu Glu Thr Leu Lys Leu Gly Arg 195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Asp Lys Pro Asn Leu Tyr Lys Gly Ser Cys Phe 225 230 235 240

Asp lie Glu Lys Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Lys Glu 245 250 255

Ser Thr Ala Leu Phe Pro Ser Val Tyr Leu Thr Ser Arg Ala Arg Ser 260 265 270

Ala Thr Ala Leu Ser Lys Leu Tyr Val Val Arg Asn Arg Val His Glu 275 280 285

Ala lie Arg Val Ser Lys lie Pro Asp Asp Lys Ser Pro Leu Pro Asn 290 295 300

Phe Val Tyr Thr Arg Leu Val Phe Thr Asp Gin lie Phe Gin Phe Leu 305 310 315 320

Ser His His Asp Leu Val Tyr Thr lie Gly Glu lie Val Ala Leu Gly 325 330 335

Ala Ser Gly lie Val Val Trp Gly Ser Gin Ser Leu Ala Arg Ser Met 340 345 350

Lys Ser Cys Leu His Leu Asp Asn Tyr Met Lys Thr lie Leu Asn Pro 355 360 365

Tyr Leu lie Asn Val Thr Leu Ala Ala Lys Met Cys Asn Gin Val Leu 370 375 380

Cys Gin Glu Gin Gly Val Cys Thr Arg Lys Asn Trp Asn Pro Asn Asp 385 390 395 400

Tyr Leu His Leu Asn Pro Gly Asn Phe Ala lie Gin Leu Gly Ser Asn 405 410 415

Gly Thr Tyr Lys Val Asp Gly Lys Pro Thr Leu Thr Asp Leu Glu Gin 420 425 430

Phe Ser Lys Asn Phe Gin Cys Ser Cys Tyr Thr Asn Leu Asn Cys Lys 435 440 445

Glu Arg Thr Asp Met Asn Asn Val Arg Thr Val Asn Val Cys Ala Val 450 455 460

Glu Asn Val Cys lie Asp Thr Asn Val Gly Pro Gin Ala Val Thr Tyr 465 470 475 480

Ala Pro Lys Glu Lys Lys Asp Val Ala His lie Leu Ser Asn Thr Thr 485 490 495

Ser lie Asn Ser Ser Thr Thr Met Ser Leu Pro Phe Pro Arg Lys His 500 505 510

Val Ser Gly Cys Leu Leu Val Leu Cys Met Tyr Ser Gin Tyr Leu Asn 515 520 525

He Cys Tyr Arg Leu Val Ala lie Gly He Gin His Gly Tyr Tyr Leu 530 535 540

Lys 545 <210> 26 <211> 476 <212> PRT <213> Ovis aries <220> <223> hyaluronidase 2 <400> 26

I

I ) 9 9 5

Met Trp Thr Gly Leu Gly Pro Ala Val Thr Leu Ala Leu Val Leu Val 15 10 15

Val Ala Trp Ala Thr Glu Leu Lys Pro Thr Ala Pro Pro lie Phe Thr 20 25 30

Gly Arg Pro Phe Val Val Ala Trp Asp Val Pro Thr Gin Asp Cys Gly 35 40 45

Pro Arg His Lys Met Pro Leu Asp Pro Lys Asp Met Lys Ala Phe Asp 50 55 60

Val Gin Ala Ser Pro Asn Glu Gly Phe Val Asn Gin Asn lie Thr lie 1 65 70 75 80

Phe Tyr Arg Asp Arg Leu Gly Met Tyr Pro His Phe Asn Ser Val Gly 85 90 95

Arg Ser Val His Gly Gly Val Pro Gin Asn Gly Ser Leu Trp Val His 100 105 110

Leu Glu Met Leu Lys Gly His Val Glu His Tyr lie Arg Thr Gin Glu 115 120 125

Pro Ala Gly Leu Ala Val lie Asp Trp Glu Asp Trp Arg Pro Val Trp 130 135 140

Val Arg Asn Trp Gin Asp Lys Asp Val Tyr Arg Arg Leu Ser Arg Gin 145 150 155 160

Leu Val Ala Ser His His Pro Asp Trp Pro Pro Glu Arg lie Val Lys 165 170 175

Glu Ala Gin Tyr Glu Phe Glu Phe Ala Ala Arg Gin Phe Met Leu Glu 180 185 190

Thr Leu Arg Phe Val Lys Ala Phe Arg Pro Arg His Leu Trp Gly Phe 195 200 205

Tyr Leu Phe Pro Asp Cys Tyr Asn His Asp Tyr Val Gin Asn Trp Glu 210 215 220

Thr Tyr Thr Gly Arg Cys Pro Asp Val Glu Val Ser Arg Asn Asp Gin 225 230 235 240 i Leu Ser Trp Leu Trp Ala Glu Ser Thr Ala Leu Phe Pro Ser Val Tyr 245 250 255

Leu Glu Glu Thr Leu Ala Ser Ser Thr His Gly Arg Asn Phe Val Ser 260 265 270

Phe Arg Val Gin Glu Ala Leu Arg Val Ala Asp Val His His Ala Asn 275 280 285

His Ala Leu Pro Val Tyr Val Phe Thr Arg Pro Thr Tyr Ser Arg Gly 290 295 300

Leu Thr Gly Leu Ser Glu Met Asp Leu lie Ser Thr lie Gly Glu Ser 305 310 315 320

Ala Ala Leu Gly Ala Ala Gly Val lie Leu Trp Gly Asp Ala Gly Phe 1 325 330 335

Thr Thr Ser Asn Glu Thr Cys Arg Arg Leu Lys Asp Tyr Leu Thr Arg 340 345 350

Ser Leu Val Pro Tyr Val Val Asn Val Ser Trp Ala Ala Gin Tyr Cys 355 360 365

Ser Trp Ala Gin Cys His Gly His Gly Arg Cys Val Arg Arg Asp Pro 370 375 380

Asn Ala His Thr Phe Leu His Leu Ser Ala Ser Ser Phe Arg Leu Val 385 390 395 400

Pro Ser His Ala Pro Asp Glu Pro Arg Leu Arg Pro Glu Gly Glu Leu 405 410 415

Ser Trp Ala Asp Arg Asn His Leu Gin Thr His Phe Arg Cys Gin Cys 420 425 430

Tyr Leu Gly Trp Gly Gly Glu Gin Cys Gin Trp Asp Arg Arg Arg Ala 435 440 445

Ala Gly Gly Ala Ser Gly Ala Trp Ala Gly Ser His Leu Thr Gly Leu 450 455 460

Leu Ala Val Ala Val Leu Ala Phe Thr Trp Thr Ser 465 470 475 <210> 27 <211 > 114 <212> PRT <213> Ovis aries <220> <223> PH20 partial sequence <400 27

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Leu Ser Lys lie 15 10 15

Ala Ser Val Glu Ser Pro Leu Pro Val Phe Val Tyr His Arg Pro Val 20 25 30

Phe Thr Asp Gly Ser Ser Thr Tyr Leu Ser Gin Gly Asp Leu Val Asn 35 40 45

Ser Val Gly Glu lie Val Ala Leu Gly Ala Ser Gly lie lie Met Trp 50 55 60

Gly Ser Leu Asn Leu Ser Leu Thr Met Gin Ser Cys Met Asn Leu Gly 65 70 75 80

Asn Tyr Leu Asn Thr Thr Leu Asn Pro Tyr lie lie Asn Val Thr Leu 85 90 95

Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin Gly Val Cys 100 105 110 lie Arg

<210> 28 <211> 414 <212> PRT <213> Pongo pygmaeus <220> <223> hyaluronidase 3 <400 28

Met Thr Thr Arg Leu Gly Pro Ala Leu Val Leu Gly Val Ala Leu Cys 15 10 15

Leu Gly Cys Gly Gin Pro Leu Pro Gin Val Pro Glu Arg Pro Phe Ser 20 25 30

Val Leu Trp Asn Val Pro Ser Ala His Cys Lys Ser Arg Phe Gly Val 35 40 45

His Leu Pro Leu Asn Ala Leu Gly lie lie Ala Asn Arg Gly Gin His 50 55 60

Phe His Gly Gin Asn Met Thr lie Phe Tyr Lys Asn Gin Leu Gly Leu ' 65 70 75 80

Tyr Pro Tyr Phe Gly Pro Lys Gly Thr Ala His Asn Gly Gly He Pro 85 90 95

Gin Ala Leu Pro Leu Asp Arg His Leu Ala Leu Ala Ala Tyr Gin lie 100 105 110

His His Ser Leu Arg Pro Gly Phe Ala Gly Pro Ala Val Leu Asp Trp 115 120 125

Glu Glu Trp Cys Pro Leu Trp Ala Gly Asn Trp Gly Arg Arg Arg Ala 130 135 140

Tyr Gin Ala Ala Ser Trp Ala Trp Ala Gin Gin Val Phe Pro Asp Leu 145 150 155 160 1 Asp Pro Gin Glu Gin Leu Tyr Lys Ala Tyr Thr Gly Phe Glu Gin Ala 165 170 175

Ala Arg Ala Leu Met Glu Asp Thr Leu Arg Val Ala Gin Ala Leu Arg 180 185 190

Pro His Gly Leu Trp Gly Phe Tyr His Tyr Pro Ala Cys Gly Asn Gly 195 200 205

Trp His Ser Met Ala Ser Asn Tyr Thr Gly Arg Cys His Ala Ala Thr 210 215 220

Leu Ala Arg Asn Thr Gin Leu His Trp Leu Trp Ala Ala Ser Ser Ala 225 230 235 240

Leu Phe Pro Ser He Tyr Leu Pro Pro Arg Leu Pro Pro Ala His His 245 250 255

Gin Ala Phe Val Arg His Arg Leu Glu Glu Ala Phe Arg Val Ala Leu 260 265 270

Val Gly His Leu Pro Val Leu Ala Tyr Val Arg Leu Thr His Arg Arg 275 280 285

Ser Gly Arg Phe Leu Ser Gin Asp Asp Leu Val Gin Thr He Gly Val 290 295 300

Ser Ala Ala Leu Gly Ala Ala Gly Val Val Leu Trp Gly Asp Leu Ser i 305 310 315 320

Leu Ser Ser Ser Glu Glu Glu Cys Trp His Leu His Asp Tyr Leu Val 325 330 335

Asp Thr Leu Gly Pro Tyr Gly lie Asn Val Thr Arg Ala Ala Met Ala 340 345 350

Cys Ser His Gin Arg Cys His Gly His Gly Arg Cys Ala Arg Arg Asp 355 360 365

Pro Gly Gin Met Glu Ala Phe Leu His Leu Trp Pro Asp Gly Ser Leu 370 375 380

Gly Asp Trp Lys Ser Phe Ser Cys His Cys Tyr Trp Gly Trp Ala Gly 385 390 395 400

Pro Thr Cys Gin Glu Pro Arg Leu Gly Pro Lys Glu Ala Val 405 410

<210> 29 <211 > 510 <212> PRT <213> Macaca fascicularis <220> <223> PH20 <400> 29

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Arg Ser Phe Val Lys 15 10 15

Ser Ser Gly Val Ser Gin lie Val Phe Thr Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro lie lie Pro Asn Val Pro 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60

Asn Glu Pro Leu Asp Met Ser Leu Phe Thr Leu Met Gly Ser Pro Arg 65 70 75 80 lie Asn Val Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Leu Thr Thr Gly Val Thr Val His Gly 100 105 110

Gly lie Pro Gin Lys Val Ser Leu Gin Asp His Leu Asp Lys Ser Lys 115 120 125

Gin Asp lie Leu Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140 lie Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160

Lys Asp Val Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn 165 170 175

Val Gin Leu Ser Leu Pro Gin Ala Thr Asp Lys Ala Lys Gin Glu Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Met Leu Glu Thr lie Lys Leu Gly Arg 195 200 205

Ser Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Arg Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asp 225 230 235 240

Val Glu lie Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255

Thr Ala Leu Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Val Val 260 265 270

Val Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val 275 280 285

Ser Lys lie Pro Asp Ala Lys Asn Pro Leu Pro Val Phe Val Tyr Ala 290 295 300

Arg Leu Val Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Arg Glu Glu 305 310 315 320

Leu Val Ser Thr Leu Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie 325 330 335

Val lie Trp Gly Ser Leu Ser lie Thr Arg Ser Met Lys Ser Cys Leu 340 345 350

Leu Leu Asp Thr Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin 370 375 380

Gly Val Cys lie Arg Lys Asp Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400

Asn Pro Asp Asn Phe Asp lie Arg Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415

Val His Gly Lys Pro Thr Val Glu Asp Leu Glu Glu Phe Ser Glu Lys 420 425 430

Phe Tyr Cys Ser Cys Tyr Thr Asn Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys 450 455 460 lie Asp Ala Ser Leu Lys Pro Pro Val Glu Thr Glu Gly Ser Pro Pro 465 470 475 480 lie Phe Tyr Asn Thr Ser Ser Ser Thr Val Ser Thr Thr Met Phe lie 485 490 495

Val Asn lie Leu Phe Leu lie lie Ser Ser Val Ala Ser Leu 500 505 510 <210> 30 <211> 529

<212> PRT <213> Cavia porcellus <220> <223> PH20 <400> 30

Met Gly Ala Phe Thr Phe Lys His Ser Phe Phe Gly Ser Phe Val Glu 15 10 15

Cys Ser Gly Val Leu Gin Thr Val Phe He Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Ala Asp Lys Arg Ala Pro Pro Leu He Pro Asn Val Pro Leu 35 40 45

Leu Trp Val Trp Asn Ala Pro Thr Glu Phe Cys He Gly Gly Thr Asn 50 55 60

Gin Pro Leu Asp Met Ser Phe Phe Ser He Val Gly Thr Pro Arg Lys 65 70 75 80

Asn He Thr Gly Gin Ser He Thr Leu Tyr Tyr Val Asp Arg Leu Gly 85 90 95

Tyr Tyr Pro Tyr He Asp Pro His Thr Gly Ala He Val His Gly Gly 100 105 HO

Leu Pro Gin Leu Met Asn Leu Gin Gin His Leu Arg Lys Ser Arg Gin 115 120 125

Asp lie Leu Phe Tyr Met Pro Thr Asp Ser Val Gly Leu Ala Val lie 130 135 140

Asp Trp Glu Glu Trp Arg Pro Thr Trp Thr Arg Asn Trp Arg Pro Lys 145 150 155 160

Asp lie Tyr Arg Asn Lys Ser lie Glu Leu Val Lys Ser Gin His Pro 165 170 175

Gin Tyr Asn His Ser Tyr Ala Val Ala Val Ala Lys Arg Asp Phe Glu 180 185 190

Arg Thr Gly Lys Ala Phe Met Leu Glu Thr Leu Lys Leu Gly Lys Ser 195 200 205

Leu Arg Pro Ser Ser Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr 210 215 220

Asn Thr His Phe Thr Lys Pro Asn Tyr Asp Gly His Cys Pro Pro lie 225 230 235 240

Glu Leu Gin Arg Asn Asn Asp Leu Gin Trp Leu Trp Asn Asp Ser Thr 245 250 255

Ala Leu Tyr Pro Ser Val Tyr Leu Thr Ser Arg Val Arg Ser Ser Gin 260 265 270

Asn Gly Ala Leu Tyr Val Arg Asn Arg Val His Glu Ser lie Arg Val 275 280 285

Ser Lys Leu Met Asp Asp Lys Asn Pro Leu Pro lie Tyr Val Tyr lie 290 295 300

Arg Leu Val Phe Thr Asp Gin Thr Thr Thr Phe Leu Glu Leu Asp Asp 305 310 315 320

Leu Val His Ser Val Gly Glu lie Val Pro Leu Gly Val Ser Gly lie 325 330 335 lie lie Trp Gly Ser Leu Ser Leu Thr Arg Ser Leu Val Ser Cys lie 340 345 350

Gly Leu Glu Asn Tyr Met Lys Gly Thr Leu Leu Pro Tyr Leu lie Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Gly Gin Val Leu Cys Lys Asn Gin 370 375 380

Gly lie Cys Thr Arg Lys Asp Trp Asn Thr Asn Thr Tyr Leu His Leu 385 390 395 400

Asn Ala Thr Asn Phe Asp lie Glu Leu Gin Gin Asn Gly Lys Phe Val 405 410 415

Val His Gly Lys Pro Ser Leu Glu Asp Leu Gin Glu Phe Ser Lys Asn 420 425 430

Phe His Cys Ser Cys Tyr Thr Asn Val Ala Cys Lys Asp Arg Leu Asp 435 440 445

Val His Asn Val Arg Ser Val Asn Val Cys Thr Ala Asn Asn lie Cys 450 455 460 lie Asp Ala Val Leu Asn Phe Pro Ser Leu Asp Asp Asp Asp Glu Pro 465 470 475 480

Pro lie Thr Asp Asp Thr Ser Gin Asn Gin Asp Ser lie Ser Asp lie 485 490 495

Thr Ser Ser Ala Pro Pro Ser Ser His lie Leu Pro Lys Asp Leu Ser 500 505 510

Trp Cys Leu Phe Leu Leu Ser lie Phe Ser Gin His Trp Lys Tyr Leu 515 520 525

Leu

<210> 31 <211> 512 <212> PRT <213> Rattus norvegicus <220> <223> PH20 <400 31

Met Gly Glu Leu Gin Phe Lys Trp Leu Phe Trp Arg Ser Phe Ala Glu 15 10 15

Ser Gly Gly Thr Phe Gin Thr Val Leu lie Phe Leu Phe lie Pro Tyr 20 25 30

Ser Leu Thr Val Asp Tyr Arg Ala Thr Pro Val Leu Ser Asp Thr Thr 35 40 45

Phe Val Trp Val Trp Asn Val Pro Thr Glu Ala Cys Val Glu Asn Val 50 55 60

Thr Glu Pro lie Asp Leu Ser Phe Phe Ser Leu lie Gly Ser Pro Arg 65 70 75 80

Lys Thr Ala He Gly Gin Pro Val Thr Leu Phe Tyr Val Asp Arg Leu 85 90 95

Gly Asn Tyr Pro His lie Asp Ala Gin Gin Thr Glu His His Gly Gly 100 105 110

He Pro Gin Lys Gly Asp Leu Thr Thr His Leu Val Lys Ala Lys Glu 115 120 125

Asp Val Glu Arg Tyr He Pro Thr Asp Lys Leu Gly Leu Ala He He 130 135 140

Asp Trp Glu Glu Trp Arg Pro Thr Trp Met Arg Asn Trp Thr Pro Lys 145 150 155 160

Asp He Tyr Arg Asn Lys Ser He Glu Leu Val Gin Ala Ala Asp Pro 165 170 175

Ala He Asn He Thr Glu Ala Thr Val Arg Ala Lys Ala Gin Phe Glu 180 185 190

Gly Ala Ala Lys Glu Phe Met Glu Gly Thr Leu Lys Leu Gly Lys His 195 200 205

He Arg Pro Lys His Leu Trp Gly Phe Tyr Leu Phe Pro Asp Cys Tyr 210 215 220

Asn Asn Lys Phe Gin Val Asp Asn Tyr Asp Gly Gin Cys Pro Asp Val 225 230 235 240

Glu Lys Lys Arg Asn Asp Asp Leu Asp Trp Leu Trp Lys Glu Ser Thr 245 250 255

Gly Leu Tyr Pro Ser Val Tyr Leu Lys Lys Asp Leu Lys Ser Ser Arg 260 265 270

Lys Ala Thr Leu Tyr Val Arg Tyr Arg Val Leu Glu Ser He Arg Val 275 280 285

Ser Lys Val Ser Asp Glu Ser Asn Pro Val Pro He Phe Val Tyr He 290 295 300

Arg Leu Val Phe Thr Asp His Val Ser Glu Tyr Leu Leu Glu Asp Asp 305 310 315 320

Leu Val Asn Thr He Gly Glu He Val Ala Gin Gly Thr Ser Gly He 325 330 335

He He Trp Asp Ala Met Ser Leu Ala Gin Arg Ser Ala Gly Cys Pro 340 345 350

He Leu Arg Gin Tyr Met Lys Thr Thr Leu Asn Pro Tyr He Val Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Thr Leu Cys Lys Glu Lys 370 375 380

Gly Met Cys Ser Arg Lys Thr Glu Ser Ser Asp Ala Tyr Leu His Leu 385 390 395 400

Asp Pro Ser Ser Phe Ser He Asn Val Thr Glu Ala Gly Lys Tyr Glu 405 410 415

Val Leu Gly Lys Pro Glu Val Lys Asp Leu Glu Tyr Phe Ser Glu His 420 425 430

Phe Lys Cys Ser Cys Phe Ser Lys Met Thr Cys Glu Glu Thr Ser Asp 435 440 445

Met Arg Ser He Gin Asp Val Asn Val Cys Met Gly Asp Asn Val Cys 450 455 460

He Lys Ala Thr Leu Gly Pro Asn Ser Ala Phe His Leu Leu Pro Gly 465 470 475 480

Lys Gly Leu Leu Leu Met Thr Thr Leu Ala His He Leu His His Leu 485 490 495

Pro His Asp lie Phe Val Phe Pro Trp Lys Met Leu Val Ser Thr Pro 500 505 510 <210> 32 <211> 512 <212> PRT <213> Mus musculus <220> <223> PH20 <400> 32

I

I

Met Gly Glu Leu Arg Phe Lys His Leu Phe Trp Gly Ser Phe Val Glu 15 10 15

Ser Gly Gly Thr Phe Gin Thr Val Leu lie Phe Leu Leu lie Pro Cys 20 25 30

Ser Leu Thr Val Asp Tyr Arg Ala Ala Pro lie Leu Ser Asn Thr Thr 35 40 45

Phe Leu Trp lie Trp Asn Val Pro Thr Glu Arg Cys Val Gly Asn Val 50 55 60

Asn Asp Pro lie Asp Leu Ser Phe Phe Ser Leu lie Gly Ser Pro Arg 65 70 75 80

Lys Thr Ala Thr Gly Gin Pro Val Thr Leu Phe Tyr Val Asp Arg Leu 85 90 95

Gly Leu Tyr Pro His lie Asp Ala Asn Gin Ala Glu His Tyr Gly Gly 100 105 110 lie Pro Gin Arg Gly Asp Tyr Gin Ala His Leu Arg Lys Ala Lys Thr 115 120 125

Asp lie Glu His Tyr lie Pro Asp Asp Lys Leu Gly Leu Ala lie lie 130 135 140

Asp Trp Glu Glu Trp Arg Pro Thr Trp Leu Arg Asn Trp Lys Pro Lys 145 150 155 160

Asp Asn Tyr Arg Asn Lys Ser lie Glu Leu Val Gin Ser Thr Asn Pro 165 170 175

Gly Leu Ser lie Thr Glu Ala Thr Gin Lys Ala lie Gin Gin Phe Glu 180 185 190

Glu Ala Gly Arg Lys Phe Met Glu Gly Thr Leu His Leu Gly Lys Phe 195 200 205

Leu Arg Pro Asn Gin Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr 210 215 220

Asn Asn Lys Phe Gin Asp Pro Lys Tyr Asp Gly Gin Cys Pro Ala Val 225 230 235 240

Glu Lys Lys Arg Asn Asp Asn Leu Lys Trp Leu Trp Lys Ala Ser Thr 245 250 255

Gly Leu Tyr Pro Ser Val Tyr Leu Lys Lys Asp Leu Lys Ser Asn Arg 260 265 270

Gin Ala Thr Leu Tyr Val Arg Tyr Arg Val Val Glu Ala lie Arg Val 275 280 285

Ser Lys Val Gly Asn Ala Ser Asp Pro Val Pro lie Phe Val Tyr lie 290 295 300

Arg Leu Val Phe Thr Asp Arg Thr Ser Glu Tyr Leu Leu Glu Asp Asp 305 310 315 320

Leu Val Asn Thr lie Gly Glu lie Val Ala Leu Gly Thr Ser Gly lie 325 330 335 lie lie Trp Asp Ala Met Ser Leu Ala Gin Arg Ala Ala Gly Cys Pro 340 345 350 lie Leu His Lys Tyr Met Gin Thr Thr Leu Asn Pro Tyr lie Val Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Thr Leu Cys Asn Glu Lys 370 375 380

Gly Met Cys Ser Arg Arg Lys Glu Ser Ser Asp Val Tyr Leu His Leu 385 390 395 400

Asn Pro Ser His Phe Asp lie Met Leu Thr Glu Thr Gly Lys Tyr Glu 405 410 415

Val Leu Gly Asn Pro Arg Val Gly Asp Leu Glu Tyr Phe Ser Glu His 420 425 430

Phe Lys Cys Ser Cys Phe Ser Arg Met Thr Cys Lys Glu Thr Ser Asp 435 440 445

Val Lys Asn Val Gin Asp Val Asn Val Cys Val Gly Asp Asn Val Cys 450 455 460 lie Lys Ala Lys Val Glu Pro Asn Pro Ala Phe Tyr Leu Leu Pro Gly 465 470 475 480

Lys Ser Leu Leu Phe Met Thr Thr Leu Gly His Val Leu Tyr His Leu 485 490 495

Pro Gin Asp lie Phe Val Phe Pro Arg Lys Thr Leu Val Ser Thr Pro 500 505 510

<210> 33 <211> 807 <212> PRT <213> Staphylococcus aureus <220> <223> hyaluronidase <400> 33

Met Thr Tyr Arg lie Lys Lys Trp Gin Lys Leu Ser Thr lie Thr Leu 15 10 15

Leu Met Ala Gly Val lie Thr Leu Asn Gly Gly Glu Phe Arg Ser Val 20 25 30

Asp Lys His Gin lie Ala Val Ala Asp Thr Asn Val Gin Thr Pro Asp 35 40 45

Tyr Glu Lys Leu Arg Asn Thr Trp Leu Asp Val Asn Tyr Gly Tyr Asp 50 55 60

Lys Tyr Asp Glu Asn Asn Pro Asp Met Lys Lys Lys Phe Asp Ala Thr 65 70 75 80

Glu Lys Glu Ala Thr Asn Leu Leu Lys Glu Met Lys Thr Glu Ser Gly 85 90 95

Arg Lys Tyr Leu Trp Ser Gly Ala Glu Thr Leu Glu Thr Asn Ser Ser 100 105 110

His Met Thr Arg Thr Tyr Arg Asn lie Glu Lys lie Ala Glu Ala Met 115 120 125

Arg Asn Pro Lys Thr Thr Leu Asn Thr Asp Glu Asn Lys Lys Lys Val 130 135 140

Lys Asp Ala Leu Glu Trp Leu His Lys Asn Ala Tyr Gly Lys Glu Pro 145 150 155 160

Asp Lys Lys Val Lys Glu Leu Ser Glu Asn Phe Thr Lys Thr Thr Gly 165 170 175

Lys Asn Thr Asn Leu Asn Trp Trp Asp Tyr Glu lie Gly Thr Pro Lys 180 185 190

Ser Leu Thr Asn Thr Leu lie Leu Leu Asn Asp Gin Phe Ser Asn Glu 195 200 205

Glu Lys Lys Lys Phe Thr Ala Pro He Lys Thr Phe Ala Pro Asp Ser 210 215 220

Asp Lys lie Leu Ser Ser Val Gly Lys Ala Glu Leu Ala Lys Gly Gly 225 230 235 240

Asn Leu Val Asp He Ser Lys Val Lys Leu Leu Glu Cys He He Glu 245 250 255

Glu Asp Lys Asp Met Met Lys Lys Ser He Asp Ser Phe Asn Lys Val 260 265 270

Phe Thr Tyr Val Gin Asp Ser Ala Thr Gly Lys Glu Arg Asn Gly Phe 275 280 285

Tyr Lys Asp Gly Ser Tyr He Asp His Gin Asp Val Pro Tyr Thr Gly 290 295 300

Ala Tyr Gly Val Val Leu Leu Glu Gly lie Ser Gin Met Met Pro Met 305 310 315 320 lie Lys Glu Thr Pro Phe Asn Asp Lys Thr Gin Asn Asp Thr Thr Leu 325 330 335

Lys Ser Trp lie Asp Asp Gly Phe Met Pro Leu lie Tyr Lys Gly Glu 340 345 350

Met Met Asp Leu Ser Arg Gly Arg Ala lie Ser Arg Glu Asn Glu Thr 355 360 365

Ser His Ser Ala Ser Ala Thr Val Met Lys Ser Leu Leu Arg Leu Ser 370 375 380

Asp Ala Met Asp Asp Ser Thr Lys Ala Lys Tyr Lys Lys lie Val Lys 385 390 395 400

Ser Ser Val Glu Ser Asp Ser Ser Tyr Lys Gin Asn Asp Tyr Leu Asn 405 410 415

Ser Tyr Ser Asp lie Asp Lys Met Lys Ser Leu Met Thr Asp Asn Ser 420 425 430 lie Ser Lys Asn Gly Leu Thr Gin Gin Leu Lys lie Tyr Asn Asp Met 435 440 445

Asp Arg Val Thr Tyr His Asn Lys Asp Leu Asp Phe Ala Phe Gly Leu 450 455 460

Ser Met Thr Ser Lys Asn Val Ala Arg Tyr Glu Ser lie Asn Gly Glu 465 470 475 480

Asn Leu Lys Gly Trp His Thr Gly Ala Gly Met Ser Tyr Leu Tyr Asn 485 490 495

Ser Asp Val Lys His Tyr His Asp Asn Phe Trp Val Thr Ala Asp Met 500 505 510

Lys Arg Leu Ser Gly Thr Thr Thr Leu Asp Asn Glu lie Leu Lys Asp 515 520 525

Thr Asp Asp Lys Lys Ser Ser Lys Thr Phe Val Gly Gly Thr Lys Val 530 535 540

Asp Asp Gin His Ala Ser lie Gly Met Asp Phe Glu Asn Gin Asp Lys 545 550 555 560

Thr Leu Thr Ala Lys Lys Ser Tyr Phe lie Leu Asn Asp Lys lie Val 565 570 575

Phe Leu Gly Thr Gly lie Lys Ser Thr Asp Ser Ser Lys Asn Pro Val 580 585 590

Thr Thr lie Glu Asn Arg Lys Ala Asn Gly Tyr Thr Leu Tyr Thr Asp 595 600 605

Asp Lys Gin Thr Thr Asn Ser Asp Asn Gin Glu Asn Asn Ser Val Phe 610 615 620

Leu Glu Ser Thr Asp Thr Lys Lys Asn lie Gly Tyr His Phe Leu Asn 625 630 635 640

Lys Pro Lys lie Thr Val Lys Lys Glu Ser His Thr Gly Lys Trp Lys 645 650 655

Glu lie Asn Lys Ser Gin Lys Asp Thr Gin Lys Thr Asp Glu Tyr Tyr 660 665 670

Glu Val Thr Gin Lys His Ser Asn Ser Asp Asn Lys Tyr Gly Tyr Val 675 680 685 I.eu Tyr Pro Gly Leu Ser Lys Asp Val Phe Lys Thr Lys Lys Asp Glu 690 695 700

Val Thr Val Val Lys Gin Glu Asp Asp Phe His Val Val Lys Asp Asn 705 710 715 720

Glu Ser Val Trp Ala Gly Val Asn Tyr Ser Asn Ser Thr Gin Thr Phe 725 730 735

Asp lie Asn Asn Thr Lys Val Glu Val Lys Ala Lys Gly Met Phe lie 740 745 750 lieu Lys Lys Lys Asp Asp Asn Thr Tyr Glu Cys Ser Phe Tyr Asn Pro 755 760 765

Glu Ser Thr Asn Ser Ala Ser Asp lie Glu Ser Lys lie Ser Met Thr 770 775 780

Gly Tyr Ser lie Thr Asn Lys Asn Thr Ser Thr Ser Asn Glu Ser Gly 785 790 795 800

Val His Phe Glu Leu Thr Lys 805

<210> 34 <211> 371 <212> PRT <213> Streptococcus pyogenes bacteriophage H4489A <220> <223> hyaluronidase <400> 34

Met Thr Glu Asn lie Pro Leu Arg Val Gin Phe Lys Arg Met Ser Ala 15 10 15

Asp Glu Trp Ala Arg Ser Asp Val lie Leu Leu Glu Gly Glu lie Gly 20 25 30

Phe Glu Thr Asp Thr Gly Phe Ala Lys Phe Gly Asp Gly Gin Asn Thr 35 40 45

Phe Ser Lys Leu Lys Tyr Leu Thr Gly Pro Lys Gly Pro Lys Gly Asp 50 55 60

Thr Gly Leu Gin Gly Lys Thr Gly Gly Thr Gly Pro Arg Gly Pro Ala 65 70 75 80

Gly Lys Pro Gly Thr Thr Asp Tyr Asp Gin Leu Gin Asn Lys Pro Asp 85 90 95

Leu Gly Ala Phe Ala Gin Lys Glu Glu Thr Asn Ser Lys lie Thr Lys 100 105 110

Leu Glu Ser Ser Lys Ala Asp Lys Ser Ala Val Tyr Ser Lys Ala Glu 115 120 125

Ser Lys lie Glu Leu Asp Lys Lys Leu Ser Leu Thr Gly Gly lie Val 130 135 140

Thr Gly Gin Leu Gin Phe Lys Pro Asn Lys Ser Gly lie Lys Pro Ser 145 150 155 160

Ser Ser Val Gly Gly Ala lie Asn lie Asp Met Ser Lys Ser Glu Gly 165 170 175

Ala Ala Met Val Met Tyr Thr Asn Lys Asp Thr Thr Asp Gly Pro Leu 180 185 190

Met lie Leu Arg Ser Asp Lys Asp Thr Phe Asp Gin Ser Ala Gin Phe 195 200 205

Val Asp Tyr Ser Gly Lys Thr Asn Ala Val Asn lie Val Met Arg Gin 210 215 220

Pro Ser Ala Pro Asn Phe Ser Ser Ala Leu Asn lie Thr Ser Ala Asn 225 230 235 240

Glu Gly Gly Ser Ala Met Gin lie Arg Gly Val Glu Lys Ala Leu Gly 245 250 255

Thr Leu Lys lie Thr His Glu Asn Pro Asn Val Glu Ala Lys Tyr Asp 260 265 270

Glu Asn Ala Ala Ala Leu Ser lie Asp lie Val Lys Lys Gin Lys Gly 275 280 285

Gly Lys Gly Thr Ala Ala Gin Gly lie Tyr lie Asn Ser Thr Ser Gly 290 295 300

Thr Ala Gly Lys Met Leu Arg lie Arg Asn Lys Asn Glu Asp Lys Phe 305 310 315 320

Tyr Val Gly Pro Asp Gly Gly Phe His Ser Gly Ala Asn Ser Thr Val 325 330 335

Ala Gly Asn Leu Thr Val Lys Asp Pro Thr Ser Gly Lys His Ala Ala 340 345 350

Thr Lys Asp Tyr Val Asp Glu Lys lie Ala Glu Leu Lys Lys Leu lie 355 360 365

Leu Lys Lys 370

<210> 35 <211> 1628 <212> PRT <213> Clostridium perfringens <220> <223> hyaluronidase <400> 35 i

I ) }

Met Asn Lys Asn lie Arg Lys lie lie Thr Ser Thr Val Leu Ala Ala 15 10 15

Met Thr lie Ser Val Leu Pro Ser Asn Leu Val Val Phe Ala Thr Asp 20 25 30

Gly lie Thr Glu Asn Phe Tyr Glu lie Tyr Pro Lys Pro Gin Glu lie 35 40 45

Ser Tyr Ser Gly Gly Glu Phe Gin lie Ser Asp Glu lie Asn lie Val 50 55 60

Tyr Asp Asp Gly lie Asp Thr Tyr Thr Lys Lys Arg Val Asp Glu Val 65 70 75 80

Leu Glu Ala Ser Asn Leu Glu Ala Thr Val Ser Asn Glu lie Val Pro 85 90 95

Gly Lys Thr Asn Phe Leu Val Gly lie Asn Glu Ser Gly Gly Val Val 100 105 110

Asp Asn Tyr Phe Asn Lys Asn lie Pro His Asp Glu Ser Phe Phe Asp 115 120 125

Glu Lys Met Asp Ala Asn lie Val Ser Val Lys Asp Gly Val lie Gly 130 135 140

Val lie Gly Glu Asp Thr Asp Ser Ala Phe Tyr Gly Val Thr Thr Leu 145 150 155 160

Lys His Val Phe Asn Gin Leu Glu Glu Gly Asn Lys lie Gin Ser Phe 165 170 175

Arg Ala Asp Asp Tyr Ala Glu Val Ala His Arg Gly Phe lie Glu Gly 180 185 190

Tyr Tyr Gly Asn Pro Trp Ser Asn Glu Asp Arg Ala Glu Leu Met Lys 195 200 205

Phe Gly Gly Asp Tyr Lys Leu Asn Gin Tyr Val Phe Ala Pro Lys Asp 210 215 220

Asp Pro Tyr His Asn Ser Lys Trp Arg Asp Leu Tyr Pro Glu Glu Lys 225 230 235 240

Leu Ser Glu lie Lys Lys Leu Ala Gin Val Gly Asn Glu Thr Lys Asn 245 250 255

Arg Tyr Val Tyr Ala Leu His Pro Phe Met Asn Asn Pro Val Arg Phe 260 265 270

Asp Thr Glu Glu Asn Tyr Gin Asn Asp Leu Gly Val lie Lys Ala Lys 275 280 285

Phe Thr Gin Leu Leu Glu Asn Asp Val Arg Gin Phe Ala lie Leu Ala 290 295 300

Asp Asp Ala Ser Ala Pro Ala Gin Gly Ala Ser Met Tyr Val Lys Leu 305 310 315 320

Leu Thr Asp Leu Thr Arg Trp Leu Glu Glu Gin Gin Ser Thr Tyr Pro 325 330 335

Asp Leu Lys Thr Asp Leu Met Phe Cys Pro Ser Asp Tyr Tyr Gly Asn 340 345 350

Gly Ser Ser Ala Gin Leu Lys Glu Leu Asn Lys Ala Glu Asp Asn Val 355 360 365

Ser lie Val Met Thr Gly Gly Arg lie Trp Gly Glu Val Asp Glu Asn 370 375 380

Phe Ala Asn Asn Phe Met Asn Asn lie Ser Thr Glu Gly His Pro Gly 385 390 395 400

Arg Ala Pro Phe Phe Trp lie Asn Trp Pro Cys Ser Asp Asn Ser Lys 405 410 415

Gin His Leu lie Met Gly Gly Asn Asp Thr Phe Leu His Pro Gly Val 420 425 430

Asp Pro Ser Lys lie Asp Gly lie Val Leu Asn Pro Met Gin Gin Ala 435 440 445

Glu Ala Asn Lys Ser Ala Leu Phe Ala lie Ala Asp Tyr Ala Trp Asn 450 455 460 lie Trp Asp Asn Lys Glu Glu Ala Asp Glu Asn Trp Asn Asp Ser Phe 465 470 475 480

Lys Tyr Met Asp His Gly Thr Ala Glu Glu Thr Asn Ser Ser Leu Ala 485 490 495

Leu Arg Glu lie Ser Lys His Met lie Asn Gin Asn Met Asp Gly Arg 500 505 510

Val Arg Pro Leu Gin Glu Ser Val Glu Leu Ala Pro Lys Leu Glu Ala 515 520 525

Phe Lys Gin Lys Tyr Asp Ser Gly Ala Ser lie Lys Glu Asp Ala Leu 530 535 540

Glu Leu lie Ala Glu Phe Thr Asn Leu Gin Lys Ala Ala Asp Tyr Tyr 545 550 555 560

Lys Asn Asn Pro Gly Asn Glu Arg Thr Arg Asp Gin lie lie Tyr Trp 565 570 575

Leu Asn Cys Trp Glu Asp Thr Met Asp Ala Ala lie Gly Tyr Leu Lys 580 585 590

Ser Ala lie Ala lie Glu Glu Gly Asp Asp Glu Ala Ala Trp Ala Asn 595 600 605

Tyr Ser Glu Ala Gin Gly Ala Phe Glu Lys Ser Lys Thr Tyr Gly Phe 610 615 620

His Tyr Val Asp His Thr Glu Tyr Ala Glu Val Gly Val Gin His lie 625 630 635 640

Val Pro Phe lie Lys Ser Met Gly Gin Asn Leu Ser Val Val lie Gly 645 650 655

Ser lie Val Asp Pro Asn Arg lie lie Ala Thr Tyr lie Ser Asn Arg 660 665 670

Gin Asp Ala Pro Thr Gly Asn Pro Asp Asn lie Phe Asp Asn Asn Ala 675 680 685

Ser Thr Glu Leu Val Tyr Lys Asn Pro Asn Arg lie Asp Val Gly Thr 690 695 700

Tyr Val Gly Val Lys Tyr Ser Asn Pro lie Thr Leu Asn Asn Val Glu 705 710 715 720

Phe Leu Met Gly Ala Asn Ser Asn Pro Asn Asp Thr Met Gin Lys Ala 725 730 735

Lys lie Gin Tyr Thr Val Asp Gly Arg Glu Trp lie Asp Leu Glu Glu 740 745 750

Gly Val Glu Tyr Thr Met Pro Gly Ala lie Lys Val Glu Asn Leu Asp 755 760 765

Leu Lys Val Arg Gly Val Arg Leu lie Ala Thr Glu Ala Arg Glu Asn 770 775 780

Thr Trp Leu Gly Val Arg Asp lie Asn Val Asn Lys Lys Glu Asp Ser 785 790 795 800

Asn Ser Gly Val Glu Phe Asn Pro Ser Leu lie Arg Ser Glu Ser Trp 805 810 815

Gin Val Tyr Glu Gly Asn Glu Ala Asn Leu Leu Asp Gly Asp Asp Asn 820 825 830

Thr Gly Val Trp Tyr Lys Thr Leu Asn Gly Asp Thr Ser Leu Ala Gly 835 840 845

Glu Phe lie Gly Leu Asp Leu Gly Lys Glu lie Lys Leu Asp Gly lie 850 855 860

Arg Phe Val lie Gly Lys Asn Gly Gly Gly Ser Ser Asp Lys Trp Asn 865 870 875 880

Lys Phe Lys Leu Glu Tyr Ser Leu Asp Asn Glu Ser Trp Thr Thr lie 885 890 895

Lys Glu Tyr Asp Lys Thr Gly Ala Pro Ala Gly Lys Asp Val lie Glu 900 905 910

Glu Ser Phe Glu Thr Pro lie Ser Ala Lys Tyr lie Arg Leu Thr Asn 915 920 925

Met Glu Asn lie Asn Lys Trp Leu Thr Phe Ser Glu Phe Ala lie lie 930 935 940

Ser Asp Glu Leu Glu Asn Ala Gly Asn Lys Glu Asn Val Tyr Thr Asn 945 950 955 960

Thr Glu Leu Asp Leu Leu Ser Leu Ala Lys Glu Asp Val Thr Lys Leu 965 970 975 lie Pro Thr Asp Asp lie Ser Leu Asn His Gly Glu Tyr lie Gly Val 980 985 990

Lys Leu Asn Arg lie Lys Asp Leu Ser Asn lie Asn Leu Glu lie Ser 995 1000 1005

Asn Asp Thr Gly Leu Lys Leu Gin Ser Ser Met Asn Gly Val Glu Trp 1010 1015 1020

Thr Glu lie Thr Asp Lys Asn Thr Leu Glu Asp Gly Arg Tyr Val Arg 1025 1030 1035 1040

Leu He Asn Thr Ser Asn Glu Ala Val Asn Phe Asn Leu Thr Lys Phe 1045 1050 1055

Glu Val Asn Ser Asn Glu Val Tyr Glu Pro Ser Leu Val Asp Ala Tyr 1060 1065 1070

Val Gly Asp Asp Gly Ala Lys Lys Ala Val Asp Gly Asp Leu Lys Thr 1075 1080 1085

Arg Val Lys Phe Leu Gly Ala Pro Ser Thr Gly Asp Thr lie Val Tyr 1090 1095 1100

Asp Leu Gly Gin Glu lie Leu Val Asp Asn Leu Lys Tyr Val Val Leu 1105 1110 1115 1120

Asp Thr Glu Val Asp His Val Arg Asp Gly Lys He Gin Leu Ser Leu 1125 1130 1135

Asp Gly Glu Thr Trp Thr Asp Ala He Thr He Gly Asp Gly Val Glu 1140 1145 1150

Asn Gly Val Asp Asp Met Phe Ser Thr Pro Leu Lys Asn Gly Tyr Lys 1155 1160 1165

His Gly Asn Gin Ser Gly Gly He Val Pro He Asp Ser Ala Tyr Val 1170 1175 1180

Glu Gly Asp Asn Leu Asn Gin Lys Ala Arg Tyr Val Arg He Leu Phe 1185 1190 1195 1200

Thr Ala Pro Tyr Arg His Arg Trp Thr Val He Asn Glu Leu Met He 1205 1210 1215

Asn Asn Gly Glu Tyr He Ser Thr Val Asn Asp Pro Thr Tyr He Ser 1220 1225 1230

Asn Pro He Glu Glu Arg Gly Phe Ala Pro Ser Asn Leu Arg Asp Gly 1235 1240 1245

Asn Leu Thr Thr Ser Tyr Lys Pro Asn Thr Asn Asn Gly Glu He Ser 1250 1255 1260

Glu Gly Ser He Thr Tyr Arg Leu Ser Glu Lys Thr Asp Val Arg Lys 1265 1270 1275 1280

Val Thr He Val Gin Ser Gly Ser Ser He Ser Asn Ala Lys Val Met 1285 1290 1295

Ala Arg Val Gly Asp Gly Ser Glu Asn Val Thr Asp Gin Trp Val Gin 1300 1305 1310

Leu Gly Thr Leu Ser Asn Ser Leu Asn Glu Phe He Asn Arg Asp Tyr 1315 1320 1325

Asn Asn He Tyr Glu He Lys He Glu Trp Thr Asp Val Ala Pro Asn 1330 1335 1340

He Tyr Glu He He Thr Leu Asn Gin Glu Phe Glu Phe Pro Val Asn 1345 1350 1355 1360

Asp Ser Leu Lys Ala Lys Tyr Asp Glu Leu He Asn Leu Ser Gly Asp 1365 1370 1375

Glu Tyr Thr Leu Ser Ser Phe Glu Thr Leu Lys Glu Ala Leu Asn Glu 1380 1385 1390

Ala Lys Ser He Leu Asp Asp Ser Asn Ser Ser Gin Lys Lys He Asp 1395 1400 1405

Lys Ala Leu Glu Lys Leu Asn Lys Ala Glu Glu Arg Leu Asp Leu Arg 1410 1415 1420

Ala Thr Asp Phe Glu Asp Phe Asn Lys Val Leu Thr Leu Gly Asn Ser 1425 1430 1435 1440

Leu Val Glu Glu Glu Tyr Thr Ala Glu Ser Trp Ala Leu Phe Ser Glu 1445 1450 1455

Val Leu Glu Ala Ala Asn Glu Ala Asn Lys Asn Lys Ala Asp Tyr Thr 1460 1465 1470

Gin Asp Gin lie Asn Gin lie Val lie Asp Leu Asp Ala Ser lie Lys 1475 1480 1485

Ala Leu Val Lys Glu Thr Pro Glu Val Asp Lys Thr Asn Leu Gly Glu 1490 1495 1500

Leu lie Asn Gin Gly Lys Ser Leu Leu Asp Glu Ser Val Glu Gly Phe 1505 1510 1515 1520

Asn Val Gly Glu Tyr His Lys Gly Ala Lys Asp Gly Leu Thr Val Glu 1525 1530 1535 lie Asn Lys Ala Glu Glu Val Phe Asn Lys Glu Asp Ala Thr Glu Glu 1540 1545 1550

Glu lie Asn Leu Ala Lys Glu Ser Leu Glu Gly Ala lie Ala Arg Phe 1555 1560 1565

Asn Ser Leu Leu lie Glu Glu Ser Thr Gly Asp Phe Asn Gly Asn Gly 1570 1575 1580

Lys lie Asp lie Gly Asp Leu Ala Met Val Ser Lys Asn lie Gly Ser 1585 1590 1595 1600

Thr Thr Asn Thr Ser Leu Asp Leu Asn Lys Asp Gly Ser lie Asp Glu 1605 1610 1615

Tyr Glu lie Ser Phe lie Asn His Arg lie Leu Asn 1620 1625 <210> 36 <211> 435 <212> PRT <213> Homo sapiens <220 <223> Hyaluronidase-1 [Precursor] <400> 36

Met Ala Ala His Leu Leu Pro lie Cys Ala Leu Phe Leu Thr Leu Leu 15 10 15

Asp Met Ala Gin Gly Phe Arg Gly Pro Leu Leu Pro Asn Arg Pro Phe 20 25 30

Thr Thr Val Trp Asn Ala Asn Thr Gin Trp Cys Leu Glu Arg His Gly 35 40 45

Val Asp Val Asp Val Ser Val Phe Asp Val Val Ala Asn Pro Gly Gin 50 55 60

Thr Phe Arg Gly Pro Asp Met Thr lie Phe Tyr Ser Ser Gin Leu Gly 65 70 75 80

Thr Tyr Pro Tyr Tyr Thr Pro Thr Gly Glu Pro Val Phe Gly Gly Leu 85 90 95

Pro Gin Asn Ala Ser Leu lie Ala His Leu Ala Arg Thr Phe Gin Asp 100 105 110 lie Leu Ala Ala lie Pro Ala Pro Asp Phe Ser Gly Leu Ala Val lie 115 120 125

Asp Trp Glu Ala Trp Arg Pro Arg Trp Ala Phe Asn Trp Asp Thr Lys 130 135 140

Asp lie Tyr Arg Gin Arg Ser Arg Ala Leu Val Gin Ala Gin His Pro 145 150 155 160

Asp Trp Pro Ala Pro Gin Val Glu Ala Val Ala Gin Asp Gin Phe Gin 165 170 175

Gly Ala Ala Arg Ala Trp Met Ala Gly Thr Leu Gin Leu Gly Arg Ala 180 185 190

Leu Arg Pro Arg Gly Leu Trp Gly Phe Tyr Gly Phe Pro Asp Cys Tyr 195 200 205

Asn Tyr Asp Phe Leu Ser Pro Asn Tyr Thr Gly Gin Cys Pro Ser Gly 210 215 220 lie Arg Ala Gin Asn Asp Gin Leu Gly Trp Leu Trp Gly Gin Ser Arg 225 230 235 240

Ala Leu Tyr Pro Ser lie Tyr Met Pro Ala Val Leu Glu Gly Thr Gly 245 250 255

Lys Ser Gin Met Tyr Val Gin His Arg Val Ala Glu Ala Phe Arg Val 260 265 270

Ala Val Ala Ala Gly Asp Pro Asn Leu Pro Val Leu Pro Tyr Val Gin 275 280 285 lie Phe Tyr Asp Thr Thr Asn His Phe Leu Pro Leu Asp Glu Leu Glu 290 295 300

His Ser Leu Gly Glu Ser Ala Ala Gin Gly Ala Ala Gly Val Val Leu 305 310 315 320

Trp Val Ser Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Gin Ala lie 325 330 335

Lys Glu Tyr Met Asp Thr Thr Leu Gly Pro Phe lie Leu Asn Val Thr 340 345 350

Ser Gly Ala Leu Leu Cys Ser Gin Ala Leu Cys Ser Gly His Gly Arg 355 360 365

Cys Val Arg Arg Thr Ser His Pro Lys Ala Leu Leu Leu Leu Asn Pro 370 375 380

Ala Ser Phe Ser lie Gin Leu Thr Pro Gly Gly Gly Pro Leu Ser Leu 385 390 395 400

Arg Gly Ala Leu Ser Leu Glu Asp Gin Ala Gin Met Ala Val Glu Phe 405 410 415

Lys Cys Arg Cys Tyr Pro Gly Trp Gin Ala Pro Trp Cys Glu Arg Lys 420 425 430

Ser Met Trp 435 <210> 37 <211> 473

<212> PRT <213> Homo sapiens <220 <223> Hyaluronidase-2 [Precursor] <400> 37

Met Arg Ala Gly Pro Gly Pro Thr Val Thr Leu Ala Leu Val Leu Ala 15 10 15

Val Ala Trp Ala Met Glu Leu Lys Pro Thr Ala Pro Pro lie Phe Thr 20 25 30

Gly Arg Pro Phe Val Val Ala Trp Asp Val Pro Thr Gin Asp Cys Gly 35 40 45

Pro Arg Leu Lys Val Pro Leu Asp Leu Asn Ala Phe Asp Val Gin Ala 50 55 60

Ser Pro Asn Glu Gly Phe Val Asn Gin Asn lie Thr lie Phe Tyr Arg 65 70 75 80

Asp Arg Leu Gly Leu Tyr Pro Arg Phe Asp Ser Ala Gly Arg Ser Val 85 90 95

His Gly Gly Val Pro Gin Asn Val Ser Leu Trp Ala His Arg Lys Met 100 105 110

Leu Gin Lys Arg Val Glu His Tyr lie Arg Thr Gin Glu Ser Ala Gly 115 120 125

Leu Ala Val lie Asp Trp Glu Asp Trp Arg Pro Val Trp Val Arg Asn 130 135 140

Trp Gin Asp Lys Asp Val Tyr Arg Arg Leu Ser Arg Gin Leu Val Ala 145 150 155 160

Ser Arg His Pro Asp Trp Pro Pro Asp Arg lie Val Lys Gin Ala Gin 165 170 175

Tyr Glu Phe Glu Phe Ala Ala Gin Gin Phe Met Leu Glu Thr Leu Arg 180 185 190

Tyr Val Lys Ala Val Arg Pro Arg His Leu Trp Gly Phe Tyr Leu Phe 195 200 205

Pro Asp Cys Tyr Asn His Asp Tyr Val Gin Asn Trp Glu Ser Tyr Thr 210 215 220

Gly Arg Cys Pro Asp Val Glu Val Ala Arg Asn Asp Gin Leu Ala Trp 225 230 235 240

Leu Trp Ala Glu Ser Thr Ala Leu Phe Pro Ser Val Tyr Leu Asp Glu 245 250 255

Thr Leu Ala Ser Ser Arg His Gly Arg Asn Phe Val Ser Phe Arg Val 260 265 270

Gin Glu Ala Leu Arg Val Ala Arg Thr His His Ala Asn His Ala Leu 275 280 285

Pro Val Tyr Val Phe Thr Arg Pro Thr Tyr Ser Arg Arg Leu Thr Gly 290 295 300

Leu Ser Glu Met Asp Leu lie Ser Thr lie Gly Glu Ser Ala Ala Leu 305 310 315 320

Gly Ala Ala Gly Val lie Leu Trp Gly Asp Ala Gly Tyr Thr Thr Ser 325 330 335

Thr Glu Thr Cys Gin Tyr Leu Lys Asp Tyr Leu Thr Arg Leu Leu Val 340 345 350

Pro Tyr Val Val Asn Val Ser Trp Ala Thr Gin Tyr Cys Ser Arg Ala 355 360 365

Gin Cys His Gly His Gly Arg Cys Val Arg Arg Asn Pro Ser Ala Ser 370 375 380

Thr Phe Leu His Leu Ser Thr Asn Ser Phe Arg Leu Val Pro Gly His 385 390 395 400

Ala Pro Gly Glu Pro Gin Leu Arg Pro Val Gly Glu Leu Ser Trp Ala 405 410 415

Asp lie Asp His Leu Gin Thr His Phe Arg Cys Gin Cys Tyr Leu Gly 420 425 430

Trp Ser Gly Glu Gin Cys Gin Trp Asp His Arg Gin Ala Ala Gly Gly 435 440 445

Ala Ser Glu Ala Trp Ala Gly Ser His Leu Thr Ser Leu Leu Ala Leu 450 455 460

Ala Ala Leu Ala Phe Thr Trp Thr Leu 465 470 <210> 38 <211> 417 <212> PRT <213> Homo sapiens <220> <223> Hyaluronidase-3 [Precursor] <400> 38

Met Thr Thr Gin Leu Gly Pro Ala Leu Val Leu Gly Val Ala Leu Cys 15 10 15

Leu Gly Cys Gly Gin Pro Leu Pro Gin Val Pro Glu Arg Pro Phe Ser 20 25 30

Val Leu Trp Asn Val Pro Ser Ala His Cys Glu Ala Arg Phe Gly Val 35 40 45

His Leu Pro Leu Asn Ala Leu Gly lie lie Ala Asn Arg Gly Gin His 50 55 60

Phe His Gly Gin Asn Met Thr lie Phe Tyr Lys Asn Gin Leu Gly Leu , 65 70 75 80

Tyr Pro Tyr Phe Gly Pro Arg Gly Thr Ala His Asn Gly Gly lie Pro 85 90 95

Gin Ala Leu Pro Leu Asp Arg His Leu Ala Leu Ala Ala Tyr Gin lie 100 105 110

His His Ser Leu Arg Pro Gly Phe Ala Gly Pro Ala Val Leu Asp Trp 115 120 125

Glu Glu Trp Cys Pro Leu Trp Ala Gly Asn Trp Gly Arg Arg Arg Ala 130 135 140

Tyr Gin Ala Ala Ser Trp Ala Trp Ala Gin Gin Val Phe Pro Asp Leu 145 150 155 160

Asp Pro Gin Glu Gin Leu Tyr Lys Ala Tyr Thr Gly Phe Glu Gin Ala 165 170 175

Ala Arg Ala Leu Met Glu Asp Thr Leu Arg Val Ala Gin Ala Leu Arg 180 185 190

Pro His Gly Leu Trp Gly Phe Tyr His Tyr Pro Ala Cys Gly Asn Gly 195 200 205

Trp His Ser Met Ala Ser Asn Tyr Thr Gly Arg Cys His Ala Ala Thr 210 215 220

Leu Ala Arg Asn Thr Gin Leu His Trp Leu Trp Ala Ala Ser Ser Ala 225 230 235 240

Leu Phe Pro Ser lie Tyr Leu Pro Pro Arg Leu Pro Pro Ala His His 245 250 255

Gin Ala Phe Val Arg His Arg Leu Glu Glu Ala Phe Arg Val Ala Leu 260 265 270

Val Gly His Arg His Pro Leu Pro Val Leu Ala Tyr Val Arg Leu Thr 275 280 285

His Arg Arg Ser Gly Arg Phe Leu Ser Gin Asp Asp Leu Val Gin Ser 290 295 300 lie Gly Val Ser Ala Ala Leu Gly Ala Ala Gly Val Val Leu Trp Gly 305 310 315 320

Asp Leu Ser Leu Ser Ser Ser Glu Glu Glu Cys Trp His Leu His Asp 325 330 335

Tyr Leu Val Asp Thr Leu Gly Pro Tyr Val lie Asn Val Thr Arg Ala 340 345 350

Ala Met Ala Cys Ser His Gin Arg Cys His Gly His Gly Arg Cys Ala 355 360 365

Arg Arg Asp Pro Gly Gin Met Glu Ala Phe Leu His Leu Trp Pro Asp 370 375 380

Gly Ser Leu Gly Asp Trp Lys Ser Phe Ser Cys His Cys Tyr Trp Gly 385 390 395 400

Trp Ala Gly Pro Thr Cys Gin Glu Pro Arg Pro Gly Pro Lys Glu Ala 405 410 415

Val <210> 39 <211> 481 <212> PRT <213> Homo sapiens <220> <223> Hyaluronidase-4 <400> 39

Met Lys Val Leu Ser Glu Gly Gin Leu Lys Leu Cys Val Val Gin Pro 15 10 15

Val His Leu Thr Ser Trp Leu Leu lie Phe Phe lie Leu Lys Ser lie 20 25 30

Ser Cys Leu Lys Pro Ala Arg Leu Pro lie Tyr Gin Arg Lys Pro Phe 35 40 45 lie Ala Ala Trp Asn Ala Pro Thr Asp Gin Cys Leu lie Lys Tyr Asn 50 55 60

Leu Arg Leu Asn Leu Lys Met Phe Pro Val lie Gly Ser Pro Leu Ala 65 70 75 80

Lys Ala Arg Gly Gin Asn Val Thr lie Phe Tyr Val Asn Arg Leu Gly 85 90 95

Tyr Tyr Pro Trp Tyr Thr Ser Gin Gly Val Pro lie Asn Gly Gly Leu 100 105 110

Pro Gin Asn lie Ser Leu Gin Val His Leu Glu Lys Ala Asp Gin Asp 115 120 125 lie Asn Tyr Tyr lie Pro Ala Glu Asp Phe Ser Gly Leu Ala Val lie 130 135 140

Asp Trp Glu Tyr Trp Arg Pro Gin Trp Ala Arg Asn Trp Asn Ser Lys 145 150 155 160

Asp Val Tyr Arg Gin Lys Ser Arg Lys Leu lie Ser Asp Met Gly Lys 165 170 175

Asn Val Ser Ala Thr Asp lie Glu Tyr Leu Ala Lys Val Thr Phe Glu 180 185 190

Glu Ser Ala Lys Ala Phe Met Lys Glu Thr lie Lys Leu Gly lie Lys 195 200 205

Ser Arg Pro Lys Gly Leu Trp Gly Tyr Tyr Leu Tyr Pro Asp Cys His 210 215 220

Asn Tyr Asn Val Tyr Ala Pro Asn Tyr Ser Gly Ser Cys Pro Glu Asp 225 230 235 240

Glu Val Leu Arg Asn Asn Glu Leu Ser Trp Leu Trp Asn Ser Ser Ala 245 250 255

Ala Leu Tyr Pro Ser lie Gly Val Trp Lys Ser Leu Gly Asp Ser Glu 260 265 270

Asn lie Leu Arg Phe Ser Lys Phe Arg Val His Glu Ser Met Arg lie 275 280 285

Ser Thr Met Thr Ser His Asp Tyr Ala Leu Pro Val Phe Val Tyr Thr 290 295 300

Arg Leu Gly Tyr Arg Asp Glu Pro Leu Phe Phe Leu Ser Lys Gin Asp 305 310 315 320

Leu Val Ser Thr lie Gly Glu Ser Ala Ala Leu Gly Ala Ala Gly lie 325 330 335

Val lie Trp Gly Asp Met Asn Leu Thr Ala Ser Lys Ala Asn Cys Thr 340 345 350

Lys Val Lys Gin Phe Val Ser Ser Asp Leu Gly Ser Tyr lie Ala Asn 355 360 365

Val Thr Arg Ala Ala Glu Val Cys Ser Leu His Leu Cys Arg Asn Asn 370 375 380

Gly Arg Cys lie Arg Lys Met Trp Asn Ala Pro Ser Tyr Leu His Leu 385 390 395 400

Asn Pro Ala Ser Tyr His lie Glu Ala Ser Glu Asp Gly Glu Phe Thr 405 410 415

Val Lys Gly Lys Ala Ser Asp Thr Asp Leu Ala Val Met Ala Asp Thr 420 425 430

Phe Ser Cys His Cys Tyr Gin Gly Tyr Glu Gly Ala Asp Cys Arg Glu 435 440 445 lie Lys Thr Ala Asp Gly Cys Ser Gly Val Ser Pro Ser Pro Gly Ser 450 455 460

Leu Met Thr Leu Cys Leu Leu Leu Leu Ala Ser Tyr Arg Ser lie Gin 465 470 475 480

Leu <210> 40 <211 >467

<212> PRT <213> Homo sapiens <220> <223> sHuPH20 precursor 1-467 <400> 40

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Arg Ser Phe Val Lys 15 10 15

Ser Ser Gly Val Ser Gin lie Val Phe Thr Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60

Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg 65 70 75 80 lie Asn Ala Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly 100 105 110

Gly lie Pro Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys 115 120 125

Lys Asp lie Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140 lie Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160

Lys Asp Val Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn 165 170 175

Val Gin Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys 195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240

Val Glu lie Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255

Thr Ala Leu Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val 260 265 270

Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val 275 280 285

Ser Lys lie Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300

Arg lie Val Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu 305 310 315 320

Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie 325 330 335

Val lie Trp Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu 340 345 350

Leu Leu Asp Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin 370 375 380

Gly Val Cys lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400

Asn Pro Asp Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415

Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys 420 425 430

Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys 450 455 460 lie Asp Ala 465 <210 41 <211> 477 <212> PRT <213> Homo sapiens <220 <223> sHuPH20 precursor 1-477 <400> 41

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Arg Ser Phe Val Lys 15 10 15

Ser Ser Gly Val Ser Gin lie Val Phe Thr Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60

Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg 65 70 75 80 lie Asn Ala Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly 100 105 110

Gly lie Pro Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys 115 120 125

Lys Asp lie Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140 lie Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160

Lys Asp Val Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn 165 170 175

Val Gin Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys 195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240

Val Glu lie Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255

Thr Ala Leu Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val 260 265 270

Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val 275 280 285

Ser Lys lie Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300

Arg lie Val Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu 305 310 315 320

Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie 325 330 335

Val lie Trp Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu 340 345 350

Leu Leu Asp Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin 370 375 380

Gly Val Cys lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400

Asn Pro Asp Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415

Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys 420 425 430

Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys 450 455 460 lie Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu 465 470 475 <210> 42 <211> 478

<212> PRT <213> Homo sapiens <220 <223> sHuPH20 precursor 1-478 <400 42

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Arg Ser Phe Val Lys 15 10 15

Ser Ser Gly Val Ser Gin lie Val Phe Thr Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60

Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg 65 70 75 80 lie Asn Ala Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly 100 105 110

Gly lie Pro Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys 115 120 125

Lys Asp lie Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140 lie Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160

Lys Asp Val Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn 165 170 175

Val Gin Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys 195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240

Val Glu lie Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255

Thr Ala Leu Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val 260 265 270

Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val 275 280 285

Ser Lys lie Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300

Arg lie Val Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu 305 310 315 320

Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie 325 330 335

Val lie Trp Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu 340 345 350

Leu Leu Asp Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin 370 375 380

Gly Val Cys lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400

Asn Pro Asp Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415

Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys 420 425 430

Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys 450 455 460

He Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro 465 470 475 <210> 43 <211> 479 <212> PRT <213> Homo sapiens <220> <223> sHuPH20 precursor 1-479 <400> 43

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Arg Ser Phe Val Lys 15 10 15

Ser Ser Gly Val Ser Gin lie Val Phe Thr Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60

Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg 65 70 75 80 lie Asn Ala Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly 100 105 110

Gly lie Pro Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys 115 120 125

Lys Asp lie Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140 lie Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160

Lys Asp Val Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn 165 170 175

Val Gin Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys 195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240

Val Glu lie Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255

Thr Ala Leu Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val 260 265 270

Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val 275 280 285

Ser Lys lie Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300

Arg lie Val Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu 305 310 315 320

Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie 325 330 335

Val lie Trp Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu 340 345 350

Leu Leu Asp Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin 370 375 380

Gly Val Cys lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400

Asn Pro Asp Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415

Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys 420 425 430

Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys 450 455 460 lie Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin 465 470 475 <210> 44 <211> 480 <212> PRT <213> Homo sapiens <220> <223> sHuPH20 precursor 1-480 <400> 44

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Arg Ser Phe Val Lys 15 10 15

Ser Ser Gly Val Ser Gin lie Val Phe Thr Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60

Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg 65 70 75 80 lie Asn Ala Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly 100 105 110

Gly lie Pro Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys 115 120 125

Lys Asp lie Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140 lie Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160

Lys Asp Val Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn 165 170 175

Val Gin Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys 195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240

Val Glu lie Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255

Thr Ala Leu Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val 260 265 270

Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val 275 280 285

Ser Lys lie Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300

Arg lie Val Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu 305 310 315 320

Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie 325 330 335

Val lie Trp Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu 340 345 350

Leu Leu Asp Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin 370 375 380

Gly Val Cys lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400

Asn Pro Asp Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415

Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys 420 425 430

Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys 450 455 460 lie Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin lie 465 470 475 480 <210> 45 <211> 481 <212> PRT <213> Homo sapiens <220> <223> sHuPH20 precursor 1-481 <400> 45

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Arg Ser Phe Val Lys 15 10 15

Ser Ser Gly Val Ser Gin lie Val Phe Thr Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60

Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg 65 70 75 80 lie Asn Ala Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly 100 105 110

Gly lie Pro Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys 115 120 125

Lys Asp lie Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140 lie Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160

Lys Asp Val Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn 165 170 175

Val Gin Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys 195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240

Val Glu lie Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255

Thr Ala Leu Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val 260 265 270

Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val 275 280 285

Ser Lys lie Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300

Arg lie Val Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu 305 310 315 320

Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie 325 330 335

Val lie Trp Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu 340 345 350

Leu Leu Asp Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin 370 375 380

Gly Val Cys lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400

Asn Pro Asp Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415

Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys 420 425 430

Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys 450 455 460 lie Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin lie 465 470 475 480

Phe <210> 46 <211> 483 <212> PRT <213> Homo sapiens <220> <223> sHuPH20 precursor 1-483 <400> 46

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Arg Ser Phe Val Lys 15 10 15

Ser Ser Gly Val Ser Gin lie Val Phe Thr Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60

Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg 65 70 75 80 lie Asn Ala Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly 100 105 110

Gly lie Pro Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys 115 120 125

Lys Asp lie Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140 lie Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160

Lys Asp Val Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn 165 170 175

Val Gin Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys 195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240

Val Glu lie Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 1 245 250 255

Thr Ala Leu Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val 260 265 270

Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val 275 280 285

Ser Lys lie Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300

Arg lie Val Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu 305 310 315 320

Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie 325 330 335

Val lie Trp Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu 340 345 350

Leu Leu Asp Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin 370 375 380

Gly Val Cys lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400

Asn Pro Asp Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415

Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys 420 425 430

Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys 450 455 460 lie Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin lie 465 470 475 480

Phe Tyr Asn <210> 47 <211> 432 <212> PRT <213> Homo sapiens <220> <223> sHuPH20 mature 36-467 <400> 47

Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro Phe Leu Trp 15 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro 20 25 30

Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg lie Asn Ala 35 40 45

Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu Gly Tyr Tyr 50 55 60

Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly Gly lie Pro 65 70 75 80

Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys Lys Asp lie 85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val lie Asp Trp 100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val 115 120 125

Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn Val Gin Leu 130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe Glu Lys Ala 145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys Leu Leu Arg 165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His 180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu lie 195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu 210 215 220

Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val Ala Ala Thr 225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val Ser Lys lie 245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg lie Val 260 265 270

Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu Leu Val Tyr 275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie Val lie Trp 290 295 300

Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp 305 310 315 320

Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn Val Thr Leu 325 330 335

Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin Gly Val Cys 340 345 350 lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp 355 360 365

Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly 370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys Phe Tyr Cys 385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp 405 410 415

Thr Asp Ala Val Asp Val Cys He Ala Asp Gly Val Cys lie Asp Ala 420 425 430 <210> 48 <211> 448 <212> PRT <213> Homo sapiens <220> <223> sHuPH20 mature 36-483 <400> 48

Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro Phe Leu Trp 15 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro 20 25 30

Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg lie Asn Ala 35 40 45

Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu Gly Tyr Tyr 50 55 60

Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly Gly lie Pro 65 70 75 80

Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys Lys Asp lie 85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val lie Asp Trp 100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val 115 120 125

Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn Val Gin Leu 130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe Glu Lys Ala 145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys Leu Leu Arg 165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His 180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu lie 195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu 210 215 220

Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val Ala Ala Thr 225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val Ser Lys lie 245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg lie Val 260 265 270

Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu Leu Val Tyr 275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie Val lie Trp 290 295 300

Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp 305 310 315 320

Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn Val Thr Leu 325 330 335

Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin Gly Val Cys 340 345 350 lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp 355 360 365

Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly 370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys Phe Tyr Cys 385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp 405 410 415

Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys lie Asp Ala 420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin lie Phe Tyr Asn 435 440 445 <210> 49 <211> 1446

<212> DNA <213> Homo sapiens <220> <223> DNA encoding soluble rHuPH20 "precursor" <400> 49 atgggagtgc taaaattcaa gcacatcttt ttcagaagct ttgttaaatc aagtggagta 60 tcccagatag ttttcacctt ccttctgatt ccatgttgct tgactctgaa tttcagagca 120 cctcctgtta ttccaaatgt gcctttcctc tgggcctgga atgccccaag tgaattttgt 180 cttggaaaat ttgatgagcc actagatatg agcctcttct ctttcatagg aagcccccga 240 ataaacgcca ccgggcaagg tgttacaata ttttatgttg atagacttgg ctactatcct 300 tacatagatt caatcacagg agtaactgtg aatggaggaa tcccccagaa gatttcctta 360 caagaccatc tggacaaagc taagaaagac attacatttt atatgccagt agacaatttg 420 ggaatggctg ttattgactg ggaagaatgg agacccactt gggcaagaaa ctggaaacct 480 aaagatgttt acaagaatag gtctattgaa ttggttcagc aacaaaatgt acaacttagt 540 ctcacagagg ccactgagaa agcaaaacaa gaatttgaaa aggcagggaa ggatttcctg 600 gtagagacta taaaattggg aaaattactt cggccaaatc acttgtgggg ttattatctt 660 tttccggatt gttacaacca tcactataag aaacccggtt acaatggaag ttgcttcaat 720 gtagaaataa aaagaaatga tgatctcagc tggttgtgga atgaaagcac tgctctttac 780 ccatccattt atttgaacac tcagcagtct cctgtagctg ctacactcta tgtgcgcaat 840 cgagttcggg aagccatcag agtttccaaa atacctgatg caaaaagtcc acttccggtt 900 tttgcatata cccgcatagt ttttactgat caagttttga aattcctttc tcaagatgaa 960 cttgtgtata catttggcga aactgttgct ctgggtgctt ctggaattgt aatatgggga 1020 accctcagta taatgcgaag tatgaaatct tgcttgctcc tagacaatta catggagact 1080 atactgaatc cttacataat caacgtcaca ctagcagcca aaatgtgtag ccaagtgctt 1140 tgccaggagc aaggagtgtg tataaggaaa aactggaatt caagtgacta tcttcacctc 1200 aacccagata attttgctat tcaacttgag aaaggtggaa agttcacagt acgtggaaaa 1260 ccgacacttg aagacctgga gcaattttct gaaaaatttt attgcagctg ttatagcacc 1320 ttgagttgta aggagaaagc tgatgtaaaa gacactgatg ctgttgatgt gtgtattgct 1380 gatggtgtct gtatagatgc ttttctaaaa cctcccatgg agacagaaga acctcaaatt 1440 ttctac 1446 <210> 50 <211> 509 <212> PRT <213> Homo sapiens <220> <223> PH20 variant P48A <400> 50

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Arg Ser Phe Val Lys 15 10 15

Ser Ser Gly Val Ser Gin lie Val Phe Thr Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Ala 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60

Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg i 65 70 75 80 lie Asn Ala Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly 100 105 110

Gly lie Pro Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys 115 120 125

Lys Asp lie Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140 lie Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160

I

I

I

I

Lys Asp Val Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn 165 170 175

Val Gin Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr lie Lys Leu Gly Lys 195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240

Val Glu lie Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255

Thr Ala Leu Tyr Pro Ser lie Tyr Leu Asn Thr Gin Gin Ser Pro Val 260 265 270

Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala lie Arg Val 275 280 285

Ser Lys lie Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300

Arg lie Val Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu 305 310 315 320

Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly lie 325 330 335

Val lie Trp Gly Thr Leu Ser lie Met Arg Ser Met Lys Ser Cys Leu 340 345 350

Leu Leu Asp Asn Tyr Met Glu Thr lie Leu Asn Pro Tyr lie lie Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin 370 375 380

Gly Val Cys lie Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400

Asn Pro Asp Asn Phe Ala lie Gin Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415

Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys 420 425 430

Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys lie Ala Asp Gly Val Cys 450 455 460 lie Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin lie 465 470 475 480

Phe Tyr Asn Ala Ser Pro Ser Thr Leu Ser Ala Thr Met Phe lie Val 485 490 495

Ser lie Leu Phe Leu lie lie Ser Ser Val Ala Ser Leu 500 505 <210> 51 <211> 509

<212> PRT <213> Homo sapiens <220> <223> precursor PH20 variant L499W <400> 51

Met Gly Val Leu Lys Phe Lys His lie Phe Phe Arg Ser Phe Val Lys 15 10 15

Ser Ser Gly Val Ser Gin lie Val Phe Thr Phe Leu Leu lie Pro Cys 20 25 30

Cys Leu Thr Leu Asn Phe Arg Ala Pro Pro Val lie Pro Asn Val Pro 35 40 45

Phe Leu Trp Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe 50 55 60

Asp Glu Pro Leu Asp Met Ser Leu Phe Ser Phe lie Gly Ser Pro Arg 65 70 75 80 lie Asn Ala Thr Gly Gin Gly Val Thr lie Phe Tyr Val Asp Arg Leu 85 90 95

Gly Tyr Tyr Pro Tyr lie Asp Ser lie Thr Gly Val Thr Val Asn Gly 100 105 110

Gly lie Pro Gin Lys lie Ser Leu Gin Asp His Leu Asp Lys Ala Lys 115 120 125

Lys Asp lie Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val 130 135 140

He Asp Trp Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro 145 150 155 160

Lys Asp Val Tyr Lys Asn Arg Ser lie Glu Leu Val Gin Gin Gin Asn 165 170 175

Val Gin Leu Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gin Glu Phe 180 185 190

Glu Lys Ala Gly Lys Asp Phe Leu Val Glu Thr He Lys Leu Gly Lys 195 200 205

Leu Leu Arg Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys 210 215 220

Tyr Asn His His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn 225 230 235 240

Val Glu He Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser 245 250 255

Thr Ala Leu Tyr Pro Ser He Tyr Leu Asn Thr Gin Gin Ser Pro Val 260 265 270

Ala Ala Thr Leu Tyr Val Arg Asn Arg Val Arg Glu Ala He Arg Val 275 280 285

Ser Lys He Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr 290 295 300

Arg He Val Phe Thr Asp Gin Val Leu Lys Phe Leu Ser Gin Asp Glu 305 310 315 320

Leu Val Tyr Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly He 325 330 335

Val He Trp Gly Thr Leu Ser He Met Arg Ser Met Lys Ser Cys Leu 340 345 350

Leu Leu Asp Asn Tyr Met Glu Thr He Leu Asn Pro Tyr He He Asn 355 360 365

Val Thr Leu Ala Ala Lys Met Cys Ser Gin Val Leu Cys Gin Glu Gin 370 375 380

Gly Val Cys He Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu 385 390 395 400

Asn Pro Asp Asn Phe Ala He Gin Leu Glu Lys Gly Gly Lys Phe Thr 405 410 415

Val Arg Gly Lys Pro Thr Leu Glu Asp Leu Glu Gin Phe Ser Glu Lys 420 425 430

Phe Tyr Cys Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp 435 440 445

Val Lys Asp Thr Asp Ala Val Asp Val Cys He Ala Asp Gly Val Cys 450 455 460

He Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin He 465 470 475 480

Phe Tyr Asn Ala Ser Pro Ser Thr Leu Ser Ala Thr Met Phe He Val 485 490 495

Ser He Trp Phe Leu He He Ser Ser Val Ala Ser Leu 500 505

<210> 52 <211> 6630 <212> DNA <213> Artificial Sequence <220 <223> HZ24 vector <400> 52 ) 3 5 0 5 '0 15 50 55 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 660 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 720 tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat 780 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc 840 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa 900 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac 960 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta 1020 aggctagagt acttaatacg actcactata ggctagcatg ggagtgctaa aattcaagca 1080 catctttttc agaagctttg ttaaatcaag tggagtatcc cagatagttt tcaccttcct 1140 tctgattcca tgttgcttga ctctgaattt cagagcacct cctgttattc caaatgtgcc 1200 tttcctctgg gcctggaatg ccccaagtga attttgtctt ggaaaatttg atgagccact 1260 agatatgagc ctcttctctt tcataggaag cccccgaata aacgccaccg ggcaaggtgt 1320 tacaatattt tatgttgata gacttggcta ctatccttac atagattcaa tcacaggagt 1380 aactgtgaat ggaggaatcc cccagaagat ttccttacaa gaccatctgg acaaagctaa 1440 gaaagacatt acattttata tgccagtaga caatttggga atggctgtta ttgactggga 1500 agaatggaga cccacttggg caagaaactg gaaacctaaa gatgtttaca agaataggtc 1560 tattgaattg gttcagcaac aaaatgtaca acttagtctc acagaggcca ctgagaaagc 1620 aaaacaagaa tttgaaaagg cagggaagga tttcctggta gagactataa aattgggaaa 1680 attacttcgg ccaaatcact tgtggggtta ttatcttttt ccggattgtt acaaccatca 1740 ctataagaaa cccggttaca atggaagttg cttcaatgta gaaataaaaa gaaatgatga 1800 tctcagctgg ttgtggaatg aaagcactgc tctttaccca tccatttatt tgaacactca 1860 gcagtctcct gtagctgcta cactctatgt gcgcaatcga gttcgggaag ccatcagagt 1920 ttccaaaata cctgatgcaa aaagtccact tccggttttt gcatataccc gcatagtttt 1980 tactgatcaa gttttgaaat tcctttctca agatgaactt gtgtatacat ttggcgaaac 2040 tgttgctctg ggtgcttctg gaattgtaat atggggaacc ctcagtataa tgcgaagtat 2100 gaaatcttgc ttgctcctag acaattacat ggagactata ctgaatcctt acataatcaa 2160 cgtcacacta gcagccaaaa tgtgtagcca agtgctttgc caggagcaag gagtgtgtat 2220 aaggaaaaac tggaattcaa gtgactatct tcacctcaac ccagataatt ttgctattca 2280 acttgagaaa ggtggaaagt tcacagtacg tggaaaaccg acacttgaag acctggagca 2340 attttctgaa aaattttatt gcagctgtta tagcaccttg agttgtaagg agaaagctga 2400 tgtaaaagac actgatgctg ttgatgtgtg tattgctgat ggtgtctgta tagatgcttt 2460 tctaaaacct cccatggaga cagaagaacc tcaaattttc tactgaggat ccatagctaa 2520 cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg 2580 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc 2640 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa 2700 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 2760 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc 2820 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc 2880 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac 2940 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg 3000 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac 3060 ggggacgtgg ttttcctttg aaaaacacga tgataagctt gccacaaccc acagcggccg 3120 ctgccatcat ggttcgacca ttgaactgca tcgtcgccgt gtcccaaaat atggggattg 3180 gcaagaacgg agacctaccc tggcctccgc tcaggaacga gttcaagtac ttccaaagaa 3240 tgaccacaac ctcttcagtg gaaggtaaac agaatctggt gattatgggt aggaaaacct 3300 ggttctccat tcctgagaag aatcgacctt taaaggacag aattaatata gttctcagta 3360 gagaactcaa agaaccacca cgaggagctc attttcttgc caaaagtttg gatgatgcct 3420 taagacttat tgaacaaccg gaattggcaa gtaaagtaga catggtttgg atagtcggag 3480 gcagttctgt ttaccaggaa gccatgaatc aaccaggcca cctcagactc tttgtgacaa 3540 ggatcatgca ggaatttgaa agtgacacgt ttttcccaga aattgatttg gggaaatata 3600 aacttctccc agaataccca ggcgtcctct ctgaggtcca ggaggaaaaa ggcatcaagt 3660 ataagtttga agtctacgag aagaaagact aaacgcgtgg tacctctaga gtcgacccgg 3720 gcggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag 3780 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac 3840 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt 3900 tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat 3960 cgataaggat ccgggctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 4020 gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt 4080 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 4140 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 4200 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 4260 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 4320 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 4380 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 4440 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt 4500 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 4560 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 4620 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 4680 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 4740 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 4800 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 4860 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 4920 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 4980 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 5040 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 5100 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 5160 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 5220 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 5280 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 5340 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 5400 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 5460 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 5520 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 5580 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 5640 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 5700 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 5760 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 5820 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 5880 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 5940 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 6000 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 6060 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 6120 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 6180 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 6240 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 6300 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 6360 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 6420 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 6480 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 6540 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 6600 ggccttttgc tcacatggct cgacagatct 6630 <210> 53 <211 > 186

<212> PRT <213> Mus musculus <220> <223> dihydrofolate reductase <400 53

Val Arg Pro Leu Asn Cys lie Val Ala Val Ser Gin Asn Met Gly lie 15 10 15

Gly Lys Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Phe Lys 20 25 30

Tyr Phe Gin Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Gin Asn 35 40 45

Leu Val lie Met Gly Arg Lys Thr Trp Phe Ser lie Pro Glu Lys Asn 50 55 60

Arg Pro Leu Lys Asp Arg lie Asn lie Val Leu Ser Arg Glu Leu Lys 65 70 75 80

Glu Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser Leu Asp Asp Ala 85 90 95

Leu Arg Leu lie Glu Gin Pro Glu Leu Ala Ser Lys Val Asp Met Val 100 105 110

Trp lie Val Gly Gly Ser Ser Val Tyr Gin Glu Ala Met Asn Gin Pro 115 120 125

Gly His Leu Arg Leu Phe Val Thr Arg lie Met Gin Glu Phe Glu Ser 130 135 140

Asp Thr Phe Phe Pro Glu lie Asp Leu Gly Lys Tyr Lys Leu Leu Pro 145 150 155 160

Glu Tyr Pro Gly Val Leu Ser Glu Val Gin Glu Glu Lys Gly He Lys 165 170 175

Tyr Lys Phe Glu Val Tyr Glu Lys Lys Asp 180 185

<210> 54 <211>6 <212> PRT <213> HisArtificial Sequence <220 <223> His tag <400 54

His His His His His His 1 5

<210> 55 <211>8 <212> PRT <213> Artificial Sequence <220> <223> Flag tag <400> 55

Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 56 <211 > 1449 <212> DNA <213> Homo sapiens <220> <223> Gen2 mRNA sequence <400> 56 atgggagtgc taaaattcaa gcacatcttt ttcagaagct ttgttaaatc aagtggagta 60 tcccagatag ttttcacctt ccttctgatt ccatgttgct tgactctgaa tttcagagca 120 cctcctgtta ttccaaatgt gcctttcctc tgggcctgga atgccccaag tgaattttgt 180 cttggaaaat ttgatgagcc actagatatg agcctcttct ctttcatagg aagcccccga 240 ataaacgcca ccgggcaagg tgttacaata ttttatgttg atagacttgg ctactatcct 300 tacatagatt caatcacagg agtaactgtg aatggaggaa tcccccagaa gatttcctta 360 caagaccatc tggacaaagc taagaaagac attacatttt atatgccagt agacaatttg 420 ggaatggctg ttattgactg ggaagaatgg agacccactt gggcaagaaa ctggaaacct 480 aaagatgttt acaagaatag gtctattgaa ttggttcagc aacaaaatgt acaacttagt 540 ctcacagagg ccactgagaa agcaaaacaa gaatttgaaa aggcagggaa ggatttcctg 600 gtagagacta taaaattggg aaaattactt cggccaaatc acttgtgggg ttattatctt 660 tttccggatt gttacaacca tcactataag aaacccggtt acaatggaag ttgcttcaat 720 gtagaaataa aaagaaatga tgatctcagc tggttgtgga atgaaagcac tgctctttac 780 ccatccattt atttgaacac tcagcagtct cctgtagctg ctacactcta tgtgcgcaat 840 cgagttcggg aagccatcag agtttccaaa atacctgatg caaaaagtcc acttccggtt 900 tttgcatata cccgcatagt ttttactgat caagttttga aattcctttc tcaagatgaa 960 cttgtgtata catttggcga aactgttgct ctgggtgctt ctggaattgt aatatgggga 1020 accctcagta taatgcgaag tatgaaatct tgcttgctcc tagacaatta catggagact 1080 atactgaatc cttacataat caacgtcaca ctagcagcca aaatgtgtag tcaagtgctt 1140 tgccaggagc aaggagtgtg tataaggaaa aactggaatt caagtgacta tcttcacctc 1200 aacccagata attttgctat tcaacttgag aaaggtggaa agttcacagt acgtggaaaa 1260 ccgacacttg aagacctgga gcaattttct gaaaaatttt attgcagctg ttatagcacc 1320 ttgagttgta aggagaaagc tgatgtaaaa gacactgatg ctgttgatgt gtgtattgct 1380 gatggtgtct gtatagatgc ttttctaaaa cctcccatgg agacagaaga acctcaaatt 1440 ttctactga 1449

Claims 1. A combination of compositions of immune globulin (IG) and a soluble hyaluronidase for use for treating an IG-treatable disease or condition in a subject, wherein: the IG and soluble hyaluronidase are formulated separately for subcutaneous administration; the IG and soluble hyaluronidase are formulated for single dosage administration once a month; the soluble hyaluronidase is administered separately from the IG prior to administration of the IG and at the same site as the IG; the concentration of IG is 5 to 15% w/v and the amount of IG in the composition is 0.5 grams (g) to 70 g; the IG is formulated in a volume of liquid that is 50 mL to 700 mL; the soluble hyaluronidase is formulated for administration as a ratio of hyaluronidase to IG of 10 U/gram (g) to 500 U/g of IG, whereby the amount of hyaluronidase in the composition is sufficient to effect increased bioavailability of the IG when administered in combination with the IG to at least 90% of the bioavailability of the same single dosage of IG administered via intravenous administration for treatment of the same IG-treatable disease or condition; the soluble hyaluronidase is formulated in a volume of liquid that is 5 to 30 mL; and the IG-treatable disease or condition is selected from among immunodeficiency; acquired hypogammaglobulinemia secondary to hematological malignancies; Kawasaki’s disease; chronic inflammatory demyelinating polyneuropathy (CIDP); Guillain-Barre Syndrome; Idiopathic thrombocytopenic purpura; inflammatory myopathies; Lambert-Eaton myasthenic syndrome; multifocal motor neuropathy; Myasthenia Gravis; Moersch-Wolt-mann syndrome; secondary hypogammaglobulinaemia, including iatrogenic immunodeficiency; specific antibody deficiency; Acute disseminated encephalomyelitis; ANCA-positive systemic necrotizing vasculitis; Autoimmune haemolytic anaemia; Bullous pemphigoid; Cicatricial pemphigoid; Evans syndrome, including autoimmune haemolytic anaemia with immune thrombocytopenia; Foeto-maternal/neonatal alloimmune thrombocytopenia (FMAIT/NAIT); Flaemophagocytic syndrome; High-risk allogeneic haemopoietic stem cell transplantation; IgM paraproteinaemic neuropathy; kidney transplantation; multiple sclerosis; Opsoclonus myoclonus ataxia; Pemphigus foliaceus; Pemphigus vulgaris; Post-transfusion purpura; Toxic epidermal necrolysis/Steven Johnson syndrome (TEN/SJS); Toxic shock syndrome; Alzheimer’s Disease; Systemic lupus erythematosus; multiple myeloma; sepsis; B cell tumors; trauma; and a bacterial, viral or fungal infection. 2. The combination of claim 1, wherein the amount of IG in the composition is 5g, 10 g, 15 g, 20 g, 21 g, 22 g, 23 g, 24 g, 25 g, 26 g, 27 g, 28 g, 29 g, 30 g, 31 g, 32 g, 33 g, 34 g, 35 g, 36 g, 37 g, 38 g, 39 g or 40 g. 3. The combination of claim 1, wherein the IG in the composition is formulated for subcutaneous infusion by gravity, pump infusion or injection in an amount that is 20 to 30 grams. 4. The combination of claim 1, wherein the IG composition is a 10% liquid IG formulation. 5. The combination of any of claims 1-4, wherein the hyaluronidase in the composition is 5000 Units to 7500 Units or 1,000 Units to 10,000 Units. 6. The combination of any of claims 1-5, wherein the hyaluronidase in the composition is in an amount that is at a ratio (units hyaluronidase/grams of IG) of 10 U/gram (g), 20 U/g, 30 U/g, 35 U/g, 40 U/g, 50 U/g, 60 U/g, 70 U/g, 80 U/g, 90 U/g, 100 U/g, 150 U/g, or 300 U/g. 7. The combination of claim 6, wherein the hyaluronidase in the composition is administered at a ratio of 50 U/gram IG. 8. The combination of any of claims 1-7, wherein the hyaluronidase in the composition is a PH20, or a truncated form thereof. 9. The combination or composition of claim 8, wherein the truncated human PH20: retains a hyaluronidase activity and is soluble; and is selected from among polypeptides having a sequence of amino acids set forth in any of SEQ ID NOS:4-9, or a sequence of amino acids comprising at least 91% sequence identity to the sequence of amino acids set forth in any of SEQ ID NOS: 4-9. 10. The combination of any of claims 1-9, wherein the IG in the composition is purified from human plasma. 11. The combination of any of claims 1-10, wherein the IG in the composition contains greater than 95% IgG. 12. The combination of any of claims 1-11, wherein the concentration of the IG is 6 to 15% w/v, or 8 to 12% w/v of the IG composition. 13. The combination of claim 12, wherein the IG concentration is 10% w/v. 14. The combination of claim 1, wherein the IG-treatable disease or condition is selected from among: an immunodeficiency selected from among common variable immunodeficiency (CVID), congenital agammaglobulinemia, Wiskott-Aldrich syndrome, severe combined immunodeficiency (SCID), primary hypogammaglobulinemia, primary immunodeficiency diseases with antibody deficiency, X-linked agammaglobulinemia (XLA), hypogammaglobulinemia of infancy, and paraneoplastic cerebellar degeneration with no antibodies; acquired hypogammaglobulinemia secondary to hematological malignancies selected from among chronic lymphocytic leukemia (CLL), multiple myeloma (MM) and non-Hodgkin’s lymphoma (NHL); an inflammatory myopathy selected from among polymyositis, dermatomyositis and inclusion body myositis; and a bacteria, viral or fungal condition selected from among Haemophilus influenzae type B, Pseudomonas aeruginosa types A and B, Staphylococcus aureus, Group B Streptococcus, Streptococcus pneumoniae types 1, 3, 4, 6, 7, 8, 9, 12, 14, 18, 19, and 23, Adenovirus types 2 and 5, Cytomegalovirus, Epstein Barr virus VCA, Hepatitis A virus, Hepatitis B virus, Herpes simplex virus-1, Herpes simplex virus-2, Influenza A, Measles, Parainfluenza types 1,2 and 3, Polio, Varicella zoster virus, Aspergillus and Candida albicans.

Patentansprüche 1. Kombination von Zusammensetzungen von Immunglobulin (IG) und einer löslichen Hyaluronidase zur Verwendung zur Behandlung einer IG-behandelbaren Erkrankung oder eines IG-behandelbaren Zustandes in einem Subjekt, wobei: das IG und die lösliche Hyaluronidase getrennt zur subkutanen Verabreichung formuliert sind, das IG und die lösliche Hyaluronidase für eine Einzeldosisverabreichung einmal monatlich formuliert sind, die lösliche Hyaluronidase getrennt von dem IG vor der Verabreichung des IGs und an dieselbe Stelle wie das IG verabreicht wird, die Konzentration des IGs 5-15% Gew./Vol. beträgt und die Menge des IGs in der Zusammensetzung 0,5

Gramm (g) bis 70 g beträgt, das IG in einem Volumen einer Flüssigkeit formuliert ist, welches 50 mL bis 700 mL beträgt, die lösliche Hyaluronidase zur Verabreichung als ein Verhältnis von Hyaluronidase zu IG von 10 U/Gramm (g) bis 500 U/g IG formuliert ist, wobei die Menge der Hyaluronidase in der Zusammensetzung ausreichend ist, um eine erhöhte Bioverfügbarkeit des IGs zu bewirken, wenn es in Kombination mit dem IG verabreicht wird, bis wenigstens 90% der Bioverfügbarkeit derselben Einzeldosierung von IG, welche durch intravenöse Verabreichung zur Behandlung derselben IG-behandelbaren Erkrankung oderdesselben IG-behandelbaren Zustands verabreicht wird, die lösliche Hyaluronidase in einem Volumen einer Flüssigkeit formuliert ist, welches 5 bis 30 mL beträgt, und die IG-behandelbare Erkrankung oder der IG-behandelbare Zustand ausgewählt ist aus Immundefizienz, erworbener Hypogammaglobulinämie sekundär zu hämatologischen Malignitäten, Kawasaki-Syndrom, chronischer inflammatorischer demyelinisierender Polyneuropathie (CIDP), Guillain-Barre-Syndrom, idiopathischer thrombocytopenischer Purpura, inflammatorischen Myopathien, myasthenischem Lambert-Eaton-Syndrom, multifokaler motorischer Neuropathie, Myasthenia gravis, Moersch-Woltmann-Syndrom, sekundärer Hypogam-maglubolinämie einschließend iatrogene Immundefizienz, spezifischer Antikörperdefizienz, akuter disseminier-ter Enzephalomyelitis, ANCA-positiver systemischer nekrotisierender Vaskulitis, hämolytischer Autoimmunanämie, bullösem Pemphigoid, zikatrizialem Pemphigoid, Evans-Syndrom einschließend hämolytische Autoimmunanämie mit Immunthrombocytopenie, fetomaternaler/neonataler Alloimmun-Thrombocytopenie (FMAIT/NAIT), hämophagocytischem Syndrom, allogener hämopoietischer Hochrisiko-Stammzelltransplanta-tion, IgM-paraproteinämischer Neuropathie, Nierentransplantation, multipler Sklerose, opsoklonischer myoklo-nischer Ataxie, Pemphigus foliaceus, Pemphigus vulgaris, posttransfusioneller Purpura, toxischer epidermaler Nekrolyse/Steven-Johnson-Syndrom (TEN/SJS), toxischem Schocksyndrom, Alzheimer-Erkrankung, systemischem Lupus erythematosus, multiplem Myelom, Sepsis, B-Zelltumoren, Trauma und einer bakteriellen, viralen oder Pilzinfektion. 2. Kombination nach Anspruch 1, wobei die Menge des IGs in der Zusammensetzung 5 g, 10 g, 15 g, 20 g, 21 g, 22 g, 23 g, 24 g, 25 g, 26 g, 27 g, 28 g, 29 g, 30 g, 31 g, 32 g, 33 g, 34 g, 35 g, 36 g, 37 g, 38 g, 39 g oder 40 g beträgt. 3. Kombination nach Anspruch 1, wobei das IG in der Zusammensetzung zur subkutanen Infusion durch Schwerkraft, Pumpeninfusion oder Injektion in einer Menge formuliert ist, welche 20 bis 30 Gramm beträgt. 4. Kombination nach Anspruch 1, wobei die IG-Zusammensetzung eine 10%ige flüssige IG-Formulierung ist. 5. Kombination nach einem der Ansprüche 1-4, wobei die Hyaluronidase in der Zusammensetzung in 5.000 Einheiten bis 7.500 Einheiten oder 1.000 Einheiten bis 10.000 Einheiten vorliegt. 6. Kombination nach einem der Ansprüche 1-5, wobei die Hyaluronidase in der Zusammensetzung in einer Menge vorliegt, welche bei einem Verhältnis (Einheiten Hyaluronidase/Gramm IG) von 10 U/Gramm (g), 20 U/g, 30 U/g, 35 U/g, 40 U/g, 50 U/g, 60 U/g, 70 U/g, 80 U/g, 90 U/g, 100 U/g, 150 U/g oder 300 U/g liegt. 7. Kombination nach Anspruch 6, wobei die Hyaluronidase in der Zusammensetzung in einem Verhältnis von 50 U/Gramm IG verabreicht wird. 8. Kombination nach einem der Ansprüche 1-7, wobei die Hyaluronidase in der Zusammensetzung ein PH20 oder eine trunkierte Form davon ist. 9. Kombination oder Zusammensetzung nach Anspruch 8, wobei das trunkierte humane PH20: eine Hyaluronidaseaktivität behält und löslich ist, und

ausgewählt ist aus Polypeptiden, welche eine Sequenz von Aminosäuren aufweisen, welche in einer aus SEQ ID NO: 4-9 dargestellt ist, oder eine Sequenz von Aminosäuren umfassend wenigstens 91 % Sequenzidentität zu der Sequenz von Aminosäuren, welche in einer der SEQ ID NO: 4-9 dargestellt ist. 10. Kombination nach einem der Ansprüche 1-9, wobei das IG in der Zusammensetzung aus humanem Plasma gereinigt ist. 11. Kombination nach einem der Ansprüche 1-10, wobei das IG in der Zusammensetzung mehr als 95% IgG enthält. 12. Kombination nach einem der Ansprüche 1-11, wobei die Konzentration des IGs 6 bis 15% Gew.A/ol. oder 8 bis 12% Gew./Vol. der IG-Zusammensetzung beträgt. 13. Kombination nach Anspruch 12, wobei die IG-Konzentration 10% Gew.A/ol. beträgt. 14. Kombination nach Anspruch 1, wobei die IG-behandelbare Erkrankung oder der IG-behandelbare Zustand ausgewählt ist aus: einer Immundefizienz ausgewählt aus variablem Immundefektsyndrom (OVID), kongenitaler Agammaglobulinämie, Wiskott-Aldrich-Syndrom, schwerem kombiniertem Immundefekt (SCID), primärer Hypogammaglobu-linämie, primären Immundefizienzerkrankungen mit Antikörperdefizienz, X-gebundener Agammaglobulinämie (XLA), Hypogammaglobulinämie der Kindheit und paraneoplastischer zerebellärer Degeneration ohne Antikörper, erworbener Hypogammaglobulinämie sekundär zu hämatologischen Malignitäten ausgewählt aus chronischer lymphocytischer Leukämie (CLL), multiplem Myelom (MM) und non-Hodgkin-Lymphom (NHL), einer inflammatorischen Myopathie ausgewählt aus Polymyositis, Dermatomyositis und Einschlußkörpermyositis, und einer bakteriellen, viralen oder Pilzinfektion ausgewählt aus Haemophilus influenzae Typ B, Pseudomonas aeruginosa Typ A und B, Staphylococcus aureus, Gruppe-B-Streptococcus, Streptococcus pneumoniae Typ 1,3, 4, 6, 7, 8, 9, 12, 14, 18, 19 und 23, Adenovirus Typ 2 und 5, Cytomegalovirus, Epstein-Barr-Virus VCA, Hepatitis-A-Virus, Hepatitis-B-Virus, Herpes simplex Virus-1, Herpes simplex Virus-2, Influenza A, Masern, Parainfluenza Typ 1,2 und 3, Polio, Varicellazoster-Virus, Aspergillus und Candida albicans.

Revendications 1. Combinaison de compositions d’immunoglobuline (IG) et d’une hyaluronidase soluble pour une utilisation pour le traitement d’une maladie ou d’une affection pouvant être traitée par IG chez un sujet, dans laquelle : l’IG et l’hyaluronidase soluble sont formulées séparément pour une administration sous-cutanée ; l’IG et l’hyaluronidase soluble sont formulées pour une administration de dose unique une fois par mois ; l’hyaluronidase soluble est administrée séparément de l’IG avant l’administration de l’IG et au même site que l’IG ; la concentration d’IG est de 5 à 15% p/v et la quantité d’IG dans la composition est de 0,5 gramme (g) à 70 g ; l’IG est formulée dans un volume de liquide qui est de 50 mL à 700 mL ; l’hyaluronidase soluble est formulée pour une administration en tant que rapport d’hyaluronidase sur IG allant de 10 U/gramme (g) à 500 U/g d’IG, moyennant quoi la quantité d’hyaluronidase dans la composition est suffisante pour entraîner une biodisponibilité accrue de l’IG lorsqu’elle est administrée en combinaison avec l’IG jusqu’à au moins 90% de la biodisponibilité de la même dose unique d’IG administrée via une administration intraveineuse pour le traitement de la même maladie ou affection pouvant être traitée par IG ; l’hyaluronidase soluble est formulée dans un volume de liquide qui est de 5 à 30 mL ; et la maladie ou affection pouvant être traitée par IG est choisie parmi l’immunodéficience ; l’hypogamma-globu-linémie acquise secondaire à des malignités hématologiques ; la maladie de Kawasaki; la polyneuropathie démyélinisante inflammatoire chronique (CIDP) ; le syndrome de Guillain-Barré ; le purpura thrombocytopéni-que idiopathique ; les myopathies inflammatoires ; le syndrome myasthénique de Lambert-Eaton ; la neuropathie motrice multifocale ; la myasthénie grave ; le syndrome de Moersch-Woltmann ; l’hypogammaglobulinémie secondaire, y compris l’immunodéficience iatrogène ; la déficience en anticorps spécifique ; l’encéphalomyélite aiguë disséminée ; la vascularite nécrosante systémique ANCA-positive ; l’anémie hémolytique auto-immune ; la pemphigoide bulleuse ; la pemphigoide cicatricielle ; le syndrome d’Evans, y compris l’anémie hémolytique auto-immune avec thrombocytopénie immune ; la thrombocytopénie allo-immune foeto-maternelle/néonatale (FMAIT/NAIT) ; le syndrome hémophagocytaire ; la transplantation de cellules souches hématopoïétiques al logéniques à haut risque ; la neuropathie paraproteinémique à IgM ; la transplantation rénale ; la sclérose en plaques ; l’ataxie Opsoclonie myoclonie ; le pemphigus foliacé ; le pemphigus vulgaire ; le purpura post-transfusionnel ; la nécrolyse épidermique toxique/le syndrome de Steven Johnson (TEN/SJS) ; le syndrome du choc toxique ; la maladie d’Alzheimer ; le lupus érythémateux disséminé ; le myélome multiple ; la sepsie ; des tumeurs à cellules B ; un traumatisme ; et une infection bactérienne, virale ou fongique. 2. Combinaison de la revendication 1, dans laquelle la quantité d’IG dans la composition est de 5 g, 10 g, 15 g, 20 g, 21 g, 22 g, 23 g, 24 g, 25 g, 26 g, 27 g, 28 g, 29 g, 30 g, 31 g, 32 g, 33 g, 34 g, 35 g, 36 g, 37 g, 38 g, 39 g ou 40 g. 3. Combinaison de la revendication 1, dans laquelle l’IG dans la composition est formulée pour une perfusion sous-cutanée par gravité, une injection ou une perfusion par pompe en une quantité qui est de 20 à 30 grammes. 4. Combinaison de la revendication 1, dans laquelle la composition d’IG est une formulation d’IG liquide à 10%. 5. Combinaison de l’une des revendications 1 à 4, dans laquelle l’hyaluronidase dans la composition est de 5000 unités à 7500 unités ou de 1000 unités à 10000 unités. 6. Combinaison de l’une des revendications 1 à 5, dans laquelle l’hyaluronidase dans la composition est présente en une quantité qui est à un rapport (unités d’hyaluronidase/grammes d’IG) de 10 U/5 grammes (g), 20 U/g, 30 U/g, 35 U/g, 40 U/g, 50 U/g, 60 U/g, 70 U/g, 80 U/g, 90 U/g, 100 U/g, 150 U/g, ou 300 U/g. 7. Combinaison de la revendication 6, dans laquelle l’hyaluronidase dans la composition est administrée à un rapport de 50 U/gramme IG. 8. Combinaison de l’une des revendications 1 à 7, dans laquelle l’hyaluronidase dans la composition est une PH20, ou l’une de ses formes tronquées. 9. Combinaison ou composition de la revendication 8, dans laquelle la PH20 humaine tronquée : conserve une activité hyaluronidase et est soluble ; et est choisie parmi des polypeptides ayant une séquence d’acides aminés présentée dans l’une des séquences SEC ID NO : 4 à 9, ou une séquence d’acides aminés comprenant au moins 91% d’identité de séquence par rapport à la séquence d’acides aminés présentée dans l’une des séquences SEQ ID NO : 4 à 9. 10. Combinaison de l’une des revendications 1 à 9, dans laquelle l’IG dans la composition est purifiée à partir de plasma humain. 11. Combinaison de l’une des revendications 1 à 10, dans laquelle l’IG dans la composition contient plus de 95% d’IgG. 12. Combinaison de l’une des revendications 1 à 11, dans laquelle la concentration de l’IG est de 6 à 15% p/v, ou de 8 à 12% p/v de la composition d’IG. 13. Combinaison de la revendication 12, dans laquelle la concentration d’IG est de 10% p/v. 14. Combinaison de la revendication 1, dans laquelle la maladie ou affection pouvant être traitée par IG est choisie parmi : une immunodéficience choisie parmi l’immunodéficience commune variable (CVID), l’agammaglobulinémie congénitale, le syndrome de Wiskott-Aldrich, l’immunodéficience combinée sévère (SCID), l’hypogammaglo-bulinémie primaire, les maladies immunodéficitaires primaires avec déficience en anticorps, l’agammaglobulinémie liée au chromosome X (XLA), l’hypogammaglobulinémie infantile, et la dégénérescence cérébelleuse paranéoplasique sans anticorps ; l’hypogammaglobulinémie acquise secondaire à des malignités hématologiques choisies parmi la leucémie lymphoïde chronique (CLL), le myélome multiple (MM) et le lymphome non hodgkinien (NHL) ; une myopathie inflammatoire choisie parmi la polymyosite, la dermatomyosite et la myosite à corps d’inclusion ; et une affection bactérienne, virale ou fongique choisie parmi l’affection par Haemophilus influenzae de type B, Pseudomonas aeruginosa de types A et B, Staphylococcus aureus, Streptococcus du groupe B, Streptococcus pneumoniae de types 1, 3, 4, 6, 7, 8, 9, 12, 14, 18, 19 et 23, Adénovirus de types 2 et 5, Cytomégalovirus, le virus d’Epstein Barr VCA, le virus de I’hepatite A, le virus de I’hepatite B, le virus Herpes simplex-1, le virus Herpes simplex-2, le virus de la grippe A, de la rougeole, le Para-influenza de types 1, 2 et 3, le Poliovirus, le virus varicelle-zona, Aspergillus et Candida albicans.

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Claims (7)

1. KöPsblrGdök és e|É%Dkimffluiigfóbiéinés loalnronidáz sztÉkmátí 'beadására szabadam: Btesmíc 1, l^í««:g!0bu!kí (IG) és oldható hiak^atááx^PÍi^éay^Éömbisiáoíója, «p$r I£r*vél felélhető betegség: vagy állapot kezelésfc egy alanyban, ahol* M IGA ás az oldliátá láaliifOPldlEt Ml^küi^föraíuliz^k tólteátllfeeáiáste;. m Ι0Φ;^,μ #Ι#ι«0\ΜιΙ«ί·0Βΐ#Ι«:;Ι^0η{^ cg>s#ei agy adagban ttm«nü tedáihoz lorrnu-llzzuk; az oldható hlalumnidázt az IG-töi küldő adjuk be. az IG beadása élői, ugyanabban a pontban, mint az KM; az IG koncentrációja 5-15 v%, és a készítményben az IG mennyisége 0,5 gramm (g) és 70 g között van; az KI 50-700 ml térfogatú folyadékban van klszerelvé; az oldható hialuromáázt jlgy' Jfairail&amp;ttfc, diogy: a hialorönldáznak az IG-hez vlszüuylfotí azÉtya 10 l/graömt (g) 500 1% IG-re számítva, miáltal a, készítményben a, Maluronldáz tnéitlylaégé: elegendő ahhoz,, liógy legalább 00%-kal megnövelje az KI biológiai hózzáíérhe-tőségét, ha az IGwel Jköufolnleióbsn: adjuk be, egyetlen IG dózis imrmmnls «tön íbítárfo leadásihoz viszonyítva, ugyanannak azIG-vel kezelhető betegségnek vagy áll apóinak a kezelésére; és m IG-véí kezelhető betegséget vagy állapptpt a következők közül váiaszthzüfok II: immun-deléiencia; szerzett hípöga.mmaglöbtdinémía rosszindulatú: hematológiai daganatok miatt; Kawasaki betegseg: krónikus, gyulladásos denbelinezö polineuropátia tCIDP}, Guillalfi-Bane szindróma, khüpátiás trombocitopeniás purpura; gyulladásos oúelopábák; Lambéria Eaton miasztemas szindróma, muitifbkaüs motoros nenropaüa; izomsorvadás; Móerséla Wöltmarm szindróma; szekunder hipogammagiobulinénia, heleGive a. jztxogéo Mimandeíl-ciánéi át ; apeeífikna ellenanyag dedeiehela* akut disszetnlnált ágyvelőgyulládái; AMCA" pozitív szisztémásinekrötízálő vaszkuliisz; autoimmun hemollilins vérszegénység; Builöus pemphigoid, Cicáiddá! pemphígoid; Evans szindróma, beleértve az autoimmun beuMdiikus vérszegénységét InununoremfooeJ'yenKnah ?Vu«maiernáiis / neomtialis alloim:mUilitöm.b^ eltopénia {EMAJT/NAÍ% tiemoiagcfoitás szindróma; nagy kockázatú ajiogén hermvmeu\n$ ov*m Pans/p ama. n\ KM t\n mu'iemmmas ^outopatu, veseátültetés; szkle- tőzh multiplex; Opsociomtft myoclonua ataxia, Fempfcigus foHaceus; Pemphigus WtigfKUg bmv.lnziö utáni purpura* toxikus epklermális nekroUzis/Stevens-dohnson szmnróma (TEN/SJS); toxikus sokk gztMrómá:;. AUhetnuar kór szisztémás iupus eryfbémafosuáí tnielónta nitdiplefo szepszis;: i~séjí tummk; kmmn M mráis fortózéá.
2. 2, Az i. igénypont szerinti kombjnleiíl, Iái kfoátménybea mtÚ mmmimm ^ g 15 g, 20 g, 21 g, 22 24 g, 25 g, 2§g, 22 g, 2S g. 29 g, 30&amp; 31 g, 32 g, 33 g, 34 g, 33 g, 36 g:1 37 g, 38 g, 39 g vagy 40 g.
3. 3, Az L igénypont szeflníi kombináció, ahol a készítményben az 1G szohkután Infúzió-hoz van kisxeínlve, grigiáclos öióA pumgás infozióval vagy injekcióval 20-30 iranM íncnnyinég-ben 4 Az L igénypont szerinti fcpnbi»á#ióí::am#|3^e«!'#? IGkonfoiMoid ^ dék !ö kiszerelési forma.
4. 5. Az 'M. igénypontok bármelyike szerinti kombináció, ahol a készítményben a biamronidáz 5000 · 7500 egység, vagy 1000 -40.000 egység. ő, Az 1 -5. igénypontok bátme lyike szeri ni kombinseió, ahol a készitnénybsm a hialto ronidáz olyan mennyiségben van jelem ahol a (hialuronidáz egység/gramm 10} arány 10 B/gramm tg), 20 B/g: 30 l/g:l 3$ E% 401¾ 50il% 00 B/g, 70 E/g, 80 E/g, 90 E/g, 100 E/g, ISO E/g, vagy 300 E/g. % A. i. igénypont szerinti konfoinániü, ahol a késxlioiénvhón :» hialotonidázt: 50 E/gramm ÍG menns bégben adjuk be. B. Az 1-7; igénypontok bármelyike szerinti bontbináníőv üköt ,$ ikészfonényhen: &amp; bialnronidáz egy Pl!2IE vagy annak ésonkiloti formája. 9. A. Ji Igénypont szeáihiiiléSÉitmény vagy komMaáóió, zhól a esottkitott: humán FIÍI20; megtart egy blaluronidlz aktivitást éa oinlafo; és azok tezol a poiipeptiílek közül választjuk ki, amelyeknél az aminoaav szekvenciáját a 4-9 szán# szekvéneia-vázlamn niutannk be, vagy az aohnosav szekvencia legalább 9Í%~oa szekvencia azondss^ot inntat bármelyik, a 4*2. atninosav szekvendáMl.
5. 10. Az 1-9. igénypontok bármelyike szerinti kombinációd tlfol &amp;'lés^ltrnéíiyberr Jogé: JPO-humán plazmából van úszóivá ! E \? i-10 igénypontok bármelyike sz^rípöombináció, ahol a készítményben levő ÍG több mint 95% IgG-t tartalmaz,
6. 12. Αχ MI. igénypoöix%MgTimi köutbintóéy á!@l az: lg koncenirádó|«A Uikvitsnm o-|A' »-a ^l'\' <-a, 13 \ ' KOi'gnisOM! ko'plMKV *'en a K? Nomcvauo* 10 v%
7. 14, Ak I. igénypont szerinti konibináeiő, ahol ázICi-vel ItoélheÉl betegséget vagy álla potot a következők közül választjuk ki: egy lmtu und ebei énei a, amelyet a következők közöl választunk ki: általános variábilis aMMintleSpeacia (CVIDk öröklött agMtmagiobuknenna. Wtsktut-Aidrtch szindróma, súlyos kombinált ttnmundeftetáácm (SC1D), primer Mpöpmötsglóbultfíitiiá, gölnes Imáim> Moienda betegségek ellenanyag áeikieneiávab X-kapcsolt agarmoagkmulmérala tXt.A}, és pmmeoplasxtllöís eerebelllrls Jegenetielé ellen- anyagok nélkül; szerzett tsipogaromaglobulinémia rosszindulatú hematológiai daganatok miatt, amelyet a következők közül választhatunk 11: kmöikös iintfoésás leukémia (CLkk mieléma multiplex (MM) es nem-Hodgkin lintfimafMíLI; gyulladáson ndopátia, amelyet a következők közül választhatunk ki. pölimmiltlsx, dermatonnoziüsz és Inlíuzlős test miozltiszy -is egy bakteriális vitális vagy gotnba^áinpot, amelpt a következo^te választhatunk ki. Hmmophiíus m/kumzae type B, Pmm^&amp;monm A ék B típss mir&amp;u&amp; Síoop Ü Streptoeoeens, Brepfmmmm pmmnűmae I, 3, 4:. 6, 7, 8, 9. 12, 14, IS, 19,. és 23 típus, Adenővirüs 2~es és 5-ös típus, CI-ioetegaiövlrua, Hpsiein Bán vírus VCA, Hepatitis A vírus, Bepauős B vlmS;: Herpes ehrspiex vírus-1, Herpes simplex virus~2, Influenza A. kanyaré^ Batainflüenza I 2, 3-as tipeg, Vancella xoster vaus, Asperpiíux és Contiuki albiccm&amp;