NZ624489B2 - Companion diagnostic for anti-hyaluronan agent therapy and methods of use thereof - Google Patents

Abstract

Disclosed is a TSG-6-link module (LM) multimer, comprising: - a first polypeptide containing a TSG-6-LM linked directly or indirectly via a linker to a first multimerisation domain; and - a second polypeptide containing a TSG-6-LM linked directly or indirectly via a linker to a second multimerisation domain, wherein: - the first and second multimerisation domains each comprise a sequence of amino acids, whereby the first and second polypeptides form a multimer; and - the TSG-6-LM multimer has a binding affinity to hyaluronan (HA) with an association constant of at least 10^7 M^-1. ation domain, wherein: - the first and second multimerisation domains each comprise a sequence of amino acids, whereby the first and second polypeptides form a multimer; and - the TSG-6-LM multimer has a binding affinity to hyaluronan (HA) with an association constant of at least 10^7 M^-1.

Description

COMPANION DIAGNOSTIC FOR YALURONAN AGENT THERAPY AND METHODS OF USE THEREOF RELATED APPLICATIONS Benefit of priority is claimed to US. Provisional Application Serial No. 61/628,187, filed October 24, 2011, entitled nion Diagnostic For Hyaluronan— Degrading Enzyme Therapy and s ofUse Thereof,” to US. Provisional Application No. ,011, filed November 11, 2011, entitled “Companion stic for Hyaluronan-Degrading Enzyme Therapy and Methods of Use Thereof,” to US. Provisional ation No. 61/630,765, filed December 16, 2011, entitled “Companion Diagnostic for Hyaluronan-Degrading Enzyme Therapy and Methods ofUse f,” and to US. Provisional Application No. 61/714,700, filed October 16, 2012, entitled nion Diagnostic for Anti—Hyaluronan Agent Therapy and Methods of Use Thereof. The subject matter of each of the above-noted applications is incorporated by reference in its entirety.

This application is related to US. Patent Application No. l3/694,071, filed the same day herewith, entitled nion Diagnostic for Anti-Hyaluronan Agent Therapy and Methods of Use Thereof,” which claims priority to US. Provisional Application Serial Nos. 61/628,187; 61/559,011; 61/630,765 and ,700.

The subject matter of the noted related application is incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their ty. The electronic file was created on October 24, 2012, is 1827 kilobytes in size, and titled 3096seqPC1.txt.

FIELD OF INVENTION Methods and diagnostic agents for identification of subjects for cancer treatment with a onan—degrading enzyme are provided. Diagnostic agents for the detection and quantification of hyaluronan in a biological sample and monitoring cancer treatment with a hyaluronan-degrading enzyme are provided. Combinations and kits for use in practicing the methods also are provided.

RECTIFIED SHEET (RULE 91) ISAIEP 2012/061743 BACKGROUND Hyaluronan-degrading enzymes have been used therapeutically, typically as dispersing and spreading agents in combination with other therapeutic agents.

Hyaluronan-degrading enzymes also can be used in single-agent therapy for the ent of hyaluronan-associated es and disorders. For example, tumors and s are associated with accumulation of hyaluronan and treatment with a hyaluronan-degrading enzyme inhibits the grth of tumor and increases vascular perfusion and es delivery of chemotherapeutic agents to the tumor. There exists a need for methods and reagents for improving treatment of patients who are treated with hyaluronan-degrading enzymes.

SUMMARY Provided herein is a method for selecting a subject for treatment of a tumor with an anti-hyaluronan agent, for example, a hyaluronan-degrading enzyme. In the provided method, a tissue or body fluid sample from a subject with a tumor or cancer is ted with a hyaluronan binding protein (HABP) that has not been prepared from or ed from animal cartilage. Binding of the hyaluronan binding protein to the sample is detected, y determining the amount of hyaluronan in the sample, wherein if the amount of hyaluronan in the sample is at or above a ermined threshold level, selecting the subject for treatment with an anti-hyaluronan agent, for example, a hyaluronan degrading enzyme.

Provided herein is a method for selecting a subject for ent of a tumor with an yaluronan agent, for example a hyaluronan-degrading enzyme, wherein a body fluid from a subject with a tumor or cancer is contacted with a hyaluronan binding protein (HABP) that has not been prepared from or isolated from animal cartilage and binding of the HABP to the sample is effected by a solid-phase binding assay with a colorimetric or fluorescent signal, thereby determining the amount of hyaluronan in the sample, wherein a subject is selected for treatment with am antihyaluronan agent, for e a hyaluronan degrading enzyme, when the predetermined threshold level is high HA. In some examples of the method, the predetermined threshold level is at least or above 0.025 ug HA/ml of sample, 0.030 ug/ml, 0.035 ug/ml, 0.040 ug/ml, 0.045 ug/ml, 0.050 ug/ml, 0.055 ug/ml, 0.060 ug/ml, 0.065 rig/ml, 0.070 ug/ml, 0.08 ug/ml, 0.09 pig/ml. 0.1 ug/ml, 0.2 rig/ml, 0.3 pg/ml or higher.

Provided herein is a method for selecting a subject for treatment of a tumor with an anti-hyaluronan agent, for example a hyaluronan—degrading enzyme, wherein a tumor tissue sample from a subject with a tumor or cancer is contacted with a hyaluronan binding n (HABP) that has not been prepared from or isolated from animal cartilage and binding of the HABP to the sample is effected by histochemistry, thereby ining the amount of hyaluronan in the sample, wherein a subject is selected for treatment with an yaluronan agent, for example a hyaluronan degrading enzyme, when the predetermined threshold level is an HA score of at least +2 (HA+2) or at least +3 (HAH). In some examples, the ermined threshold level is an HA score of at least +3 (HA8) (high levels). In other es, the predetermined threshold level is at least a percent HA positive pixels in tumor (cells and stroma) to total stain in tumor tissue of at least 10%, 10% to 25% or greater than 25%. For example, the predetermined threshold level is at least a percent HA positive pixels in tumor (cells and stroma) to total stain in tumor tissue of at least 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40%.

Also provided herein is a method for selecting a subject for ent of a tumor with an anti~hyaluronan agent, for example a hyaluronan-degrading enzyme, wherein the subject is then treated with the anti-hyaluronan agent, for example a hyaluronan-degrading enzyme. In some es, the anti-hyaluronan agent is a hyaluronan—degrading enzyme that is administered in a dosage range amount of between or about between 0.01 ug/kg (of the subject) to 50 pig/kg, 0,01 ug/kg to 20 ug/kg, 0.01 pig/kg to 15 ug/kg, 0.05 ug/kg to 10 ug/kg, 0.75 ug/kg to 7.5 gig/kg or 1.0 pig/kg to 3.0 ng/kg and a frequency of administration is twice weekly, once weekly, once every 14 days, once every 21 days or once every month. In particular examples ofthe method, a osteroid is administered prior to administration of a onan~degrading enzyme or after administration of the hyaluronan~degrading , typically in an amount sufficient to ameliorate an adverse effect in the subject from the administered hyaluronan-degrading enzyme. For example, the RECTIFIED SHEET (RULE 91) ISA/EP amount of corticosteroid administered is between at or about 0.1 to 20 mgs, 0.1 to 15 mgs, 0.1 to 10 mgs, 0.] to 5 mgs, 0.2 to 20 mgs, 0.2 to 15 mgs, 0.2 to 10 mgs, 0.2 to 5 mgs, 0.4 to 20 mgs, 0.4 to 15 mgs, 0.4 to 10 mgs, 0.4 to 5 mgs, 0.4 to 4 mgs, 1 to 20 mgs, 1 to 15 mgs or 1 to 10 mgs.

Also provided herein is a method for predicting cy of treatment of a t with an anti-hyaluronan agent, for example a hyaluronan degrading .

In the provided method, a tissue or body fluid sample from a subject who is or has been treated with an anti-hyaluronan agent, for example a hyaluronan degrading. enzyme, is contacted with a hyaluronan g protein (HABP) that has not been prepared from or isolated from animal cartilage and binding of the hyaluronan binding protein to the sample is ed, thereby determining the amount ofhyaluronan in the sample, wherein detection of a decrease in hyaluronan compared to before treatment with the anti-hyaluronan agent (e. g. hyaluronan-degrading ) or the last dose of anti-hyaluronan agent (e.g. onan—degrading enzyme) indicates that the treatment 1.5 is effective.

Also provided herein is a method for monitoring treatment of a subject with an anti-hyaluronan agent (6.g. a hyaluronan degrading enzyme). In the provided method, a tissue or body fluid sample from a subject with a tumor or cancer is contacted with a hyaluronan binding protein (HABP) that has not been prepared from or isolated from animal age and the amount ofhyaluronan binding protein that binds to the sample is detected, thereby determining the amount of hyaluronan in the sample, and the level of hyaluronan is compared to a control or reference sample to thereby determine the amount of hyaluronan in the sample relative to the control or reference sample, wherein the amount of hyaluronan is an indicator ofthe progress of treatment.

Also ed herein are methods for predicting y of treatment of a subject with an anti-hyaluronan agent (e. g. a hyaluronan degrading enzyme) and monitoring treatment of a subject with an yaluronan agent (2.g. a hyaluronan degrading enzyme) wherein treatment is altered based on the determined amount of hyaluronan in the sample relative to the control or reference sample, such that ifthe amount ofhyaluronan in the sample is at or above the amount in the control or reference sample, ent is continued or ted by increasing the dosage and/or dose schedule; or if the amount of hyaluronan in the sample is below the amount in RECTIFIED SHEET (RULE 91) ISA/EP the control or reference sample, treatment is continued, reduced by decreasing the dosage and/or dose schedule, or terminated. In some examples, the control or reference sample is a sample from a healthy subject, is a baseline sample from the subject prior to treatment with an yaluronan agent (6.g. hyaluronan-degrading enzyme) or is a sample from a subject prior to the last dose of anti-hyaluronan agent (6.g. hyaluronan-degrading enzyme). In some examples, the subject has a tumor or cancer and the sample is a tumor tissue sample and detection is effected by histochemistry. In other examples, the subject has a tumor or cancer and the sample is a body fluid and ion is effected by a solid-phase binding assay. In some examples, the solid-phase binding assay is a microtiter plate assay and binding is detected colorimetrically or via fluorescence.

In any of the methods provided herein, the step of contacting the sample with a HABP can be ed at n or about between pH 5.6 to 6.4. For example, the step of contacting the sample with a HABP is effected at a pH of about 5.8, 5.9, 6.0, 6.1 or 6.2. In some examples, the HABP specifically binds to HA with a binding affinity represented by the dissociation constant (Kd) of at least less than or less than or 1 x 10'7M, 9 x lO'SM, 8 x lO'SM, 7 x lO'SM, 6 x lO'SM, 5 x lO'SM, 4 x lO'SM, 3 x ‘8 M, 2 x 10‘8 M, 1 x 10‘8 M, 9 x 10‘9 M, 8 x 10-9 M, 7 x 10-9 M, 6 x 10‘9 M, 5 x 10-9 M, 4 x 10-9 M, 3 x 10-9 M, 2 x 10‘9 M, 1 x 10'9M or lower Kd.

In any of the methods provided herein, the HABP can be generated recombinantly or synthetically. In some examples, the HABP contains a link module.

In other examples, the HABP ns two or more link s. In further examples, the link module or modules are the only HABP portion of the molecule.

Thus, provided herein are methods wherein the HABP ns a link module selected from among CD44, LYVE-l, HAPLNl/link protein, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, Stabilin-l, Stabilin-2, CAB6l358 and KIAAO527. In some examples of the method, the HABP contains a n of a CD44, LYVE-l, HAPLNl/link protein, , , , aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, Stabilin-l, Stabilin-2, CAB6l358 or KIAAO527 including a link module or a sufficient portion of a link module to bind HA. In a particular example, the HABP is a tumor necrosis -stimulated Gene (TSG-6) protein or a link module of TSG-6 or a sufficient portion of a link module of TSG-6 to bind HA. In other examples of the method herein, the HABP ns a G1 domain of a type C hyaluronan binding protein, for example, a G1 domain selected from among Aggrecan G1, Versican G1, Neurocan G1 and BreVican G1. In particular examples, the G1 domain is the only HABP portion of the molecule.

In some examples of the methods provided herein, the HABP contains the sequence of amino acids set forth in any of SEQ ID NOS: 207, 222, 360, 361, 371- 394, 416-418 and 423-426 or has a ce of amino acids that ts at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or more sequence ty to a sequence of amino acids set forth in any of SEQ ID NOS: 207, 360, 361, 371-394, 416-418 and 423-426 and specifically binds HA, or is an HA- binding domain thereof or a sufficient portion thereof to specifically bind to HA. In an exemplary example, the HABP contains a TSG-6 link module (LM) or a sufficient portion thereof that specifically binds HA. For example, the TSGLM ns the sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 418, or a ce of amino acids comprising at least 65% amino acid sequence identity to the sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 418 and specifically binds HA. In specific examples of the method, the HABP contains a link module set forth in SEQ ID NO:207 or a sequence of amino acids comprising at least 65% amino acid sequence identity to the ce of amino acids set forth in SEQ ID NO:207 and specifically binds HA. In other examples, the HABP contains a link module that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 418, whereby the HABP specifically binds HA.

In some examples of the method provided herein, the TSG-6 link module is modified to reduce or eliminate binding to heparin. For example, binding to heparin is reduced at least 1.2-fold, ld, 2-fold, 3-fold, 4-fold, , 6-fold, 7-fold, 8- fold, 9-fold, d, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold or more. In some examples of the method provided herein, TSG-6 link module contains an amino acid replacement at an amino acid position ponding to amino acid residue 20, 34, 41, 54, 56, 72 or 84 set forth in SEQ ID NO:360, whereby a corresponding amino acid residue is identified by alignment to a TSGLM set forth in SEQ ID NO:360. For example, the amino acid ement is in a TSGLM set forth in SEQ ID NO:207 and the amino acid replacement or ements is at amino acid residue 21, 35, 42, 55, 57, 73 or 85. The amino acid replacement can be to a non—basic amino acid residue selected from among Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Ala (A), Val (V), Ile (I), Leu (L), Met (M), Phe (F), Tyr (Y) and Trp (W). In a further example, the TSG—6 link module contains an amino acid replacement corresponding to amino acid replacement KZOA, K34A or K41 A in a TSG—6—LM set forth in SEQ ID NO:360 or the replacement at the corresponding e in another TSG—6—LM. In another example, the TSG-6 link module contains amino acid replacements corresponding to amino acid replacements KZOA, K34A and K41A in a TSG—6~LM set forth in SEQ ID NO:36O or the replacement at the corresponding residue in another TSGLM. For example, the HABP contains a link module set forth in SEQ ID NO:361 or 416 or a sequence of amino acids comprising at least 65% amino acid sequence identity to the sequence of amino acids set forth in SEQ ID N02361 or 416 that specifically binds HA.

In some examples of the methods provided herein, the I-IABP contains a link module that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids set forth in SEQ ID NO:361 or 416, whereby the HABP cally binds HA. In ular es, the HABP contains a link module set forth in SEQ ID NO:361 or SEQ ID NO:416. In other examples, the link module is the only TSG~6 portion of the HABP.

In some examples ofthe methods ed herein, the HABP is a multimer containing a first HA—binding domain linked directly or indirectly via a linker to a multimerization domain and a second HA-binding domain linked directly or indirectly via a linker to a multimerization domain. For example, the PIA-binding domain is a link module or a G1 domain. The first and second PIA-binding domain can be the same or different. In a particular example, the first and second HA-binding domain is a TSG-6 link module, a variant thereof or a sufficient portion thereofthat specifically binds to HA. For example, —6—LM contains a sequence of amino acids set forth in SEQ ID NO: 207, 360, 36], 416, 417 or 418 or a sequence of amino acids comprising at least 65% amino acid sequence identity to the sequence of amino acids set forth in SEQ ID NO: 207, 360, 361, 416, 417 or 418 that specifically binds RECTIFIED SHEET (RULE 91) ISA/EP HA. For example, the link module exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids set forth in SEQ ID NO: 207, 360, 361, 416, 417 or 418, whereby the HABP cally binds HA. In some methods provided herein, the link module contains a sequence of amino acids set forth in SEQ ID NO: 207, 360, 361, 416, 417 or 4 1 8.

In the provided s, the HABPS, HA-binding domains, link modules or portions thereof can be linked to a multimerization domain that is selected from among an immunoglobulin constant region (Fe), a leucine zipper, complementary hydrophobic s, complementary hydrophilic regions, compatible protein~protein interaction domains, free thiols that form an intermolecular disulfide bond between two molecules, and a protuberance—into—cavity and a satory cavity of identical or similar size that form stable multimers. In a particular example, the multimerization domain is an Fe domain or a variant thereof that effects multimerization. For example, the Fe domain is from an IgG, IgM or an IgE, or the Fe domain has a sequence of amino acids set forth in SEQ ID NO:359. In some ces of the methods provided , the HABP is a fusion protein that contains a TSG—6 link module and an globulin Fe domain. For example, the HABP is TSG—6-LM-Fc that has a sequence of amino acids set forth in SEQ ID NO: 212 or a sequence of amino acids that ts at least 65% amino acid sequence identity to SEQ ID NO:212 and specifically binds HA, such as a sequence of amino acids that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids set forth in SEQ ID N022 12, whereby the HABP specifically binds HA. In particular examples, the HABP has a sequence of amino acids set forth in SEQ ID NO:212 or 215. In any of the s provided herein, the HABP can be LM-Fc/AHep that has a sequence of amino acids set forth in SEQ ID NO: 215 or a sequence of amino acids that exhibits at least 65% amino acid sequence identity to SEQ ID NO:215 and specifieallyqbinds HA, such as a sequence of amino acids that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids set forth in SEQ ID NO:215, whereby the HABP specifically binds HA.

RECTIFIED SHEET (RULE 91) ISA/EP In particular methods provided herein, the HABP is a TSG-6 or hyaluronan- binding region thereof. In some examples of the methods provided herein, the HABP or TSG-6 has a binding affinity to HA of at least 1 x 108 M'l, 2 x 108 M'l, 3 x 108 M'l, 4 x 108M'1, 5 x 108M'1,6 x ,7 x ,8 x 108M'1, 9 1,lx109M'1 or higher. In other examples, the HABP or TSG-6 is conjugated to a detectable moiety that is detectably labeled or that can be detected. For example, the HABP or TSG-6 is biotinylated.

In some examples of the methods provided herein the sample is a stromal tissue sample, such as a stromal tissue sample from a tumor. The tissue sampled in the methods herein can be fixed, paraffin-embedded, fresh, or frozen. In some examples, the sample is taken from a biopsy from a solid tumor, for example, obtained by needle biopsy, CT-guided needle biopsy, aspiration biopsy, endoscopic biopsy, oscopic biopsy, bronchial lavage, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy, skin biopsy, bone marrow biopsy, and the Loop Electrosurgical on Procedure (LEEP). In other examples, the sample is a fluid sample that is a blood, serum, urine, sweat, semen, saliva, cerebral spinal fluid, or lymph sample. In any of the methods provided herein the sample can be obtained from a . In an exemplary example, the mammal is a human.

In any of the methods provided herein, the tumor can be of a cancer selected from among breast cancer, pancreatic cancer, ovarian cancer, colon cancer, lung cancer, all cell lung cancer, in situ carcinoma (ISC), us cell carcinoma (SCC), thyroid cancer, cervical cancer, uterine cancer, prostate cancer, testicular cancer, brain cancer, bladder cancer, stomach cancer, hepatoma, melanoma, glioma, retinoblastoma, mesothelioma, myeloma, lymphoma, and leukemia. In some examples, the tumor is of a cancer that is a late-stage cancer, a metastatic cancer and an undifferentiated cancer.

In any of the methods provided herein, the anti-hyaluronan agent can be an agent that degrades hyaluronan or can be an agent that inhibits the sis of hyaluronan. For example, the anti-hyaluronan agent can be a onan ing enzyme. In another example, the anti-hyaluronan agent or is an agent that inhibits onan synthesis. For e, the anti-hyaluronan agent is an agent that inhibits hyaluronan synthesis such as a sense or antisense c acid molecule against an HA synthase or is a small molecule drug. For example, an yaluronan agent is 4- methylumbelliferone (MU) or a derivative thereof, or leflunomide or a tive thereof. Such derivatives include, for example, a tive of 4-methylumbelliferone (MU) that is hydroxymethyl coumarin or 5,7-dihydroxymethyl coumarin.

In fiarther examples of the methods provided herein, the hyaluronan degrading enzyme is a hyaluronidase. In some examples, the hyaluronan-degrading enzyme is a PH20 hyaluronidase or truncated form thereof to lacking a C-terminal glycosylphosphatidylinositol (GPI) attachment site or a portion of the GPI attachment site. In specific es, the hyaluronidase is a PH20 selected from a human, , bovine, ovine, rat, mouse or guinea pig PH20. For example, the hyaluronan- degrading enzyme is a human PH20 hyaluronidase that is neutral active and N- glycosylated and is ed from among (a) a hyaluronidase polypeptide that is a hilllength PH20 or is a C-terminal truncated form of the PH20, wherein the truncated form includes at least amino acid es 36-464 of SEQ ID NO:1, such as 36-481, 36-482, 36-483, where the full-length PH20 has the sequence of amino acids set forth in SEQ ID NO:2; or (b) a hyaluronidase polypeptide comprising a sequence of amino acids having at least 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 9l %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with the polypeptide or truncated form of sequence of amino acids set forth in SEQ ID NO:2; or (c) a hyaluronidase polypeptide of (a) or (b) comprising amino acid substitutions, whereby the hyaluronidase polypeptide has a sequence of amino acids having at least 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 9l %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence ty with the polypeptide set forth in SEQ ID NO:2 or the with the corresponding truncated forms f. In exemplary examples, the hyaluronan- degrading enzyme is a PH20 that comprises a composition designated rHuPH20.

In other examples, the anti-hyaluronan agent is a hyaluronan degrading enzyme that is d by conjugation to a polymer. The polymer can be a PEG and the anti-hyaluronan agent a PEGylated hyaluronan degrading enzyme. Hence, in some examples of the methods provided herein the hyaluronan-degrading enzyme is modified by conjugation to a polymer. For example, the hyaluronan-degrading enzyme is conjugated to a PEG, thus the hyaluronan degrading enzyme is PEGylated.

In an exemplary example, the hyaluronan-degrading enzyme is a PEGylated PH20 WO 63155 enzyme (PEGPH20). In the methods provided herein, the corticosteroid can be a glucocorticoid that is selected from among cortisones, dexamethasones, hydrocortisones, prednisolones, prednisolones and prednisones.

Also provided herein is a kit containing a hyaluronan binding agent (HABP) for detecting the amount of hyaluronan in a sample, n the HABP has not been prepared from animal cartilage and a hyaluronan-degrading enzyme. The HABP can be generated recombinantly or tically. In some examples, the HABP contains one link module. In other examples, the HABP contains two or more link modules.

In some examples, the link module or modules are the only HABP portion of the le. For example, the HABP contains a link module selected from among CD44, LYVE-l, HAPLNl/link protein, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan, breVican, phosphacan, TSG-6, Stabilin-l, Stabilin-2, CAB61358 and KIA0527 or a portion thereof comprising a link module or a sufficient portion of a link module to bind HA. In other examples, the HABP contains a G1 domain of a type C hyaluronan g protein, for example, a G1 domain selected from among an G1, Versican G1, Neurocan G1 and BreVican G1. In particular examples, the G1 domain is the only HABP portion of the molecule.

In some examples, the kit contains a HABP containing the sequence of amino acids set forth in any of SEQ ID NOS: 207, 222, 360, 361, 371-394 and 416-418, and 423-426 or a ce of amino acids that exhibits at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a ce of amino acids set forth in any of SEQ ID NOS: 207, 360, 361, 371-394, 416-418 and 423-426 and specifically binds HA, or an HA-binding domain thereof or a sufficient portion f to specifically bind to HA. In an exemplary example, the HABP contains a TSG-6 link module (LM) or a sufficient portion thereof that specifically binds HA. For example, the TSGLM contains the sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 418, or a sequence of amino acids comprising at least 65% amino acid sequence identity to the sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 418 and specifically binds HA. In some examples, the HABP contains a link module that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids set forth in SEQ ID NO:360, .12- whereby the HABP binds HA. In particular examples, the HABP contains a link module set forth in SEQ ID NO: 207, 360, 417 or 418.

In some examples of the kits ed herein, the TSG—6 link module is modified to reduce or eliminate binding to heparin. For example, binding to n is reduced at least d, 15-fold, 2-fold, 3-fold, , 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, d, 20-fold, 30-fold, 40-fold, 50-fold, lob-fold or more. In some examples, TSG-6 link module contains an amino acid replacement at an amino acid position corresponding to amino acid residue 20, 34, 41, S4, 56, 72 or 84 set forth in SEQ ID NOz360, whereby a corresponding amino acid residue is identified by ent to a TSG-6—LM set forth in SEQ ID NO:360. For e, the amino acid replacement is~ to a non-basic amino acid residue selected from among Asp (D), Glu (B), Set (S), Thr (T), Asn (N), Gln (Q), Ala (A), Val (V), lie (1), Leu (L), Met (M), Phe (F), Tyr (Y) and Trp (W). Thus, provided herein is a kit wherein the TSG-6tlink module contains an amino acid replacement corresponding to amino acid replacement K20A, K34A or K41A in a TSGLM set forth in SEQ ID NO:36O or the replacement at the corresponding residue in another TSGLM. For example, the TSG-6 link module contains amino acid replacements corresponding to amino acid replacements K20A, K34A and K41A in a TSG—6-LM set forth in SEQ ID NO:36O or the replacement at the corresponding residue in another LM. Also provided herein, are kits wherein the HABP contains a link module set forth in SEQ ID N01361 or 416 or a sequence of amino acids comprising at least 65% amino acid sequence identity to the sequence of amino acids set forth in SEQ ID NO:361 or 416 that specifically binds HA, For example, the HABP contains a link module that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence ty to the sequence of amino acids set forth in SEQ ID N0:361 or 416, whereby the HABP specifically binds HA. In ular examples, the HABP contains a link module set forth in SEQ ID —NO:361 or 416. In other examples, the link module is the only TSG—6 portion of the HABP.

In some examples ofthe kits provided herein, the HABP is a multimer containing a first PIA-binding domain linked directly or indirectly via a linker to a multimerization domain and a second nding domain linked directly or indirectly via a linker to a multimerization domain. For example, the HA-binding domain is a RECTIFIED SHEET (RULE 91) ISA/EP -13.. link module or a G1 . The first and second HA-binding domain can be the same or ent. In a ular example, the first and second HA-binding domain is a TSG-6 link module, a variant thereof or a sufficient portion f that specifically binds to HA. For example, the TSG—6-LM contains a ce of amino acids set forth in SEQ ID NO: 207, 360, 361, 416, 417 or 418 or a sequence of amino acids comprising at least 65% amino acid sequence identity to the sequence of amino acids set forth in SEQ ID NO: 207, 360, 361, 416, 417 or 418 that specifically binds HA.

For example, the link module exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids set forth in SEQ ID NO: 207, 360, 361, 416, 417 or 418, whereby the HABP specifically binds HA. In some methods ed herein, the link module contains a ce of amino acids set forth in SEQ ID NO: 207, 360, 361, 416, 417 or 418.

In the provided kits, the HABPs can be linked by a multimerization domain that is selected fiom among an immunoglobulin constant region (Fe), a leucine zipper, complementary hydrophobic regions, complementary hydrophilic regions, compatible protein-protein interaction domains, free thiols that form an intermolecular disulfide bond between two mo1oculoa, and a protuberance into cavity and a oomponaatory cavity of identical or similar size that form stable multimers. In a particular example, the multimerization domain is an Fc domain or a variant thereof that effects multimerization. For example, the Fc domain is from an IgG, IgM or an IgE, or the Fc domain has a sequence of amino acids set forth in SEQ ID N01359. In some instances of the methods provided herein, the HABP is a fusion protein that contains a TSG-6 link module and an innnunoglobulin Fc . For example, the HABP is TSG-6—LM-Fc that has a sequence of amino acids set forth in SEQ ID NO:212 or 215 or a ce of amino acids that exhibits at least 65% amino acid sequence identity to SEQ ID NO:212 or 215 and specifically binds HA, such as a sequence ofamino acids that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids set forth in SEQ ID NO:212 or 215, whereby the HABP specifically binds HA. In particular ‘20 examples, the HARP has a sequence nt'aminn acids set forth in SEQ ID N012] '2 (W 215. In any of the methods provided herein the, HABP can be TSGLM-Fc/AHep that has a ce of amino acids set forth in SEQ ID NO: 215 or a sequence of RECTIFIED SHEET (RULE 91) ISA/EP amino acids that ts at least 65% amino acid sequence identity to SEQ ID NO:215 and specifically binds HA, such as a sequence of amino acids that exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence ty to the sequence of amino acids set forth in SEQ ID NO:215, whereby the HABP specifically binds HA.

In particular examples of kits provided herein, the HABP is a TSG-6 or- hyaluronan—binding region thereof. In some examples of the methods provided herein, the HABP has a binding y, represented by the association constant (Ka), to HA of at least 107 M'I, for example, at least 1 x 108 M‘l, 2 x 108M'1, 3 x 108 M", 4 x108M'1,5 x 103M“, 6 x 108M", 7 x 103M“, 8 x 103M", 9 x108M'1,1x109M'10r higher, For example, the HABP has a binding affinity, represented by the dissociation . constant (Kd), to HA of at least less than or less than 1 x 10‘7 M, 9 x 10'8 M, 8 x 10‘8 M, 7 x10'8M,6x10'8M,5 x 10‘8M, 4x 10'8M, 3 x10'8M,2x10‘8M,1x10'8M,9 x 10‘9M, 8 x WM, 7 x 10'9M, 6 x 10'9M, 5 x 10'9 M, 4 x 10'9M, 3 x 10‘9M, 2 x10'9 M, l x 10’9 M or lower Kd. In other examples, the HABP is conjugated to a detectable moiety that is detectably labeled or that can be detected. For example, the HABP. is biotinylated.

Also provided herein are kits containing a onan binding agent (HABP) for detecting the amount of hyaluronan in a sample, wherein the HABP has not been prepared from animal age and an anti-hyaluronan agent (ag. a hyaluronan— ing enzyme). Any of the kits provided herein can further contain reagents for detection of the HABP. In any e of the kits ed herein, the anti- hyaluronan agent can be any described above or elsewhere herein. For example, the yaluronan agent can be a hyaluronan degrading enzyme such as a hyaluronidase.

For example, the hyaluronan.-degrading enzyme is a PHZO hyaluronidase or ted form thereof lacking a C»terminal glycosylphosphatidylinositol (GPI) attachment site or a portion of the GPI attachment site. In some examples, the PH20 is selected from a human, monkey, bovine, ovine, rat, mouse or guinea pig PH20. For example, the hyaluronan-dcgrading enzyme is a human PH20 hyaluronidase that is neutral active and N-glycosylated and is selected from among (a) a hyaluronidase polypeptide that is a full-length PH20 or is a C-terminal truncated form of the PH20, wherein the truncated form includes at least amino acid residues 36-464 of SEQ ID N021, wherein RECTIFIED SHEET (RULE 91) ISA/EP the full-length PH20 comprises the sequence of amino acids set forth in SEQ ID N02; or (b) a hyaluronidase polypeptide comprising a sequence of amino acids having at least 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with the polypeptide or truncated form of sequence of amino acids set forth in SEQ ID N02; or (c) a hyaluronidase polypeptide of (a) or (b) comprising amino acid substitutions, whereby the hyaluronidase polypeptide has a sequence of amino acids having at least 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 9l %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity with the polypeptide set forth in SEQ ID NO:2 or the with the corresponding truncated forms thereof. In particular examples, the onan- degrading enzyme is a PH20 that comprises a composition designated rHuPH20. In some es, the hyaluronan-degrading enzyme is modified by conjugation to a polymer, such as, for example, a PEG, and the onan degrading enzyme is PEGylated. Therefore provided herein is a kit wherein the hyaluronan-degrading enzyme is a PEGylated PH20 enzyme (PEGPH20). Also provided herein are kits that further contain a corticosteroid. Any of the kits provided herein can r contain a label or e insert for use of its components.

Provided herein are methods of use of a hyaluronan binding n (HABP) for selecting a subject for treatment of a tumor with an anti-hyaluronan agent (6.g. a hyaluronan-degrading enzyme), wherein the HABP has not been prepared or isolated from animal cartilage. Also ed herein are pharmaceutical itions containing a hyaluronan binding protein (HABP) for use in selecting a subject for treatment of a tumor with an anti-hyaluronan agent (6.g. a hyaluronan-degrading enzyme), wherein the HABP has not been prepared or isolated from animal cartilage.

Provided herein are methods of use of a hyaluronan binding protein (HABP) for predicting efficacy of ent of a subject with an anti-hyaluronan agent (6.g. a hyaluronan-degrading enzyme), wherein the HABP has not been ed or isolated from animal cartilage. Also provided herein are ceutical compositions containing a hyaluronan binding protein (HABP) for predicting efficacy of ent of a subject with an anti-hyaluronan agent (6.g. a hyaluronan-degrading enzyme), wherein the HABP has not been prepared or isolated from animal cartilage.

In any of the uses or pharmaceutical compositions provided herein, the HABP can n a link module or modules or a G1 domain. In some examples, the HABP contains a TSG-6 link module (LM), a variant thereof or a ent portion thereof that binds HA. In a particular example, the TSG-6 link module is modified to reduce or eliminate binding to n. In some examples, the HABP contains a sequence of amino acids set forth in any of SEQ ID NOS: 207, 212, 215, 222, 360, 361, 371-394 and 416-418, and 423-426 or a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a ce of amino acids set forth in any of SEQ ID NOS: 207, 212, 215, 222, 360, 361, 371-394 and 8, and 423-426 and specifically binds HA, or an HA-binding domain thereof or a sufficient portion thereof to specifically bind to HA.

Also provided herein is a TSGLM multimer containing a first link module linked directly or indirectly via a linker to a multimerization domain and a second link module linked directly or indirectly via a linker to a multimerization domain, wherein the first and second polypeptide do not comprise the full-length sequence of TSG-6.

In some examples, the link module is the only TSG-6 portion of the first polypeptide and the second polypeptide. The first and second link module can be the same or ent. In some examples, the link module contains a sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 418 or a sequence of amino acids comprising at least 65% amino acid sequence identity to the sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 418 that specifically binds HA. For example, the link module exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 418 that cally binds HA. In some examples, the link module contains a sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 41 8.

In some examples, the TSG-6 link module is modified to reduce or ate binding to heparin. Binding to heparin can be reduced at least 1.2-fold, 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40- fold, 50-fold, 100-fold or more. In some examples, the TSG-6 link module ns an amino acid replacement at an amino acid on corresponding to amino acid residue 20, 34, 41, 54, 56, 72 or 84 set forth in SEQ ID , whereby a corresponding amino acid residue is fied by alignment to a TSG—6—LM set forth in SEQ ID N02360. For example, the amino acid replacement is in a LM set forth in SEQ ID NO:207 and the amino acid replacement or replacements is at amino acid e 21, 35, 42, 55, 57, 73 or 85. In some examples, the amino acid replacement is to a non-basic amino acid residue selected from among Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Ala (A), Val (V), lie (I), Leu (L), Met (M), Phe (F), Tyr (Y) and Trp (W). For example, the TSG—6 link module contains an amino acid replacement corresponding to amino acid replacement K20A, K34A or K4lA in a TSG—6~LM set forth in SEQ ID NO:360 or the replacement at the corresponding residue in another TSG-6—LM. In a particular example, the TSG—6 link module contains amino acid replacements corresponding to amino acid replacements KZOA, K34A and K41A in a TSGLM setforth in SEQ'ID NO:360 or the replacement at the corresponding residue in another TSG—6—LM. In some es, the link module contains a sequence of amino acids set forth in SEQ ID NO: 361 or 416 or a sequence of amino acids sing at least 65% amino acid sequence identity to the sequence of amino acids set forth in SEQ ID NO: 361 or 416 that specifically binds HA. For example, the link module exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of amino acids set forth in SEQ ID NO: 361 or 416 and specifically binds HA. In particular examples, the link module contains a sequence of amino acids set forth in SEQ ID NO: 361 or 416.

Also provided herein is a TSG—6-LM multimer wherein the multimerization domain is selected from among an immunoglobulin constant region (Fc), a leucine , complementary hydrophobic regions, complementary hilic regions, ible protein-protein interaction domains, free thiols that forms an intermolecular disulfide bond between two molecules, and a protuberance-into-cavity and a compensatory cavity of identical or similar size that form stable multimers. In some examples, the multimerization domain is an F0 domain or a variant thereof that effects multimerization. For example, the Fc domain is from an IgG, IgM or an IgE.

In a particular example, the Fc domain has a sequence of amino acids set forth in SEQ ID NO:359.

RECTIFIED SHEET (RULE 91) ISA/EP Also provided herein is a TSG—6-LM multimer ning a TSG-6 link module and an immunoglobulin Fc domain. In some examples, the TSG—6-LM multimer contains a sequence of amino acids set forth in SEQ ID NO: 212 or 215 or a ce of amino acids that exhibits at least 65% amino acid ce identity to SEQ ID NO:212 or 215. For example, the TSG—6 multimer contains a sequence of amino acids that exhibits at least , 75%, 80%, 850/, o, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ce identity to the sequence of amino acids set forth in SEQ ID NO:212 or 215 and that specifically binds HA. In particular examples, the TSGLM multirner contains a sequence of amino acids set forth in SEQ ID N02212 or 215. In some examples, the LM er has a binding affinity to HA of at least 107 M", for example at least 1 x 103M'1,2 x 108 M", 3 x 108 M'1,4 x 103M“, 5 x 108 M", 6 x103 M'1,7 x108 M", s x108M‘1,9 x108 M‘1,1x 109 M"1 or lower.

Also provided herein are methods of ing a subject, predicting efficacy and/or monitoring treatment using any of the above provided HABP to detect HA by in vivo imaging methods. The in vivo g method can be magnetic resonance imaging (MRI), single—photon emission computed tomography (SPECT), computed tomography (CT), computed axial tomography (CAT), electron beam computed tomography (EBCT), high resolution computed tomography (HRCT), hypocycloidal tomography, positron emission tomography (PET), scintigraphy, gamma camera, a [3+ detector, at 7 detector, fluorescence imaging, low-light imaging, , and/or bioluminescence imaging. In such methods, the HABP is conjugated, directly or indirectly, to a moiety that provides a signal or s a signal that is detectable in vivo.

DETAILED DESCRIPTION Outline A. DEFINITIONS B. HYALURONAN BINDING PROTEIN AND COMPANION DIAGNOSTIC 1. Hyaiuronan Accumulation in Disease And Correlation to Prognosis 2. Therapy of Tumors with An Anti-Hyaluronan Agent (e.g. Hyaluronan- Degrading Enzyme) and Responsiveness to Treatment 3. Hyaluronau Binding Proteins ) Reagent and Diagnostic 4. Companion Diagnostic and Prognostic Methods C. HYALURONAN BINDING PROTEINS (HABPs) FOR USE AS A COMPANION DIAGNOSTIC 1. HA g Proteins with Link Modules or G] domains RECTIFIED SHEET (RULE 91) ISA/EP WO 63155 PCT/U82012/061743 a. Type A: TSG-6 sub-group i. TSG-6 ii Stabilinl and Stabilin-Z b. Type B: CD44 sub-group i. CD44 ii LYVE-I c. Type C: Link Protein sub-group i. HAPLN/Link Protein family (1) HAPLNl (2) HAPLNZ (3) HAPLN3 (4) HAPLN4 (S) Aggrecan (6) Brevican (.7) Versican (8) an (9) Phosphacan 2. HA Binding Proteins Without Link Modules a. HABPI/CIQBP b. Layilin c. M d. Others 3. Modifications ofHA Binding Proteins a. Multimers of HABP i. Peptide Linkers ii. Heterobit‘unetional linking agents iii. Polypeptide Multimerization domains (1) Immunoglobulin domain (a) Fe domain (2) Leucine Zipper (3) n-Protein Interaction between Subunits iv. Other multimerization domains b. Mutations to Improve HA Binding c. Modifications of HA Binding Proteins for Detection i. ation to able Proteins or es 4. Selection of HA g Proteins for Diagnostic Use ASSAYS AND CLASSIFICATION l. Assays for Measuring Hyaluronan a. hemicai and Immunohistochemical Methods 40 b. Solid Phase Binding Assays c. In vivo Imaging Assays 2. Classification of Subjects TREATMENT OF SELECTED SUBJECT WITH AN ANTI- HYALURONAN AGENT 45 1. Anti-Hyaiuronan Agent a. Agents that Inhibit Hyaluronan Synthesis b. Hyaluronan-degrading Enzymes i. Hyaluronidases (I) Mammalian-type hyaluronidases 50 (a) PHZO (2) Other hyaluronidases (3) Other hyaluronan degrading enzymes ii. Soluble hyaluronan-degrading enzymes (1) Soluble Human PH20 55 (2) rHuPH20 iii. Glycosyiation of hyaluronan-degrading enzymes RECTIFIED SHEET (RULE 91) ISA/EP _ 20 _ iv. Modified (Polymer-Conjugated) onan degrading enzymes 2. Pharmaceutical Compositions and Formulations 3. Dosages and Administration a. Administration of a PEGylated onan-degrading enzyme 4. Combination Treatments F. METHODS OF PRODUCING NUCLEIC ACIDS AND ENCODED POLYPEPTIDES OF HYALURONAN-DEGRADING ENZYMES AND HYALURONAN BINDING PROTEINS 1. Vectors and Cells 2. Expression a Prokaryotic Cells b Yeast Cells c. Insect Cells d ian Cells e Plants 3. Purification Techniques 4. PEGylation of Hyaluronan-degrading Enzyme Polypeptides G. METHODS OF ASSESSING ACTIVITY AND MONITORING EFFECTS OF ANTI-HYALURONAN AGENTS 1. Methods to Assess Side Effects 2. Evaluating Biomarkers Associated With Activity of an Anti- Hyaluronan Agent (e.g. Hyaluronan-Degrading Enzyme Activity) a. Assays to assess the activity of a Hyaluronan Degrading Enzyme b. Measurement of HA catabolites c. Tumor metabolic activity d. Increased apparent diffusion and enhanced tumor perfusion 3. Tumor Size and Volume 4. Pharmacokinetic and Pharmacodynamic Assays H. KITS AND ARTICLES OF MANUFACTURE I. EXAMPLES A. DEFINITIONS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is ly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, K sequences, websites and other published als referred to throughout the entire disclosure , unless noted otherwise, are incorporated by 40 reference in their entirety. In the event that there is a plurality of definitions for terms , those in this section l. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but lent information is known and can be y accessed, such as by searching the internet and/or appropriate 45 databases. nce thereto evidences the availability and public dissemination of such information. 2012/061743 As used herein, a companion diagnostic refers to a diagnostic method and or reagent that is used to identify subjects tible to treatment with a particular treatment or to monitor treatment and/or to fy an effective dosage for a subject or sub-group or other group of ts. For purposes herein, a companion diagnostic refers to reagents, such as modified TSG-6 proteins, that are used to detect hyaluronan in a sample. The companion diagnostic refers to the reagents and also to the test(s) that is/are performed with the reagent.

As used herein, hyaluronan (HA; also known as hyaluronic acid or onate) refers to a naturally occurring polymer of repeated disaccharide units of ylglucosamine and D-glucuronic acid. Hyaluronan is ed by certain As used herein, “high HA” with reference to the amount or level ofHA in a tissue or body fluid sample refers to the degree or extent ofHA in the tissue or body fluid sample as compared to a normal or healthy tissue or body fluid sample. The amount ofHA is high if the amount is at least or at least about 2.5-fold, 3-fold, , -fold, 6-fold, 7-fold, 8-fold, 9-fold, lO-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60- fold, 70-fold or higher than the amount or level ofHA in a corresponding normal or healthy tissue. It is understood that the amount ofHA can be determined and quantitated or semi-quantitated using methods such as phase binding assays or histochemistry. For example, the amount can be based on comparison of plasma levels or comparison of staining intensity (6.g. percent positive pixels) as determined by histochemistry. For example, high HA exists if the HA score by histochemistry or other method is HA+3 and/or if there is HA staining over 25% of tumor section. For e, high HA exists if there is a ratio of strong positive stain (such as brown stain) to the sum of total stained area that is more than 25% strong positive stain to total stain the tumor tissue.

As used herein, an HA score refers to a semi-quantitative score ofHA positivity levels on cell members and stroma of tumors. The score can be determined by detection ofHA in tumor tissue, such as formalin-fixed and paraffin-embedded tissue, by hemistry methods, such as immunohistochemistry or pseudo immunohistochemistry methods, for HA using an HABP. The degree of stain on cells and stroma can be determined visually under a microscope or by available computer -22.. thm programs and sofiware. For example, images can be quantitatively analyzed using a pixel count thm for HA stain (e. g. Aperio Spectrum Software and other standard methods that measure or quantitate or semi-quantitate the degree of staining). A tumor is graded or scored as HAHigh (HA8) at strong HA staining over % oftumor section; as HAM‘mlm"e (HA+2) at strong HA staining between 10 and % oftumor n; and as HAL°W)HA+1) at strong HA staining under 10% of tumor section. For example, a ratio of strong positive stain (such as brown stain) to the sum of total stained area can be calculated and scored, where if the ratio is more than 25% strong positive stain to total stain the tumor tissue is scored as HA”, if the ratio is 10-25% of strong positive stain to total stain the tumor tissue is scored as HA”, if the ratio is less than 10% of strong ve stain to total. stain the tumor tissue is scored as HA“, and if the ratio of strong positive stain to total stain is 0 the tumor tissue is scored as 0. The Aperio method, as well as software therefor, are known to those of skill in the art (see, e. g. ,U.S. Patent No. 8,023,714; US. Patent No.7,257,268). , .

As used herein, a hyaluronan binding n (HA binding protein; HABP) or hyaladherin refers to any protein that specifically binds to HA to permit detection of the HA. The binding affinity is one that has as an association constant Ka that is at least about or is at least 107 M‘l. For the methods and companion diagnostic products provided herein, the HA binding protein is a recombinantly produced or synthetic protein(s), not a protein derived from a biological source or physiologic source, such as cartilage. HA binding proteins include HA binding domains, including link modules that bind to HA and ent portions thereof that specifically binds to HA to permit detection thereof. Hence, HABPs include any protein that contains a onan binding region or domain or a sufficient portion f to specifically bind HA. Exemplary hyaluronan binding regions are link s (link domains) or G1 domains. A sufficient portion includes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or more contiguous amino acids of a g domain or link module. HA binding proteins also include fusion proteins containing an HA binding protein and one or more additional polypeptides, including multimerization domains. Exemplary HA g proteins include but are not limited to an, versican, neurocan, brevican, phosphacan, TSG—6, TSG-6 mutants, such as those ed herein, RECTIFIED SHEET (RULE 91) ISA/EP including polypeptides containing HA binding domains and link modules thereof that bind to HA.

As used herein, hyaluronan-binding domain or HA-binding domain refers to a region or domain of an HABP polypeptide that specifically binds to onan with a binding y that has as an association constant Ka that is at least about or is at least 106 M'1 or 107 M'1 or greater or a dissociation constant Kd that is less than 10'6 M or 10'7 M or less. Exemplary hyaluronan-binding domains include, for e, link modules (also called link domains herein) or G1 domains, or sufficient portions of a link module or G1 domain that specifically binds to HA.

As used herein, reference that “the only portion of an HABP” is a link module or G1 domain or grammatical variations thereof means that the HABP molecule (e.g. a TSG-6 link module) ts or consists essentially of the link module or G1 domain but does not include the complete full-length sequence of amino acids of the reference HABP. Hence, the HABP only contains a hyaluronan-binding region or a sufficient portion thereof to specifically bind to HA. It is understood that the HABP can contain additional non-HABP amino acid sequences, including but not limited to, sequences that correspond to a detectable moiety or moiety capable of detection or a multimerization domain.

As used herein, modified, with respect to modified HA binding proteins refers to ations to alter, typically e, one more properties of an HA binding protein for detection in the diagnostic methods provided herein. ations include mutations that increase y and/or specificity of the protein for HA.

As used herein, a domain refers to a portion (a sequence of three or more, generally 5 or 7 or more amino acids) of a polypeptide that is a structurally and/or functionally distinguishable or definable. For example, a domain includes those that can form an independently folded structure Within a protein made up of one or more structural motifs (e.g. combinations of alpha helices and/or beta strands ted by loop regions) and/or that is recognized by Virtue of a functional actiVity, such as kinase actiVity. A protein can have one, or more than one, distinct . For e, a domain can be identified, defined or guished by homology of the sequence therein to related family members, such as homology and motifs that define an ellular domain. In another example, a domain can be distinguished by its on, such as by enzymatic activity, cg. kinase activity, or an ability to interact with a biomolecule, such as DNA binding, ligand binding, and dimerization. A domain independently can exhibit a function or activity such that the domain independently or fused to another molecule can perform an activity, such as, for U) example proteolytic ty or ligand binding. A domain can be a linear sequence of amino acids or a non-linear sequence of amino acids from the polypeptide. Many polypeptides n a plurality of domains.

As used herein, a filsion n refers to a chimeric protein containing two or more ns from two more proteins or es that are linked directly or indirectly via peptide bonds.

As used herein, a multimerization domain refers to a sequence of amino acids that promotes stable interaction of a polypeptide molecule with another polypeptide molecule containing a complementary multimerization domain, which can be the same or a different erization domain to form a stable multimer with the first domains. Generally, a polypeptide is joined directly or indirectly to the multimerization domain. Exemplary multimerization domains include the immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic s, compatible protein—protein interaction domains such as, but not limited to an R subunit of PKA and an anchoring domain (AD), a free thiol that forms an intermolecular disulfide bond between two molecules, and a protuberance-into- cavity (1'. e., knob into hole) and a compensatory cavity of identical or similar size that form stable multimers. The multimerization domain, for example, can be an immunoglobulin constant region. The immunoglobulin sequence can be an immunoglobulin constant domain, such as the Fc domain or portions thereof from IgGl, IgGZ, IgG3 or IgG4 subtypes, IgA, IgE, IgD and IgM.

As used , “knobs into holes” (also ed to herein as protuberance- into-cavity) refers to particular multimerization domains engineered such that steric interactions between and/or among such s, not only promote stable interaction, but also promote the formation of heterodimers (or multimers) over homodimers (or ltimers) from a mixture of monomers. This can be achieved, for example by constructing erances and es. Protuberances can be constructed by replacing small amino acid side chains from the interfge ofthe first polypeptide with RECTIFIED SHEET (RULE 91) ISA/EP larger side chains (e. g. ne or tryptophan). Compensatory ies” of identical or similar size to the protuberances optionally are created on the interface of a second polypeptide by replacing large amino acid side chains with smaller ones (cg, alanine or threonine).

As used herein, complementary multimerization domains refer to two or more multimerization domains that interact to form stable multimers of polypeptides linked to each such domain. Complementary multimerization domains can be the same domain or a member of a family of domains, such as for example, Fc regions, leucine zippers, and knobs and holes.

As used herein, “PC” or “Fc region” or “Fc domain” refers to a polypeptide containing the constant region of an antibody heavy chain, excluding the first constant region immunoglobulin domain. Thus, F0 refers to the last two constant region irrnnunoglobulin domains of IgA, IgD, and lgE, or the last three constant region immunoglobulin domains of IgE and lgM. Optionally, an Fc domain can include all or part of the flexible hinge inal to these domains. For IgA and IgM, Fc can include the J chain. For an exemplary Fe domain of IgG contains immunoglobulin domains C72 and C313, and optionally all or part of the hinge between Cyl and C72.

The ries of the Fc region can vary, but lly, include at least part of the hinge . In addition, Fc also includes any c or species variant or any variant or modified form, such as any t or modified form that alters the binding to an FcR or alters an Fc—mediated effector function. Exemplary sequences of other Fc domains, including modified Fc domains are known.

As used herein, “Fc chimera” refers to a chimeric polypeptide in which one or more ptides is linked, directly or indirectly, to an Fc region or a derivative thereof. Typically, an Fc chimera combines the Fc regiori ofan immunoglobulin with r polypeptide, such as for example an ECD ptide. Derivatives of or modified Fc polypeptides are known to those of skill in the art.

As used herein, “multimer” with reference to a hyaluronan binding protein refers to an HABP that contains multiple HA binding sites, for example, at least 2, 3, or 4 HA binding sites. For example, an HABP multimer refers to a HABP that contains at least 2 link modules that are each e ofbinding to HA. For example, a multimcr can be generated by linking, directly or ctly, two or more link RECTIFIED SHEET (RULE 91) ISA/EP WO 63155 modules (6.g. TSG-6 link module). The linkage can be facilitated using a multimerization domain, such as an Fc protein.

As used , an allelic variant or c variation references to a polypeptide encoded by a gene that differs from a reference form of a gene (lie. is encoded by an allele). 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 s. 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 pecies or intraspecies.

Generally, pecies 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 r identity with a wildtype and/or predominant form of a polypeptide.

As used herein, species variants refer to variants of the same polypeptide n and among species. Generally, interspecies variants have at least about 60%, 70%, 80%, 85%, 90%, or 95% identity or greater with a wildtype and/or predominant form from r species, including 96%, 97%, 98%, 99% or greater ty with a wildtype and/or predominant form of a polypeptide.

As used herein, modification in reference to ation 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.

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

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 fianctionally associated or related. A kit is a ed combination that optionally includes instructions for use of the combination or elements thereof.

As used herein, normal levels or values can be defined in a variety of ways known to one of skill in the art. Typically, normal levels refer to the expression levels of a HA across a healthy population. The normal levels (or nce levels) are based on measurements of healthy subjects, such as from a specified source (1'. e. blood, serum, tissue, or other source). Often, a normal level will be specified as a “normal range”, which typically refers to the range of values of the median 95% of the healthy population. Reference value is used interchangeably herein with normal level but can be different fiom normal levels ing on the subjects or the source. Reference levels are typically dependent on the normal levels of a particular segment of the population. Thus, for purposes herein, a normal or reference level is a predetermined standard or control by which a test patient can be compared.

As used herein, elevated level refers to the any level of amount or expression ofHA above a recited or normal threshold.

As used herein, biological sample refers to any sample obtained from a living or viral source or other source of macromolecules and biomolecules, and includes any cell type or tissue of a subject from which c acid or protein or other macromolecule can be obtained. The biological sample can be a sample obtained directly fiom a ical source or to sample that is processed. For example, isolated nucleic acids that are amplified constitute a biological . Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ s from animals, including biopsied tumor samples.

As used , detection includes methods that permit visualization (by eye or equipment) of a protein. A protein can be ized using an antibody specific to the protein. Detection of a protein can also be tated by fusion of a protein with a tag ing an epitope tag or label.

As used herein, a label refers to a detectable compound or composition which is conjugated directly or indirectly to a polypeptide so as to generate a labeled polypeptide. The label can be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can catalyze chemical alteration of a substrate nd ition which is detectable. Non-limiting examples of labels ed fluorogenic moieties, green fluorescent protein, or luciferase.

As used herein, affinity refers to the strength of interaction between two molecules such as between a hyaluronan binding protein and hyaluronan. Affinity is often measured by equilibrium association constant (Ka) or brium dissociation constant (Kd). The binding affinity between the molecules described herein, typically has a binding affinity with an association constant (Ka) of at least about 106 l/mol, lO7 l/mol, lO8 l/mol, lO9 l/mol or greater (generally 107-108 l/mol or greater).

The binding affinity of molecules herein also can be described based on the dissociation constant (Kd) of at least less than or less than or 10'7 M, 10'8 M, 10'9 M,10'10 M, 10‘11 M, 10‘12 M or lower.

As used herein, nce to a sufficient portion thereof that binds to HA means that the binding molecule ts a Ka of at least or at least about 107 to 108 M"1 or a dissociation constant (Kd) of l x 10'7 M or 1 x 10'8 M or less to HA.

As used herein, city (also referred to herein as selectively) with respect to two molecules, such as with respect to a hyaluronan binding protein and HA, refers to the r affinity the two molecules exhibit for each other compared to affinity for other molecules. Thus, a hyaluronan binding protein (HABP) with greater specificity for HA means that it binds to other molecules, such as heparin, with lower affinity than it binds to HA. Specific binding typically results in ive binding.

As used herein, a “G1 domain” refers to an HA binding domain of a Type C HA g protein. The G1 domain contains an Ig module and two link modules.

Exemplary proteins that n a G1 domain e HAPLNl/link n, HAPLNl, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, brevican, neurocan and phosphacan.

As used herein, link modules or link domain, used hangeably herein, are hyaluronan-binding domains that occur in proteins and facilitate binding to HA and that are involved in the assembly of ellular matrix, cell adhesion and migration.

For example, the link module from human TSG-6 ns two alpha helices and two antiparallel beta sheets arranged around a hydrophobic core. This defines the consensus fold for the Link module superfamily, which includes CD44, TSG-6, cartilage link protein, aggrecan and others as described herein.

As used herein, an “Ig module” refers to the portion ofthe G1 domain of Type C HABPs that is involved in. the g between Type C HABPs. Ig modules of Type C hyaluronans interact with one another to form a stable tertiary structure with hyaluronan.

As used herein, a “solid phase binding assay” refers to an in vitro assay in which an antigen is contacted with a ligand, where one of the antigen or ligand are bound to a solid support. The solid phase can be one in which components are physically immobilized to a solid support. For example, solid supports include, but are not limited to, a microtiter plate, a membrane (e. g., nitrocellulose), a bead, a dipstick, a thin-layer chromatographic plate, or other solid medium. Upon antigen- ligand interaction, the unwanted or non-specific components can be removed (e.g. by washing) and the antigen—ligand complex detected.

As used herein, predicting efficacy of treatment with an anti-hyaluronan agent, such as a hyaluronan-degrading enzyme, means that the companion diagnostic can be a prognostic tor of treatment with an anti-hyaluronan agent, such as a hyaluronan ing enzyme. For example, based on the s of detection of hyaluronan or other marker with the companion diagnostic, it can be determined that an anti-hyaluronan agent, such as a hyaluronan—degrading enzyme, will likely have some effect in treating subject.

As used herein, a prognostic indicator refers to a ter that indicates the probability of a particular outcome, such as the probability that a treatment will be effective for a particular disease or subject.

As used herein, elevated HA in a sample refers to an amount ofHA in a sample that is sed compared to the level present in a corresponding sample from a healthy sample or compared to a predetermined standard.

As used herein, elevated hyaluronan levels refers to amounts of hyaluronan in particular , body fluid or cell, dependent upon the disease or condition, as a consequence of or ise observed in the disease. For example, as uence of the ce of a onan-rich tumor, hyaluronan (HA) levels can be elevated in body fluids, such as blood, urine, saliva and serum, and/or in the tumorous tissue or cell. The level can be compared to a standard or other suitable control, such as a RECTIFIED SHEET (RULE 91) ISA/EP comparable sample from a subject who does not have the ociated disease, such as a subject that does not have a tumor.

As used herein, corresponding residues refers to residues that occur at aligned loci. d or variant polypeptides are aligned by any method known to those of skill in the art. Such methods typically maximize matches, and e methods such as using manual alignments and by using the us alignment programs available (for example, BLASTP) and others known to those of skill in the art. By aligning the sequences of polypeptides, one skilled in the art can identify corresponding residues, using conserved and identical amino acid residues as guides. Corresponding positions also can be based on structural alignments, for example by using er simulated alignments of protein structure. In other instances, corresponding s can be identified.

As used herein, an anti-hyaluronan agent refers to any agent that modulates hyaluronan (HA) sis or degradation, thereby ng hyaluronan levels in a tissue or cell. For purposes herein, anti-hyaluronan agents reduce hyaluronan levels in a tissue or cell compared to the absence of the agent. Such agents include compounds that modulate the sion of genetic material encoding HA synthase (HAS) and other enzymes or receptors involved in hyaluronan metabolism, or that modulate the proteins that synthesize or degrade hyaluronan including HAS function or activity. The agents include small-molecules, nucleic acids, peptides, proteins or other compounds. For example, anti-hyaluronan agents include, but are not limited to, antisense or sense molecules, antibodies, enzymes, small le inhibitors and HAS substrate analogs.

As used herein, a hyaluronan-degrading enzyme refers to an enzyme that catalyzes the cleavage of a hyaluronan polymer (also referred to as hyaluronic acid or HA) into smaller lar weight nts. Exemplary of hyaluronan-degrading s are hyaluronidases, and particular chondroitinases and lyases that have the ability to depolymerize hyaluronan. Exemplary chondroitinases that are hyaluronandegrading enzymes include, but are not limited to, chondroitin ABC lyase (also known as chondroitinase ABC), chondroitin AC lyase (also known as chondroitin sulfate lyase or chondroitin sulfate eliminase) and chondroitin C lyase. Chondroitin ABC lyase comprises two enzymes, chondroitin-sulfate-ABC endolyase (EC 4.2.2.20) and oitin-sulfate-ABC exolyase (EC 4.2.2.21). An exemplary chondroitin- sulfate-ABC ases and oitin-sulfate-ABC exolyases include, but are not limited to, those from Proteus vulgaris and Pedobacter heparinus (the Proteus vulgaris chondroitin-sulfate-ABC endolyase is set forth in SEQ ID NO:98; Sato et al. (1994) Appl. Microbiol. Biotechnol. 41(1):39-46). ary chondroitinase AC enzymes from the bacteria include, but are not limited to, those from Pedobacter heparinus, set forth in SEQ ID NO: 99, Victivallis vadensis, set forth in SEQ ID NO: 100, and Arthrobacter aurescens (Tkalec et al. (2000) Applied and Environmental Microbiology 66(1):29-35; Ernst et al. (1995) al Reviews in Biochemistry and Molecular Biology 30(5):387-444). Exemplary chondroitinase C enzymes from the bacteria include, but are not limited to, those from Streptococcus and Flavobacterium (Hibi et al. (1989) FEMS-Microbiol—Lett. 48(2): 121-4; Michelacci et al. (1976) J. Biol. Chem. 251 :1 154-8; Tsuda et al. (1999) Eur. J.

Biochem. 262:127-133).

As used herein, hyaluronidase refers to a class of hyaluronan-degrading s. Hyaluronidases include bacterial hyaluronidases (EC 4.2.2.1 or EC 4.2.99.1), hyaluronidases from leeches, other parasites, and crustaceans (EC 3.2.1.36), and mammalian-type hyaluronidases (EC 35). Hyaluronidases include any of non-human origin including, but not limited to, murine, canine, , leporine, aVian, bovine, ovine, porcine, equine, piscine, ranine, bacterial, and any from leeches, other parasites, and crustaceans. ary non-human hyaluronidases e, hyaluronidases from cows (SEQ ID NOS:10, 11, 64 and BH55 (US. Pat. Nos. ,747,027 and 5,827,721), 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 NOS:26, 27, 63 and 65), chimpanzee (SEQ ID NO: 101), Rhesus monkey (SEQ ID NO: 102), orangutan (SEQ ID , cynomolgus monkey (SEQ ID NO:29), guinea pig (SEQ ID NO:30), Arthrobacter sp. (strain FB24 (SEQ ID NO:67)), Bdellovibrio bacteriovorus (SEQ ID NO:68), Propionibacterium acnes (SEQ ID NO:69), ococcus agalactiae ((SEQ ID NO:70); 18RS21 (SEQ ID NO:71); serotype Ia (SEQ ID NO:72); serotype III (SEQ ID NO:73)), Staphylococcus aureus (strain COL (SEQ ID NO:74); strain MRSA252 (SEQ ID NOS:75 and 76); strain 6 (SEQ ID NO:77); strain NCTC 8325 (SEQ ID NO:78); strain bovine RF122 (SEQ ID NOS:79 and 80); strain USA300 (SEQ ID NO:81)), Streptococcus niae ((SEQ ID NO:82); strain ATCC BAA- 255 /R6 (SEQ ID NO:83); serotype 2, strain D39 / NCTC 7466 (SEQ ID NO:84)), Streptococcus pyogenes (serotype M1 (SEQ ID NO:85); pe M2, strain MGAS10270 (SEQ ID NO:86); serotype M4, strain MGAS10750 (SEQ ID NO:87); serotype M6 (SEQ ID NO:88); serotype M12, strain MGAS2096 (SEQ ID NOS:89 and 90); serotype M12, strain MGAS9429 (SEQ ID NO:91); serotype M28 (SEQ ID NO:92)), Streptococcus suz's (SEQ ID NOS:93-95); ofischerz’ (strain ATCC 700601/ ES114 (SEQ ID NO:96)), and the Streptomyces hyaluronolytz'cus hyaluronidase enzyme, which is specific for hyaluronic acid and does not cleave chondroitin or chondroitin sulfate (Ohya, T. and Kaneko, Y. (1970) Biochim. Biophys.

Acta 198:607). Hyaluronidases also include those of human origin. Exemplary human hyaluronidases include HYALl (SEQ ID NO:36), HYAL2 (SEQ ID NO:37), HYAL3 (SEQ ID NO:38), HYAL4 (SEQ ID , and PH20 (SEQ ID NO:1).

Also included amongst onidases are soluble hyaluronidases, including, ovine and bovine PH20, soluble human PH20 and soluble rHuPH20. Examples of commercially available bovine or ovine soluble hyaluronidases e Vitrase® (ovine hyaluronidase), Amphadase® (bovine hyaluronidase) and HydaseTM e hyaluronidase).

As used , “purified bovine testicular hyaluronidase” refers to a bovine hyaluronidase purified from bovine testicular extracts (see US. Patent Nos. 2,488,564, 2,488,565, 2,806,815, 2,808,362, 139, 2,795,529, 5,747,027 and ,827,721). Examples of cially available purified bovine testicular hyaluronidases include Amphadase® and HydaseTM, and bovine hyaluronidases, including, but not limited to, those available from Sigma Aldrich, Abnova, EMD Chemicals, GenWay Biotech, Inc., Raybiotech, Inc., and Calzyme. Also included are recombinantly produced bovine hyaluronidases, such as but not limited to, those generated by expression of a c acid molecule set forth in any of SEQ ID NOS:190-192.

As used herein, “purified ovine testicular hyaluronidase” refers to an ovine hyaluronidase purified from ovine testicular extracts (see US. Patent Nos. 2,488,564, 2,488,565 and 2,806,815 and International PCT Application No. WO2005/118799).

Examples of cially available purified ovine testicular extract include Vitrase®, and ovine hyaluronidases, including, but not limited to, those available from Sigma Aldrich, Cell Sciences, EMD Chemicals, GenWay Biotech, Inc., Mybiosource.com and tech, Inc. Also included are inantly produced ovine hyaluronidases, such as, but not limited to, those generated by expression of a nucleic acid molecule set forth in any of SEQ ID NOS:66 and 193-194.

As used herein, “PH20” refers to a type of hyaluronidase that occurs in sperm and is neutral-active. PH-20 occurs on the sperm surface, and in the lysosome- derived acrosome, where it is bound to the inner acrosomal membrane. PH20 includes those of any origin including, but not d to, human, chimpanzee, Cynomolgus monkey, Rhesus monkey, , bovine, ovine, guinea pig, rabbit and rat origin. Exemplary PH20 polypeptides e those from human (SEQ ID NO: 1), chimpanzee (SEQ ID NO: 101), Rhesus monkey (SEQ ID NO: 102), Cynomolgus monkey (SEQ ID NO:29), cow (SEQ ID NOS: 11 and 64), mouse (SEQ ID NO:32), rat (SEQ ID NO:31), rabbit (SEQ ID NO:25), sheep (SEQ ID NOS:27, 63 and 65) and guinea pig (SEQ ID NO:30).

Reference to hyaluronan-degrading enzymes includes precursor hyaluronan- degrading enzyme polypeptides and mature hyaluronan-degrading enzyme polypeptides (such as those in which a signal sequence has been removed), ted 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%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the precursor ptides set forth in SEQ ID NOS: 1 and 10-48, 63-65, 67-102, or the mature forms thereof. For example, reference to hyaluronan-degrading enzyme also includes the human PH20 precursor polypeptide variants set forth in SEQ ID NOS:50-51. Hyaluronan-degrading enzymes also include those that n chemical or posttranslational modifications and those that do not contain chemical or posttranslational modifications. Such ations include, but are not d to, PEGylation, albumination, glycosylation, famesylation, carboxylation, hydroxylation, phosphorylation, and other ptide modifications known in the art. A truncated PH20 hyaluronidase is any C-terminal shortened form thereof, ularly forms that are truncated and neutral active when N—glycosylated.

As used herein, a “soluble PH20” refers to any form of PH20 that is soluble under physiologic conditions. A soluble PH20 can be identified, for example, by its partitioning into the aqueous phase of a ® X-l 14 solution at 37 0C (Bordier et al., (1981).]. Biol. Chem, 256: 1604-7). Membrane-anchored PH20, such as lipid- anchored PH20, including GPI-anchored PH20, 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 PH20 are membrane-anchored PH20 in which one or more s associated with anchoring of the PH20 to the membrane has been removed or modified, where the soluble form retains hyaluronidase activity.

Soluble PH20 also include recombinant soluble PH20 and those contained in or purified from natural sources, such as, for example, testes extracts from sheep or cows. ary of such soluble PH20 is soluble human PH20.

As used herein, soluble human PH20 or sHuPH20 es PH20 polypeptides lacking all or a portion of the ylphosphatidylinositol (GPI) anchor sequence at the C-terminus such that upon expression, the polypeptides are soluble under physiological conditions. Solubility can be assessed by any suitable method that demonstrates solubility under physiologic conditions. Exemplary of such s is the Triton® X-l l4 assay, that assesses partitioning into the aqueous phase and that is described above and in the examples. In addition, a e human PH20 polypeptide is, if produced in CHO cells, such as CHO-S cells, a polypeptide that is sed and is secreted into the cell culture medium. Soluble human PH20 polypeptides, however, are not limited to those ed in CHO cells, but can be produced in any cell or by any method, including inant expression and polypeptide synthesis. Reference to secretion in CHO cells is definitional. Hence, if a polypeptide could be expressed and secreted in CHO cells and is soluble, z'. e. partitions into the aqueous phase when extracted with ® X-l 14, it is a soluble PH20 polypeptide whether or not it is so- produced. The sor ptides for sHuPH20 polypeptides can include a signal sequence, such as a heterologous or non-heterologous (lie. native) signal sequence.

Exemplary of the precursors are those that include a signal sequence, such as the native 35 amino acid signal sequence at amino acid positions 1-35 (see, e.g., amino acids 1-35 of SEQ ID NO:1).

As used herein, an "extended soluble PH20" or "esPH20" includes soluble PH20 polypeptides that contain residues up to the GPI anchor-attachment signal sequence and one or more contiguous residues from the GPI—anchor attachment signal sequence such that the esPH20 is soluble under physiological conditions. Solubility under physiological conditions can be determined by any method known to those of skill in the art. For example, it can be ed by the Triton® X-114 assay described above and in the examples. In on, as discussed above, a soluble PH20 is, if produced in CHO cells, such as CHO-S cells, a polypeptide that is expressed and is secreted into the cell culture medium. Soluble human PH20 ptides, r, are not limited to those produced in CHO cells, but can be produced in any cell or by any method, including recombinant expression and polypeptide synthesis. Reference to secretion in CHO cells is ional. Hence, if a polypeptide could be expressed and secreted in CHO cells and is soluble, z'. e. partitions into the s phase when extracted with Triton® X-114, it is a soluble PH20 polypeptide whether or not it is so- produced. Human e esPH20 polypeptides e, in on to residues 36- 490, one or more contiguous amino acids from amino acid residue position 491 of SEQ ID NO: 1, inclusive, such that the resulting polypeptide is e. Exemplary human esPH20 soluble polypeptides are those that have amino acids residues corresponding to amino acids 36-491, 36-492, 36-493, 36-494, 36-495, 36-496 and 36-497 of SEQ ID NO: 1. Exemplary of these are those with an amino acid sequence set forth in any of SEQ ID NOS: 15 1-154 and 185-187. Also included are allelic variants and other variants, such as any with 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with the corresponding polypeptides of SEQ ID NOS: 15 1-154 and 185-187 that retain neutral activity and are soluble. Reference to sequence identity refers to variants with amino acid substitutions.

As used herein, reference to 0s” includes sor esPH20 polypeptides and mature esPH20 polypeptides (such as those in which a signal sequence has been removed), truncated forms thereof that have enzymatic activity (retaining at least 1%, 10%, 20%, 30%, 40%, 50% or more of the full-length form) and are soluble, and includes allelic variants and species variants, variants encoded by splice ts, and other variants, ing polypeptides that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the precursor polypeptides set forth in SEQ ID NOS:l and 3, or the mature forms thereof.

As used herein, reference to “esPH20s” also include those that contain chemical or posttranslational ations and those that do not contain chemical or posttranslational modifications. Such modifications include, but are not limited to, PEGylation, albumination, glycosylation, famesylation, carboxylation, hydroxylation, orylation, and other polypeptide modifications known in the art.

As used herein, “soluble recombinant human PH20 (rHuPH20)” refers to a composition containing solubles form of human PH20 as recombinantly expressed and secreted in Chinese Hamster Ovary (CHO) cells. Soluble 0 is encoded by nucleic acid molecule that includes a signal sequence and is set forth in SEQ ID NO:49. The nucleic acid encoding soluble rHuPH20 is expressed in CHO cells which e 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 amino acids 36-481 and 36-482 of PH20 (e.g., SEQ ID NO:4 to SEQ ID NO:9) in various abundance.

Similarly, for other forms of PH20, such as the s, inantly expressed polypeptides and compositions thereof can include a plurality of species whose C-terminus exhibits heterogeneity. For example, compositions of recombinantly expressed esPH20 ed by expression of the polypeptide of SEQ ID NO: 151, which encodes an esPH20 that has amino acids , can include forms with fewer amino acids, such as 36-496, 36-495.

As used herein, an “N-linked moiety” refers to an asparagine (N) amino acid residue of a ptide that is capable of being glycosylated by post-translational modification of a polypeptide. ary N-linked moieties of human PH20 include amino acids N82, N166, N235, N254, N368 and N393 of human PH20 set forth in SEQ ID NO: 1.

As used herein, an “N-glycosylated polypeptide” refers to a PH20 polypeptide or truncated form thereto containing oligosaccharide e of at least three N-linked amino acid residues, for example, N-linked moieties corresponding to amino acid residues N235, N368 and N393 of SEQ ID NO: 1. An N-glycosylated polypeptide can include a polypeptide where three, four, five and up to all of the N-linked moieties are linked to an accharide. The N-linked oligosaccharides can include oligomannose, complex, hybrid or sulfated oligosaccharides, or other oligosaccharides and monosaccharides.

As used herein, an “N-partially ylated polypeptide” refers to a polypeptide that minimally contains an N-acetylglucosamine glycan linked to at least three N-linked moieties. A partially glycosylated polypeptide can include various glycan forms, including monosaccharides, oligosaccharides, and branched sugar forms, including those formed by treatment of a polypeptide with EndoH, , EndoF2 and/or EndoF3.

As used herein, a “deglycosylated PH20 ptide” refers to a PH20 polypeptide in which fewer than all possible glycosylation sites are glycosylated.

Deglycosylation can be effected, for example, by ng glycosylation, by preventing it, or by modifying the polypeptide to eliminate a glycosylation site. ular osylation sites are not required for ty, whereas others are.

As used herein, “PEGylated” refers to covalent or other stable attachment of polymeric les, such as polyethylene glycol (PEGylation moiety PEG) to hyaluronan-degrading enzymes, such as hyaluronidases, typically to increase half-life of the hyaluronan-degrading enzyme.

As used herein, a “conjugate” refers to a polypeptide linked ly or indirectly to one or more other polypeptides or al moieties. Such conjugates include fusion proteins, those produced by chemical conjugates and those produced by any other methods. For example, a conjugate refers to e PH20 polypeptides linked directly or indirectly to one or more other polypeptides or chemical moieties, whereby at least one soluble PH20 polypeptide is linked, directly or indirectly to another polypeptide or chemical moiety so long as the ate retains hyaluronidase activity.

As used herein, a “fusion” protein refers to a polypeptide encoded by a c acid sequence containing a coding sequence from one nucleic acid molecule and the coding sequence from another nucleic acid molecule in which the coding sequences are in the same g frame such that when the fusion construct is ribed and translated in a host cell, the protein is produced containing the two proteins. The two molecules can be adjacent in the construct or separated by a linker polypeptide that contains, 1, 2, 3, or more, but typically fewer than 10, 9, 8, 7, or 6 amino acids. The protein product encoded by a fusion construct is referred to as a fusion polypeptide.

As used herein, a “polymer” refers to any high molecular weight natural or synthetic moiety that is conjugated to, z'.e. stably linked directly or indirectly via a , to a polypeptide. Such polymers, typically increase serum half-life, and include, but are not limited to sialic moieties, PEGylation moieties, dextran, and sugar and other moieties, such as for glycosylation. For example, hyaluronidases, such as a soluble PH20 or rHuPH20, can be conjugated to a polymer.

As used herein, a hyaluronidase substrate refers to a substrate (e.g. protein or polysaccharide) that is cleaved and/or depolymerized by a hyaluronidase enzyme. lly, a hyaluronidase substrate is a glycosaminoglycan. An exemplary hyaluronidase substrate is hyaluronan (HA).

As used herein, a hyaluronan-associated disease, disorder or condition refers to any e or condition in which hyaluronan levels are elevated as cause, consequence or otherwise observed in the disease or ion. Hyaluronan- associated diseases and conditions are ated with elevated hyaluronan expression in a tissue or cell, increased interstitial fluid pressure, decreased vascular volume, and/or increased water t in a tissue. Hyaluronan-associated es, disorders or conditions can be treated by administration of a ition containing an anti- hyaluronan agent, such as a hyaluronan-degrading , such as a hyaluronidase, for example, a soluble hyaluronidase, either alone or in combination with or in addition to another treatment and/or agent. ary diseases and conditions, e, but are not limited to, inflammatory diseases and hyaluronan-rich cancers. onan rich cancers include, for example, tumors, including solid tumors such as late-stage cancers, a metastatic cancers, undifferentiated cancers, ovarian cancer, in situ carcinoma (ISC), squamous cell carcinoma (SCC), prostate cancer, atic cancer, non-small cell lung cancer, breast cancer, colon cancer and other cancers.

Also exemplary of hyaluronan-associated diseases and conditions are diseases that are associated with elevated interstitial fluid pressure, such as diseases associated with disc pressure, and edema, for example, edema caused by organ transplant, stroke, brain trauma or other injury. Exemplary hyaluronan-associated diseases and conditions include diseases and conditions associated with elevated titial fluid pressure, decreased ar volume, and/or increased water content in a tissue, including cancers, disc pressure and edema. In one example, treatment of the hyaluronan-associated condition, disease or disorder includes amelioration, reduction, or other beneficial effect on one or more of increased interstitial fluid pressure (IFP), decreased vascular volume, and increased water content in a .

As used herein, “activity” refers to a fianctional activity or activities of a polypeptide or portion thereof associated with a full-length (complete) protein. For example, active nts of a polypeptide can exhibit an activity of a fiJll-length protein. Functional activities include, but are not limited to, biological activity, catalytic or enzymatic activity, antigenicity ty 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.

As used herein, ronidase activity” refers to the ability to enzymatically ze the cleavage of hyaluronic acid. The United States Pharmacopeia (USP) XXII assay for hyaluronidase determines hyaluronidase activity indirectly by measuring the amount of higher molecular weight hyaluronic acid, or hyaluronan, (HA) substrate remaining after the enzyme is d to react with the HA for 30 min at 37 °C (USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention, Inc, Rockville, MD). A Reference Standard solution can be used in an assay to ascertain the relative activity, in units, of any hyaluronidase. In vitro assays to determine the onidase activity of hyaluronidases, such as PH20, ing e PH20 and esPH20, are known in the art and described herein. Exemplary assays include the microturbidity assay that measures cleavage of hyaluronic acid by hyaluronidase indirectly by detecting the insoluble precipitate formed when the uncleaved hyaluronic acid binds with serum albumin and the biotinylated-hyaluronic acid assay that measures the ge of onic acid indirectly by detecting the remaining biotinylated-hyaluronic acid non-covalently bound to iter plate wells with a streptavidin-horseradish peroxidase conjugate and a chromogenic substrate. 2012/061743 Reference Standards can be used, for example, to generate a standard curve to determine the activity in Units of the hyaluronidase being tested.

As used herein, specific activity refers to Units of activity per mg protein. The milligrams of hyaluronidase is defined by the absorption of a solution of at 280 nm assuming a molar extinction coefficient of approximately 1.7, in units of M"1 cm'l.

As used herein, “neutral active” refers to the ability of a PH20 polypeptide to enzymatically catalyze the cleavage of hyaluronic acid at neutral pH (6.g. at or about pH 7.0). Generally, a neutral active and soluble PH20, e.g., C-terminally truncated or N-partially glycosylated PH20, has or has about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500%, 1000% or more activity compared to the hyaluronidase activity of a corresponding neutral active PH20 that is not inally truncated or N-partially glycosylated.

As used herein, a “GPI-anchor attachment signal sequence” is a C-terminal sequence of amino acids that directs addition of a med GPI-anchor to the polypeptide within the lumen of the ER. GPI-anchor attachment signal sequences are present in the precursor polypeptides of GPI-anchored polypeptides, such as GPI- anchored PH20 polypeptides. The C-terminal chor attachment signal sequence typically ns a predominantly hobic region of 8-20 amino acids, preceded by a hilic spacer region of 8-12 amino acids, immediately downstream of the oa-site, or site of GPI-anchor attachment. GPI-anchor attachment signal sequences can be identified using methods well known in the art. These include, but are not limited to, in silico methods and algorithms (see, e.g. iend et al. (1995) Methods Enzymol. 250:571-5 82, Eisenhaber et al., (1999) J. Biol. Chem. 292: 741-758, Fankhauser et al., (2005) Bioinformatics 21 1852, Omaetxebarria et al., (2007) Proteomics 7: 195 1-1960, Pierleoni et al., (2008) BMC Bioinformatics 9:392), ing those that are readily available on bioinformatic websites, such as the ExPASy Proteomics tools site (e.g., the WorldWideWeb site expasy.ch/tools/).

As used herein, “nucleic acids” e DNA, RNA and analogs thereof, including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single or double-stranded. When ing 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 ally less than 5, generally less than 3) for probing or priming a library. Generally a probe or primer contains at least l4, 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.

As used , a peptide refers to a polypeptide that is greater than or equal to 2 amino acids in , and less than or equal to 40 amino acids in length.

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 l). The nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art.

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 (z'.e., amino acids wherein the ct- carbon has a side chain).

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” ic form. Residues in the “D” isomeric form, which are so designated, can be substituted for any o acid e 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 us of a polypeptide. In keeping with standard polypeptide nomenclature described in J. Biol.

Chem., 243: 3557-3559 (1968), and adopted 37 CPR. §§ l.822, iations for amino acid residues are shown in Table l: - 42 _ —A“it _IEEIE_ Glu and/or Gln >< xo>>>3aggsans?-><= Unknown or other All amino acid residue sequences represented herein by formulae have a left to right orientation in the tional direction of amino-terminus to yl- terminus. In addition, the phrase “amino acid residue” is 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 CPR. §§ 1.8214 .822, and incorporated herein by reference. rmore, 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 NH; or to a carboxyl-terminal group such as COOH.

As used herein, the "naturally ing ino acids" are the residues of those 20 u-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. Non-naturally occurring amino acids thus include, for e, amino acids or analogs of amino acids other than the 20 naturally-occurring amino acids and include, but are not limited to, the D-stereoisomers of amino acids. Exemplary non- natural amino acids are described herein and are known to those of skill in the art.

RECTIFIED SHEET (RULE 91) ISA/EP 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 lated molecules.

As used , 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.

As used herein, the term polynucleotide means a single- or double-stranded polymer of deoxyribonucleotides or cleotide bases read from the 5’ to the 3’ end. Polynucleotides include RNA and DNA, and can be ed 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 tood to be lent 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 ly in length and that the ends thereof can be staggered; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will, in general, not exceed 20 nucleotides in length.

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 ty 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 e, in one method of assessing sequence similarity, two amino acid or nucleotide sequences are d in a manner that yields a l level of identity between the sequences. “Identity” refers to the extent to which the amino acid or tide 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 es only the most similar region or regions).

“Identity” per se has an art-recognized g and can be calculated using published techniques. (See, e.g. Computational Molecular y, 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; ce Analysis in Molecular Biology, von Heinj e, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and ux, 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 ns (Carrillo, H. and Lipton, D., (1988) SIAM J Applied Math 48: 1073).

As used herein, homologous (with respect to nucleic acid and/or amino acid sequences) means about greater than or equal to 25 % ce 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 l, for determination of the percentage homology or ty, 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 ts, 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 Heinj e, G., Academic Press, 1987; and Sequence is Primer, Gribskov, M. and Devereux, J ., eds., M Stockton Press, New York, 1991; Carrillo, H. and , D., (1988) SIAMJAppll'ed Math 48:1073). By sequence homology, the number of conserved amino acids is determined by standard WO 63155 alignment algorithms programs, and can be used with default gap penalties ished 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 les that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.

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 % ical” 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 eux, J et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Altschul, S.F., et al., JMol Biol 215:403 (1990)); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carrillo, H. and Lipton, D., (1988) SIAMJApplied 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” m (Madison, WI) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison WI). Percent gy 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., tides 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 ison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as bed by Schwartz and f, eds., ATLAS OF N SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 y for each symbol in each gap; and (3) no penalty for end gaps.

Therefore, as used , 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 ties 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 exemplif1cation 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 n test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the 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 ence (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.

As used herein, an d sequence refers to the use of homology arity 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 ty 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.

As used herein, “primer” refers to a nucleic acid molecule that can act as a point of tion of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four ent side triphosphates and a rization agent, such as DNA polymerase, RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. It will be appreciated that n 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.

As used herein, “primer pair” refers to a set of primers that includes a 5‘ (upstream) primer that hybridizeg with the complement of the 5' end of a sequence to be amplified (e.g. by PCR) and a 3' (downstream) primer that hybridizes with the 3' end of the sequence to be ed.

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 ularly relevant to in vitro hybridization r include ing and washing temperature, buffer composition and salt concentration. Exemplary washing conditions for removing non-specifically bound c 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 d person can readily adjust these ters 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 tides are capable of hybridizing, typically with less than 25 %, 15 % or 5 % mismatches n 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.

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.

As used , it also is tood that the terms “substantially identical” or “similar” varies with the context as tood by those skilled in the relevant art.

As used herein, an allelic variant or allelic ion 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 RECTIFIED SHEET (RULE 91) ISA/EP WO 63155 encoded polypeptide) or can encode polypeptides having altered amino acid sequence.

The term “allelic variant” also is used herein to denote a n 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 ptide from a population or single reference member ofa species. Typically, allelic variants, which include variants between and among species lly have at least 80 %, 9O % 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 %, 95 % or greater identity with a wildtype and/or inant 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 lly refers to variations in ns among members of the same species.

As used herein, e,” which is used hangeably 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 t 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 modifications such as substitutions, deletions and insertions of tides. An allele of a gene also can be a form of a gene containing a mutation.

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

For example for PHZO, exemplary of s ts provided herein are primate PH20, such as, but not limited to, human, chimpanzee, macaque and cynomolgus . Generally, species variants have 70 %, 75 %, 80 %, 85 %, 90 %, 9i %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or greater sequence identity. Corresponding residues between and among s variants can be determined by comparing and aligning sequences to maximize the number ofmatching nucleotides or residues, for example, such that identity between the sequences is equal to or greater than 95 %, equal to or greater than 96 %, equal to0Lgreater than 9l%£qual to or greater than RECTIFIED SHEET (RULE 91) ISA/EP 98 % or equal to greater than 99 %. The position of interest is then given the number ed in the reference nucleic acid molecule. Alignment can be effected manually or by eye, particularly, where sequence identity is greater than 80 %.

As used herein, a human protein is one encoded by a nucleic acid molecule, such as DNA, t in the genome of a human, including all c variants and conservative variations thereof. A variant or modification of a protein is a human protein if the modification is based on the wildtype or prominent sequence of a human protein.

As used herein, a splice variant refers to a variant ed by differential processing of a primary transcript of genomic DNA that results in more than one type ofmRNA.

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 ements (e.g. substitutions) of amino acids and nucleotides, respectively. Exemplary of modifications are amino acid substitutions. An amino-acid substituted ptide can exhibit 65 %, 70 %, 80 %, 85 %, 90 %, 9l %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or more sequence identity to a ptide not containing the amino acid substitutions. Amino acid substitutions can be conservative or non-conservative. Generally, any modification to a polypeptide retains an activity of the polypeptide. Methods of modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies.

As used herein, le conservative substitutions of amino acids are known to those of skill in this art and can be made generally without altering the biological ty of the resulting molecule. Those of skill in this art recognize that, in l, single amino acid substitutions in non-essential regions of a ptide do not substantially alter biological activity (see, e. g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p.224). Such tutions can be made in accordance with those set forth in TABLE 2 as follows: TABLEZ al residue | xemplary conservative substitution 1y; Ser _Iys 1n;His —Ser 1a;Pro sn;G1n —Ieu;Va1 I1e;Val _g;G1n;G1u —Ieu; Tyr; He —Iet;Leu;Tyr —Ser rp;Phe Other substitutions also are permissible and can be determined empirically or in accord with known conservative substitutions.

As used herein, the term promoter means a portion of a gene containing DNA sequences that provide for the binding ofRNA rase and initiation of ription. Promoter sequences are ly, but not always, found in the 5 ’ ding region of genes.

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 fiee from chemical precursors or other chemicals when chemically synthesized.

Preparations can be determined to be substantially fiee if they appear free of readily detectable impurities as ined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high mance liquid tography (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 -5]. compound, however, can be a mixture of stereoisomers. In such instances, r purification might increase the specific ty of the compound.

Hence, nce to a substantially purified polypeptide, such as a substantially purified soluble PH20, refers to preparations of ns that are substantially free of cellular material, which includes preparations of proteins in which the protein is separated from cellular components of the cells from which it is ed or recombinantly-produced. In one ment, the term substantially free of cellular material includes preparations of enzyme proteins having less than about 30 % (by dry weight) of zyme proteins (also referred to herein as a contaminating protein), generally less than about 20 % of non—enzyme proteins or 10% of non- enzyme proteins or less than 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.

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 ed 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.

As used herein, synthetic, with reference to, for e, a synthetic nucleic acid le or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is ed by inant methods and/or by chemical synthesis methods.

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

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 s 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 RECTIFIED SHEET (RULE 91) ISA/EP WO 63155 chromosomes and mammalian artificial somes. Selection and use of such vehicles are well known to those of skill in the art.

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 e of effecting expression of such DNA fragments. Such additional ts can include promoter and terminator sequences, and optionally can include one or more origins of ation, one or more selectable markers, an enhancer, a polyadenylation signal. Expression vectors are lly derived from plasmid or viral DNA, or can n 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 riate 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.

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

As used herein, “operably” or “operatively linked” when referring to DNA segments means that the segments are arranged so that they fianction in concert for their intended purposes, 6.g. initiates ream of the promoter and , transcription upstream of any transcribed sequences. The promoter is y the domain to which the transcriptional machinery binds to initiate transcription and ds through the coding segment to the terminator.

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 protein, such as an enzyme, or a domain f, 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. For example, the chemical species actually detected need not of course be the enzymatically cleaved product itself but can for example be a derivative thereof or some fithher substance. For example, ion of a cleavage product can be a detectable moiety such as a fluorescent moiety.

As used herein, biological activity refers to the in viva activities of a compound or physiological responses that result upon in viva 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 ed in in vitro systems designed to test or use such activities. Thus, for purposes herein a biological activity of a hyaluronidase enzyme is its degradation of hyaluronic acid.

As used herein lent, when ing to two sequences of nucleic acids, means that the two ces in question encode the same ce 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 fianction of the protein or peptide. When equivalent refers to a property, the property does not need to be t 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.

As used herein, “modulate” and “modulation” or “alter” refer to a change of an activity of a molecule, such as a protein. ary activities include, but are not limited to, biological activities, such as signal transduction. Modulation can include an increase in the activity (Le. or agonist activity), a decrease in , up-regulation activity (226. , down-regulation or inhibition) or any other alteration in an activity (such as a change in periodicity, frequency, duration, kinetics or other ter).

Modulation can be context ent and typically modulation is ed to a designated state, for example, the wildtype protein, the protein in a constitutive state, or the n as expressed in a designated cell type or condition.

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 As used herein, a combination refers to any ation between or among two or more items. The combination can be two or more separate items, such as two compositions or two tions, can be a mixture thereof, such as a single mixture of the two or more items, or any variation thereof. The elements of a ation are generally fianctionally associated or related. For example, a combination can be a ation of compositions provided herein.

As used herein a kit refers to a combination of components, such as a combination of the compositions herein and another item for a purpose including, but not limited to, reconstitution, activation, and instruments/devices for delivery, administration, diagnosis, and assessment of a biological actiVity or property. Kits optionally include instructions for use.

As used herein, “disease or disorder” refers to a ogical condition in an organism ing from cause or ion including, but not d to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms.

Diseases and disorders of interest herein are onan-associated diseases and disorders.

As used herein, “treating” a subject with a disease or condition means that the t’s symptoms are partially or totally alleViated, or remain static following treatment. Hence treatment encompasses laxis, 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.

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

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

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

As used herein, the term “subject” refers to an animal, including a mammal, such as a human being. 2012/061743 As used herein, a “patient” refers to a human subject exhibiting symptoms of a disease or disorder.

As used herein, an “individual” can be a subject.

As used , about the same means within an amount that one of skill in the art would consider to be the same or to be within an able range of error. For example, typically, for pharmaceutical compositions, an amount within at least 1%, 2%, 3%, 4%, 5% or 10% is considered about the same. Such amount can vary depending upon the tolerance for variation in the particular composition by subjects.

As used herein, dosing regime refers to the amount of agent, for e, the composition containing a hyaluronan-degrading enzyme, for example a soluble hyaluronidase or other agent, stered, and the frequency of administration. The dosing regime is a function of the disease or condition to be treated, and thus can vary.

As used herein, frequency of administration refers to the time between successive administrations of treatment. For example, frequency can be days, weeks or months. For example, frequency can be more than once weekly, for example, twice a week, three times a week, four times a week, five times a week, six times a week or daily. Frequency also can be one, two, three or four weeks. The particular frequency is on of the particular disease or condition treated. Generally, frequency is more than once weekly, and generally is twice weekly.

As used , a “cycle of administration” refers to the repeated schedule of the dosing regime of administration of the enzyme and/or a second agent that is repeated over successive administrations. For example, an exemplary cycle of administration is a 28 day cycle with administration twice weekly for three weeks, followed by one-week of discontinued dosing.

As used herein, when referencing dosage based on mg/kg of the subject, an average human subject is considered to have a mass of about 70 kg-75 kg, such as 70 As used herein, amelioration of the symptoms of a ular 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 or, adverse effects of a condition, such as, for example, WO 63155 reduction of adverse effects associated with or that occur upon administration of a hyaiuronan—degrading enzyme , such as a PEGylated hyaluronidase.

As used herein, prevention or prophylaxis refers to a reduction in the risk of developing a disease or condition.

As used , 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 .

Hence, it is the quantity necessary for preventing, Curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder.

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.

As used herein, a single dosage formulation refers to a formulation as a single dose.

As used , formulation for direct administration means that the composition does not require further dilution inistration.

As used , an “article of manufacture” is a product that is, made and sold.

As used throughout this application, the term is intended to encompass anti- onan agents, for example hyaluronan-degrading enzyme, such as hyaluronidase, and second agent itions contained in articles of packaging.

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, s, creams and other such compositions.

As used , a ar extract or lysate refers to a preparation or fraction which is made from a lysed or ted cell.

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

Generally hyaluronidases are of human origin.

RECTIFIED SHEET (RULE 91) ISA/EP As used herein, anti-cancer treatments include administration of drugs and other agents for treating cancer, and also treatment protocols, such as surgery and radiation. Anti-cancer treatments include administration of anti-cancer agents.

As used , an anti-cancer agent refers to any agents, or compounds, used in anti-cancer ent. These include any agents, when used alone or in combination with other compounds, that can alleviate, reduce, ameliorate, prevent, or place or maintain in a state of remission of clinical symptoms or diagnostic markers associated with tumors and cancer, and can be used in combinations and compositions provided herein. Exemplary anti-cancer agents include, but are not limited to, hyaluronan—degrading enzymes, such as the PEGylated hyaluronan—degrading enzymes provided herein used singly or in combination with other anti—cancer agents, such as chemotherapeutics, polypeptides, antibodies, es, small molecules or gene therapy vectors, viruses or DNA.

As used herein, a control refers to a sample that is substantially identical to the test , 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.

As used , the singular forms “a,” “an” and “the” include plural referents unlcss Lhc context clceu‘ly dictates otherwise. Thus, for example, reference to a nd comprising or ning “an extracellular domain” includes nds with one or a plurality of extracellular domains.

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.” I Generally “about” es an amount that would be expected to be within experimental error.

As used herein, “optional” or “optionally” means that the subsequently described event or stance does or does not occur, and that the description includes instances where said event or circumstance occurs and ces Where it does not. For example, an optionally substituted group means that the group is unsubstituted or is substituted.

As used herein, the iations for any protective groups, amino acids and other compounds, are, unless ted otherwise, in accord with their common usage, RECTIFIED SHEET (RULE 91) ISA/EP recognized abbreviations, or the IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. l 1:1726).

B. ONAN BINDING N AND COMPANION DIAGNOSTIC Provided herein are ive and specific methods to detect and closely monitor onan (HA) levels associated with disease, particularly in the extracellular matrix (ECM) of tumor tissues. The ion diagnostic methods provided herein are based on the finding that HA accumulation specifically correlates with and predicts aggressive disease, in particular with respect to cancers. In addition, the companion diagnostic method provided herein also is based on the finding that HA specifically provides superior prognostic and treatment selection information as compared to other markers involved in the HA metabolic pathway associated with hyaluronan-associated diseases and conditions, such as hyaluronidase synthases or hyaluronidases. Hence, the method provided herein utilizes improved hyaluronan binding protein (HABP) reagents that exhibit specificity, high affinity and low variability for specific and sensitive detection of HA. Also provided herein are improved HABP ts.

In one example, the improved HABPs provided herein, such as any described in Section C, can be a companion diagnostic for selecting patients with HA-associated diseases, for example HA-associated tumors, for treatment with an anti-hyaluronan agent or hyaluronan-degrading enzyme, such as any set forth in Section E (e.g. a hyaluronidase or modified hyaluronidase such as PEGylated PH20, z'.e. PEGPH20).

In such an example, the method is useful in classification of patients for selection of therapy, such as cancer therapy, and in particular relates to the measurement ofHA levels that correlate with responsiveness to therapy with an yaluronan agent, for e a hyaluronan-degrading enzyme therapy, such as therapy by a 0 for treatment of patients with advanced tumors.

In another example, the improved HABPs provided herein, such as any described in Section C, also can be used in methods of ring y or responsiveness to treatment with an anti-hyaluronan agent or onan-degrading enzyme such as any set forth in Section E (e.g. a hyaluronidase or modified onidase such as PEGylated PH20, z'.e. PEGPH20) by detecting levels ofHA during the treatment. Thus, the improved HABP can be used in conjunction with therapy with an anti-hyaluronan-agent, for example hyaluronan-degrading'enzyme y, to monitor HA levels and to adjust and/or alter y to personalize individual treatment of a patient depending upon the particular patient and course of disease in a manner that correlates to clinical response.

Also provided herein are combinations and kits that contain an anti- hyaluronan agent, such as a hyaluronan—degrading enzyme (for example any provided herein below in Section E) and an improved HABP (for example any provided herein below in Section C), and optionally other accompanying reagents, for use in selecting, monitoring and/or treating HA-associated diseases and ions, in particular cancer. 1. onan Accumulation in Disease And Correlation to Prognosis Hyaluronan (HA; also called hyaluronic acid or onate) is a linear 1,5 glycosaminoglycan (GAG) polymer containing repeating N—acetylglucosamine and D-glucuronic acid disaccharide subunits via GlcUA-Bl,3-GlcNAc— Bl,4—1inkages.

Hyaluronan is synthesized by a class of hyaluronan ses, HASl, HASZ and HAS3. These enzymes act by lengthening hyaluronan by adding onic and N- acetylglucosamine to the nascent polysaccharide as it is extruded through the cell. In addition to the HA syntheses, the level ofHA normally is maintained by its catabolism by hyaluronidases, cally the turnover enzyme hyaluronidase 1 (Hyall). The dynamic turnover of HA is balanced by biosynthesis and catabolism to keep a constant concentration in the normal .

HA is a component of the extracellular matrix (ECM). It is ubiquitously distributed in tissues and localized in the extracellular, pericellular matrices as well as inside cells. HA has a wide range of biological firnctions such as contributing to tissue homeostasis and biomechanics, cell proliferation, immune on and activation, and cell migration during dynamic cellular processes. These processes are ed by interaction of HA with HA-binding ns known as hyaladherins, such as TSG—6, versican, inter-alpha-trypsin inhibitor, CD44, lymphatic vessel endothelial HA or (LYVE-l -l) and RHAMM.

RECTIFIED SHEET (RULE 91) ISA/EP Hyaluronan accumulation is associated with many malignant and autoimmune disease conditions (Jarvelainen H, et a1. (2009) Pharmacol Rev 61: 198-223; Whatcott CJ, er a]. (2011) Cancer ery 1:291 -296). For example, certain diseases are associated with expression and/or production of hyaluronan, including inflammatory dice-ace: and cancers T—IA is linked tn a variety nf‘ biological nrnneseae invnlved with progression of such diseases (see e. g. Itano et al. (2008) Semin Cancer Biol 18(4):268-274; Tammi et al. (2008) Semin Cancer Biol 18(4):288—295).

In particular, HA is a ent of the tumor matrix and is present in many solid tumors. lation ofHA within a tumor focus prevents cell—cell contact, promotes epithelial-mesenchymal transitions, is involved with the p53 tumor suppressor pathway via its receptors RHAMM and CD44 and recruits tumor- associated macrophages (Itano et a1. (2008) Cancer Sci 99: 1720-1725; Camenisch et a]. (2000) J Clin Invest 9-360; Thompson. et‘ a1. (2010) M0]. Cancer Ther. 9:3052-64). The assembly of a pericellular matrix rich in HA is a prerequisite for proliferation and migration of mesenchymal cells that can promote metastatic behavior. Tumors characterized by the accumulation ofHA also exhibit tumor water uptake and have high interstitial fluid pressure (IFP) that can inhibit penetration of and accessibility of the tumor to systemically applied therapeutics, such as chemotherapeutics. Further, HA ers, generated by degradation by Hyall, also have been shown to result in angiogenesis or apoptosis that can contribute to tumor pathogenesis.

The accumulation ofHA has been correlated to HAS gene expression and/or HYAL gene expression (Kosaki et a1. (1999) Cancer Res. 59:1141—1145; Liu et al. (2001) Cancer Res. 61 :5207-5214; Wang et a1, (2008) PLoS 3:3032; Nykopp et a1. (2010) BMC Cancer 102512). Studies in the art have variously shown that HA, HAS or Hyall can be used as prognostic indicators of cancer. Also, studies have suggested that the selective inhibition of Hyall, such as by anti~sense methods, or of hyaluronan synthesis by HAS, such as by the use of 4—methylumbelliferone, are s of treating tumors (Kakizaki et al. ) J. Biol. Chem. 279:33281-33289). In addition, hyaluronidases, such as PHZO as discussed below, also have been used to treat hyaluronan-associated es and conditions (see e.g. Thompson et' al. (2010) Mol.

Cancer Ther 3064).

RECTIFIED SHEET (RULE 91) ISA/EP As shown in the Examples, it is now found herein that the HA phenotype of a cell, and in particular the ion of a tumor pericellular matrix, correlates to tumor aggressiveness, and that an assay for HA levels specifically predicts that ability of HA-synthesizing tumors cells to form a pericellular matrix. Specifically, it is found herein that among the potential markers ofHA accumulation, including HAS 1 , 2,3; Hyall or 2; or HA, that only HA determination correlated with pericellular matrix formation and thereby predicted tumor cell competence to form HA-aggrecan- mediated pericellular es and thereby tumor aggressiveness. Thus, for purposes of a diagnostic to t or prognose tumor therapy, an HA g protein (HABP) is contemplated. As described herein, tumor HA production can be measured quantitatively using an HABP as a probe, and HABP for hyaluronan shows a correlation to pericellular matrix formation while no correlation was found n pericellular matrix formation and ve levels of HAS or Hyal mRNA. These findings show that direct ement of tumor cell-associated HA, and not the other markers involved in the HA metabolic pathway, offers a reliable predictor for pericellular matrix formation. 2. Therapy of Tumors with An Anti-Hyaluronan Agent (e.g.

Hyaluronan-Degrading Enzyme) and Responsiveness to Treatment It also is found herein that the amount or extent ofHA accumulation measured also correlates with siveness to treatment with an anti-hyaluronan agent, for e a onan-degrading , such as PH20. Anti-Hyaluronan agents, for example hyaluronan-degrading enzymes, such as a PH20, exhibit properties useful for single-agent or combination therapy of diseases and conditions that exhibit the lation of hyaluronan (hyaluronic acid, HA). Such hyaluronan-associated diseases, conditions and/or disorders include s and inflammatory diseases.

Hyaluronan-rich cancers include, but are not limited to, tumors, including solid tumors, for example, late-stage cancers, a metastatic cancers, undifferentiated cancers, ovarian cancer, in situ carcinoma (ISC), squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer and other cancers.

For example, HA degrading enzymes, such a hyaluronidase, for example PH20, have been shown to remove HA from tumors ing in the reduction of tumor volume, the reduction of IFP, the slowing of tumor cell proliferation, and the enhanced efficacy of co-administered chemotherapeutic drugs and biological agents by permitting increased tumor penetration (see e.g. U.S. published application No. 20100003238 and International published PCT Appl. No ; Thompson et al. (2010) M01. Cancer Ther 93052-3064).

The ability of a hyaluronidase, such as PH20, to degrade HA to serve as a therapeutic of hyaluronan-associated diseases and ers can be exploited by modification to increase systemic half-life. The increased half-life permits not only removal of HA, but also, due to its continued presence in the plasma and its ability to degrade HA, s or decreases the extent of regeneration ofHA within ed tissues, such as the tumor. Hence, maintenance of plasma enzyme levels can remove HA, such as tumor HA, and also counteract HA resynthesis. PEGylation is an established technology used to increase the half-life of therapeutic proteins in the body thus enabling their use in ic ent protocols. PEGylation of anti- hyaluronan agents, such as hyaluronan-degrading enzymes, such as hyaluronidase extends its half-life in the body from less than a minute to approximately 48 to 72 hours and allows for the systemic treatment of tumors rich in HA (see e.g. U.S. published application No. 20100003238 and International published PCT Appl. No ; Thompson et al. (2010) M01 Cancer Ther 9: 3052-3064).

It is found herein that the growth tory activity of an anti-hyaluronan agent, and in particular a hyaluronan-degrading enzyme, for example a hyaluronidase, such as a PH20 or PEGPH20, on tumor cells is correlated with the extent ofHA . As shown in the es, tumors can be characterized into ypic groups (e.g. HAH, HA+2, HA”) based on the amount ofHA expression in the tumor.

High tumor-associated HA (scored HA+3) resulted in accelerated tumor growth in animal models and to greater tumor inhibition by a hyaluronan-degrading enzyme (6.g. PEGPH20). For e, tumor growth inhibition associated with an HA+3 phenotype was 97%, whereas it was only 44% and 16% for tumor HA+2 or HA+1 ypes, respectively. The data indicate the continued grth of some tumors is dependent upon the density and amount ofHA in the tumor microenvironment and that depletion ofHA from an HA rich (e.g. HA+3) tumor has a more pronounced effect on tumor growth than depletion ofHA from an HA moderate or poor tumor (e.g. , HA+2, HA“) or HA nt tumor. Thus, as shown herein, the degree ofHA accumulation in tumor s, as measured using an HABP, is predictive of the level of inhibition of tumor growth in vivo mediated by an anti-hyaluronan agent (e.g, 3. Hyaluronan Binding Proteins (HABPS) Reagent and Diagnostic Based on the s provided above and in the Examples herein, the biomarker HA detected using an HABP has been specifically correlated to response to an anti-hyaluronan treatment, for example a hyaluronan-degrading enzyme treatment (tag. PEGPHZO). Thus, provided herein is a method of using an HABP for prognosis and also to predict the degree of sensitivity, and thus responsiveness, to an anti- hyaluronan agent, for example a hyaluronan-degrading enzyme (6.g. a hyaluronidase or modified hyaluronidase such as PEGylated PHZO, z‘. e. PEGPI—I20).

For value as a reagent, the sensitivity and specificity of an HABP is d as well as reproducibility due to low variability. For example, the detection and measurement ofHA in tissues is limited using existing reagents. Currently, the method used to detect or measure HA in tissues via immunohistological staining is mainly dependent on the animal cartilage tissue-derived HA binding proteins or domains. These include the HABP purified from bovine nasal cartilage proteoglycan by extraction with 4 M guanidinc HC1 and then by affinity chromatography using HA coupled resin. The resulting animal-derived HA is composed of two major components: Aggrecan GI domain and link . Due to variation from batch to batch, as well as different modifications used in the method to prepare the HABP, variability exists in the an in terms of differences in the HABP staining patterns and the discrepancies in staining profiles make comparisons among studies difficult.

Thus, due to its existence as a heterogenous mixture of components and no validated procedure for its tion, alternative HABP ns are provided herein for use in companion diagnostics for prognosis of disease and predicting efficacy of treatment in conjunction with an anti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme).

Hence, the HABP reagent for use in the methods ed herein includes any HABP that is not an HABP d from animal age, for example, purified from bone nasal cartilage as used in the art using a method as described by E-Laurent et a1. (1985) Ann. Rheum. Dis 44:83—88) or modified method thereof. Exemplary ‘HABPS RECTIFIED SHEET (RULE 91) ISA/EP for use in the methods herein are bed in Section C. Such proteins include, for example, HABPs containing one or more HA binding domains, ing one or more link modules for g to HA. In some examples, the HABP contains an HA binding domain (6.g. a link module) of aggrecan, versican, neurocan, breVican, phosphacan, l, HAPLN-2, HAPLN—3, HAPLN—4, stabilin-l, stabilin-2, 58, KIAA0527, or TSG-6 protein. In some examples, the HABP contains an aggrecan Gl domain, versican Gl domain, neurocan Gl domain, breVican Gl domain, or a phosphacan Gl domain. In some es, the HABP contains a G1 domain of aggrecan, versican, neurocan, breVican, or phosphacan and a link n selected from HAPLN—l, HAPLN-2, HAPLN—3, or HAPLN—4. In some examples, the HABP contains a link module of TSG-6, stabilin-l, stabilin-2, CAB6l358, or KIAA0527.

In some examples, the HABP is a modified HABP, such as, for example a modified aggrecan, versican, neurocan, brevican, phosphacan, l, HAPLN—2, HAPLN—3, HAPLN—4, stabilin-l, stabilin-2, CAB6l358, KIAA0527, or TSG-6 protein, such as, for example TSGLM-Fc. In some examples, the HABP is a modified HABP that is modified to improve its binding to HA, such as, for example, TSGLM-FcAHep.

In particular, the HABP provided herein 1) can be produced recombinantly in an expression system, such as a mammalian expression system; 2) exhibits improved biophysical properties such as stability and/or solubility; 3) can be d by simple ation methods, such as by one-step affinity purification methods; 4) is capable of being ed by procedures compatible with binding assays, and in particular immunohistochemistry or ELISA methods; 5) can be expressed in multimeric form (6.g. Via dimerization) to exhibit increased or high affinity for HA; and/or 6) exhibits specificity for HA as compared to other GAGs.

In one example, ed herein for use in the s herein are HABPs that are single module HA proteins that can be produced recombinantly in sion systems. In particular, provided herein are HABP reagents that contain a link module.

For e, HABPs provided herein are of the type A class of HABPs containing only the link module (LM) or a sufficient portion thereof to bind to hyaluronan.

Exemplary of such HABPs are tumor necrosis factor-stimulated Gene (TSG)LM 2012/061743 (link module set forth in SEQ ID NO:360), stabilin-l-LM or stabilinLM (link module set forth in SEQ ID NO:371 or 372, tively), CAB61358-LM (link module set forth in SEQ ID NO: 373) or KIAA0527-LM (link module set forth in SEQ ID NO:374).

In another example, provided herein for use in the methods herein are HABPs that are linked directly or indirectly to a multimerization domain. HA-binding domains, such as a link module, of HABPs can be directly or indirectly linked, such as covalently-linked, non-covalently-linked or chemically linked, to form ers of two or more HA binding domains. The HA binding domains can be the same or different. In particular, the HA binding domain is a link domain or module. Hence, multimers can be formed by dimerization of two or more link domains. In one example, multimers can be linked by disulfide bonds formed between cysteine es on different HA-link domains. For example, a erization domain can include a portion of an globulin molecule, such as a portion of an immunoglobulin constant region (PC). In another example, multimers can e an HA-binding domain joined via covalent or non-covalent interactions to peptide moieties fused to the polypeptide. Such peptides can be peptide linkers (spacers), or peptides that have the property of promoting multimerization. In additional example, ers can be formed between two polypeptides through chemical linkage, such as for example, by using heterobifianctional linkers. A description of erization domains is provided below. ary of a HABP multimer is a link module (LM) fused to an PC. For e, exemplary of an HABP reagent for use in the s herein is TSGLM-Fc.

In a further example, provided herein for use in the methods herein are HABP that are modified, such as by amino acid replacement, to exhibit increased specificity for hyaluronan compared to other GAGs. For example, provided herein is a mutant TSGLM containing amino acid replacement(s) at amino acid residues 20, 34, 41, 54, 56, 72 and/or 84, and in particular at amino acid residues 20, 34, 41, and/or 54 (corresponding to amino acid residues set forth in SEQ ID NO:206). The replacement amino acid can be to any other amino acid residue, and generally is to a non-basic amino acid residue. For example, amino acid replacement can be to Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Ala (A), Val (V), Ile (I), Leu (L), Met (M), Phe (F), Tyr (Y) or Trp (W). The amino acid replacement or replacements confer decreased binding to heparin. Binding can be d at least 1.2-fold, 15-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40- fold, 50-fold, lOO-fold or more compared to binding of TSGLM to heparin not containing the amino acid replacement. Exemplary of a TSGLM mutant for use as a reagent in the method provided herein is K20A/K34A/K41A. Hence, for example, binding to heparin is reduced such that specificity to hyaluronan is increased. The mutant TSGLM can be conjugated ly or ctly to a multimerization domain to generate multimers. For example, exemplary of a reagent for use in the methods herein is TSGLM(K20A/K34A/K4lA)-Fc.

Any of the reagents can be used alone or in combination in a companion diagnostic method. For example, in a sandwich ELISA or competitive ELISA, two or more of the above reagents can be used. As described herein below, any of the HABPs provided herein can be conjugated directly or indirectly to a moiety that is capable of detection. In some es, the HABPs that bind to HA, for example in a tumor sample, can be detected using a secondary reagent, such an antibody that binds to the HABP. In some examples, the HABPs are modified to permit detection ofHA binding. For example, the HABPs can be ated to a able molecule that permits either direct detection or ion via secondary agents, such as antibodies that bind to the modified HABPs and are coupled to detectable ns, such as fluorescent probes or detectable enzymes, such as adish peroxidase. 4. Companion Diagnostic and stic Methods The HABPs provided herein can be used either individually or in combination in diagnostic, prognostic or ring methods utilizing binding assays on various biological samples of patients having a hyaluronan-associated disease or ion or at risk or suspected of having a hyaluronan-associated disease or condition. For example, the HABPs can be used in assays on patients having a solid tumor or at risk of developing a solid tumor or other cancer. In particular examples, a TSGLM, TSGLM-Fc or variant or mutant thereof such as one that exhibits reduced binding to heparin and increased specificity for hyaluronan is used in the methods herein. The diagnostic and prognostic methods can be used in conjunction with therapy with a —67- hyaluronan-degrading enzyme in order to classify and/or select patients for ent or to alter or modify the course of treatment.

In exemplary methods provided herein, the diagnostic and prognostic methods are ion methods to therapy with an anti-hyaiuronan agent, such as a hyaluronan-degrading enzyme, for example a hyaluronidase or modified hyaluronidase such as PH20 or PEGPHZO. HA detection can inform treatment selection, initiation, dose customization or termination, and thus can serve to individualize ent with an anti-hyaluronan' agent, for example a hyaluronan— degrading .

For example, an HABP companion stic method can be used to determine whether a subject who is predisposed to a hyaluronan—associated disease or condition (e.g. cancer) or who is suffering from a hyaluronan—associated e or condition (e.g. cancer) will benefit from or is predicted to be responsive to receiving treatment with an anti-hyaluronan agent, such as'a hyaluronan-degrading enzyme. In the method, the level of HA expression from samples from subjects predisposed or known to have a hyaluronan—associated disease or condition (ag. cancer) can be determined and the level ofHA expression in samples from subjects compared to predetermined HA levels that classify responsiveness to an anti-hyaluronan agent, for example a hyalnronan-degrading enzyme. It is within the level of one of skill in the art to determine the threshold level ofHA for fying responsiveness to treatment with a hyaluronan-degrading enzyme. For example, it is found herein that a significant correlation exists between elevated HA accumulation and tumor growth tion, whereby tumor growth inhibition response is correlated to an HA+3 phenotype as quantified by immunohistochemistry of tumor tissue. Thus, in the companion diagnostic method ed herein, a tumor sample is assessed for HA levels using an HABP reagent provided herein by immunohistochemistry s or other methods adaptable to scoring. If the HA phenotype is HA+3 as determined by methods known to one of skill in the art and described , then the subject is selected as a candidate for treatment with a onan-degrading enzyme, such as a hyaluronidase or modified hyaluronidase (e.g. PHZO or PEGPHZO). Similar fication and classification methods can be utilized by assessing HA in bodily RECTIFIED SHEET (RULE 91) ISA/EP fluids, such as blood or plasma. Dosages and regimens of a hyaluronan—degrading enzyme, such as PH20 or PEGPHZO, for ent are provided herein.

The HABP reagents ed herein can detect HA using any binding assay known to one of skill in the art including, but not limited to, enzyme linked sorbent assay (ELISA) or other similar immunoassay, including a sandwich ELISA or competitive ELISA assay; immunohistochemistry ); flow cytometry, or western blot. The binding assay can be performed on samples obtained from a patient body fluid, cell or tissue sample of any type, including from plasma, urine, tumor or suspected tumor tissues (including fresh, frozen, and fixed or paraffin ed tissue), lymph node tissue or bone marrow.

Once the amount ofHA in the sample is determined, the amount can be compared to a l or tlu'eshold level. For example, if the amount of HA is determined to be elevated in the sample, the subject is selected as a candidate for tumor therapy. Exemplary methods for stratification of tumor samples or bodily fluid ' samples for sis, prognosis or selection of subjects for treatment are provided herein.

In one example, a method of diagnosis es a sample of tumor tissue, tumor cells or a bodily fluid containing proteins from a patient. In the method, the presence and level of expression of HA can be ined using an HABP, for example a TSG- 6-LM, TSG~6-LM-Fc or t or mutant thereof, as provided herein. The level of expression of the HA is determined and/or scored and compared to predetermined HA phenotypes associated with disease. As described below, these predetermined values can be determined by comparison or dge ofHA levels in a corresponding normal sample as determined by the same assay of detection and using the same HABP reagent. It is within the level of one of skill in the art to determine the threshold level for disease diagnosis depending on the ular disease, the assay being used for detection ofHA and/or the HABP detection reagent being used. For example, in bodily fluids such as plasma, HA levels greater than 0.015 ug/mL, and generally greater than 0.02 ug/mL, 0.03 ug/mL, 0.04 ug/mL, 0.05 ug/mL, 0.06 ttg/mL or higher correlates to the presence of a tumor or cancer. In another example, in immunohistochemistry methods of tumor tissues with a score of HA+2 or HA+3 can be RECTIFIED SHEET (RULE 91) ISA/EP WO 63155 determinative of disease. Ifthe level is indicative of disease, then the patient is diagnosed with having a tumor.

In another example, a prognostic method utilizes a sample of tumor tissue, tumor cells or a bodily fluid containing proteins from a patient. In the method, the presence and level of expression ofHA can be determined using an HABP, for example a TSGLM, TSGLM-Fc or variant or mutant thereof, as provided herein. The level of expression of the HA is determined and/or scored and ed to predetermined HA phenotypes associated with disease. As described below, these predetermined values can be ined by ison or dge of HA levels in a corresponding normal sample or samples of disease subjects as determined by the same’assay of detection and using the same IIABP reagent. It is within the level of one of skill in the art to determine the threshold levels for prognosis of disease depending on the particular disease, the assay being used for detection ofHA and/or the HABP ion reagent being used. The level of expression ofHA indicates the expected course of disease progression in the patient. For example, high levels ofHA as assessed by inununohistochemistry methods using a quantitative score scheme (e.g.

HA”) correlate to the nce of malignant disease across a range of cancer types.

In another example, HA levels in bodily fluid such as plasma of greater than 0.06 rig/mi. HA also is associated with advanced disease stage.

In a further example of ion diagnostic methods, the level ofHA expression in samples from subjects previously treated with a hyaluronan—degrading enzyme can be monitored to determine Whether a subject being administered the agent has ed an ious blood level of the drug in order to optimize dosing or scheduling.

The following sections describe exemplary HABP reagents and assays for performing the HA detection methods for use in the diagnostic and stic methods, and in particular as companions to therapy with an anti-hyaluronan agent, for example a onan—degrading enzyme. Also described are anti-hyaluronan , including hyaluronan-degrading enzyme agents, for use in treating hyaluronan-associated diseases and disorders and kits and combinations ofHABP reagents with such agents (e. g. hyaiuronan-degrading enzymes). Any ofthe above methods can be performed using any of the described VHABP reagents and assay RECTIFIED SHEET (RULE 91) ISA/EP detection methods alone or in conjunction with therapy with an anti-hyaluronan agent (9. g. a hyaluronan—degrading enzyme).

C. onan Binding Proteins (HABPs) for Use as a Companion Diagnostic " The methods provided herein are directed to quantitative or semi-quantitative measurement of onan in a sampie, such as a tumor or fluid sample from a subject having a tumor or suspected of having a tumor, using a hyaluronan binding protein (HABP). As described herein, tumors that s elevated or high levels of hyaluronan are responsive to treatment with an anti~hyaluronan agent (e. g. hyaluronan degrading ) and the degree oftumor inhibition by an anti-hyaluronan agent (e.g. hyaluronan degrading enzyme) correlates with the degree or amount of hyaluronan accumulation, and not other markers such as expression of endogenous hyaluronan syntheses or hyaluronidases. The HABPs provided for use in the methods herein, in concert with the assays for detection thereof described in Section D, permit c and sensitive detection of HA in s.

The HABP companion diagnostics provided herein can be used in conjunction with y with an anti-hyaluronan agent, such as hyaluronan-degrading enzyme therapeutics or any described in Section E, to select or identify patients predicted to be responsive to treatment and/or to monitor ent and efficacy of ent, thereby providing an improved treatment regimen ofhyaluronan—associated diseases or conditions. For example, the HABP companion diagnostics ed herein can be used to select and/or monitor subjects or patients having a tumor or cancer. In addition, the HABP companion diagnostics also can be used in other diagnostic and prognostic methods of hyaluronan-associated disease or conditions, such as tumors or cancers.

Provided herein are hyaiuronan binding proteins for use in the methods provided herein for the detection and quantitation-ofhyaluronan in a sample. The hyaluronan g proteins can contain full length HABP polypeptides, or portions thereof ning HA binding domains of HABPs, or ent portions thereof to bind HA. Typically, the HABPS or portions f containing an HA binding domain or sufficient portion thereof that binds HA, or variants or multimers thereof exhibit a binding affinity with a dissociation constant (Kd) of at least less than or less than or 1 RECTIFIED SHEET (RULE 91) ISA/EP x lO'7M, and generally at least less than or less than or 9 x 10'8 M, 8 x 10'8 M, 7 x 10'8 M, 6 x 10‘8 M, 5 x 10‘8 M, 4 x 10'8M, 3 x 10‘8 M, 2 x 10'8M, 1 x 10‘8 M, 9 x 10-9 M, 8 x 10‘9 M, 7 x 10-9 M, 6 x 10-9 M, 5 x 10-9 M, 4 x 10-9 M, 3 x 10-9 M, 2 x 10‘9 M, 1 x 10-9 M or lower Kd. As discussed herein, the exhibited binding affinity is generally exhibited under conditions that achieve optimal or close to l g to hyaluronan. In one example, pH conditions can affect binding. For example, as a ion stic herein, binding assays using a TSG-6 reagent, including TSG- 6-LM or sufficient portions thereof to bind HA, variants thereof and multimers thereof, are generally conducted at a pH of at or about n pH 5.8 to 6.4, such as about or pH 6.0.

Hyaluronan binding proteins are of two types: hyaluronan binding proteins that have an HA binding domain that contains one or two link modules, and onan binding proteins that have an HA binding domain that is not a link module. In particular examples, the companion diagnostics provided herein are derived from HABP binding molecules that have only a single link domain that confers HA binding, which can simplify expression, production and purification methods.

The HABPs provided herein can be derived from known HABPs or can be generated synthetically. In some examples, HABPs can be generated synthetically based on conserved residues of HA-binding domains ofknown HABPs. HABPs provided herein also can be derived from HABPs generated from screening methods for HA binding ns, such as phage display or affinity-based screening methods.

The HABPs, including HA g domains of HABPs, or portions thereof that are sufficient to bind to HA, provided herein can be modified to improve one or more ties of HABPs for use in the methods provided herein. For example, the HABPs, or HA binding nts thereof, provided herein can be modified to increase protein expression in mammalian sion systems, e biophysical properties such as stability and solubility, improve protein purification and detection, increase icity for HA and/or increase affinity to HA, as long as they retain their ability to bind to HA. For example, an HABP or HA binding fragment thereof provided herein for use in the methods can be modified to increase its specificity for onan compared to other glycosaminoglycans. In another example, an HABP or HA binding fragment thereof provided herein for use in the s can be linked ly or indirectly to a multimerization domain to increase the number of HA binding sites on the molecule and therefore increase the affinity for binding to HA.

Further, for use as a companion diagnostic herein, any of the HABPs, or portions thereof (e.g, link modules or sufficient ns thereofto bind HA) can be modified to facilitate detection. For example, the companion diagnostics are d by conjugation, directly or indirectly, to , a fluorescent moiety, a radiolabel or Ulllvl Ubtbblaulb taunt.

A description of exemplary HABPs for use as companion diagnostics herein, and modifications thereof, is provided below. 1. HA Binding ns with Link Modules or G1 domains Provided herein as companion diagnostic reagents for use in the methods herein are HA binding ns (HABP) or portions f that contain at least one link module or link domain, and generally at least two or more link modules. In some examples, the HABP contains a G1 domain that contains two link modules. Binding to HA is mediated via the link module. Link modules, also called proteoglycan tandem repeats, are approximately 100 amino acids (aa) in length with four cysteines that are disulfide bonded in the pattern Cysl-Cys4 and CysZ-Cys3. The three dimensional structure of the link s are composed oftwo alpha-helices and two triple ed anti-parallel beta—sheets.

There are three categories of link module-containing proteins: A domain-type proteins that contain a single link module; B -type proteins that contain a single link module extended by an N- and a C-terminal flanking region; and C domain-type proteins that have an extended structure called a G1 domain that contains one N- terminal V-type Ig-like domain followed by a contiguous pair of two link modules.

Modeling and comparison studies have demonstrated a high degree of resolution and conservation of n amino acids between and among link module-containing proteins that correlate to interaction with HA (Blundell et al. (2005) J. Biol. Chem, 280:18189-1 8201). For example, central HA-binding amino acid residues corresponding to Tyr59 and Tyr78 with ing with reference to TSG-6—LM set forth in SEQ ID NO:360 are conserved among odule—containing HABPS via identical or conservative amino acids (ag. aromatic or large and planar faced hobic residues that can also stack against a GlcNAc ring, e.g., Phe, His, Leu or Val) at the corresponding position based on alignment with TSGLM (e.g. set forth in SEQ ID NO:360). Also, basic es at positions corresponding to positions 11 and 81 set forth in SEQ ID NO:360 also are found in other link modules as determined by alignment.

HA binding proteins ning link modules for use in the s provided herein include, but are not limited to, TSG-6 (e.g. set forth in SEQ ID NO: 206 as the precursor and in SEQ ID NO:222 as the mature protein lacking a signal sequence; or the LM set forth in SEQ ID NO:207, 360, 417 or 418, which represent various lengths of the LM as reported in the literature), stabilin-l (e.g. set forth in SEQ ID NO:223 or the mature form thereof; or the LM set forth in SEQ ID NO:37l), stabilin-2 (e.g. set forth in SEQ ID NO:224 or the mature form thereof; or the LM set forth in SEQ ID NO:372), CD44 (6.g. set forth in SEQ ID NO:227 or the mature form thereof; or the LM set forth in SEQ ID NO:375), LYVE-l (e.g. set forth in SEQ ID NO:228 or the mature form thereof; or the link module set forth in SEQ ID NO:376), HAPLNl (e. g.

HAPLNl-l and HAPLNl-2; e.g., set forth in SEQ ID NO:229 or the mature form f; or the LM or LMs set forth in SEQ ID NO:377 or 378), HAPLN2 (e.g.

HAPLN2-l and HAPLN2-2; e. g. set forth in SEQ ID NO:230 or the mature form thereof; or the LM or LMs set forth in SEQ ID NO: 379 or 380), HAPLN3 (e.g.

HAPLN3-l and HAPLN3-2; e. g. set forth in SEQ ID NO:23l or the mature form thereof; or the LM or LMs set forth in SEQ ID NO:38l or 382), HAPLN4 (e.g.

HAPLN4-l and HAPLN4-2; e. g. set forth in SEQ ID NO:232 or the mature form f; or the LM or LMs set forth in SEQ ID NO:383 or 384), aggrecan (e.g. aggrecan l, an 2, aggrecan 3 and aggrecan 4; e.g. set forth in SEQ ID NO:233 or the mature form thereof; or the LM or LMs set forth in SEQ ID NO: 385, 386, 387 or 388), versican (e.g. versican l and versican 2; e. g. set forth in SEQ ID NO:235 or the mature form thereof; or the LM or LMs set forth in SEQ ID NO:39l or 392), breVican (e.g. breVican l and breVican 2; e.g. set forth in SEQ ID NO:234 or the mature form thereof; or the LM or LMs set forth in SEQ ID NO:389 or 390), neurocan (e.g. neurocan l and neurocan 2; e. g. set forth in SEQ ID NO:236 or the mature form thereof; 6.g. the LM or LMs set forth in SEQ ID NO:393 or 394) and acan (e.g. set forth in SEQ ID NO:340 or the mature form thereof). ary of an HABP provided for use in the methods herein is TSG-6.

In particular es herein, the HABP used in the methods herein contains at least one link module, and in some cases ns at least two or at least three link modules. The HABP can be a fiJll-length HABP containing a link module. For example, the companion diagnostic reagent for use in the method herein can contain a sequence of amino acids set forth in any of SEQ ID NOS:206, and 223-236, the mature form thereof, or a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any of SEQ ID NOS: 206, and 223-236.

For e, the HABP for use as a companion diagnostic herein can be a full-length TSG-6 haVing the sequence of amino acids set forth in SEQ ID NO:222, or a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ce identity to a sequence set forth in SEQ ID NO:222.

In other examples, the companion diagnostic reagent for use in the methods herein contains only the link module or sufficient portion of a link module to bind to HA d from a fiJll-length HABP set forth in any of SEQ ID NOS: 206, and 223- 236 or the mature form thereof (lacking the signal sequence). In some examples, the HABP containing a link module or modules is not the complete sequence of an HABP set forth in any of SEQ ID NOS: 206, and 223-236 or the mature form thereof (lacking the signal sequence). It is understood that the portion of an HABP or link module is generally a contiguous sequence of amino acids that is generally at least 50 amino acids in length, 60, 70, 80, 90, 100, 200, 300 or more amino acids. In some examples, the link module or modules is the only HABP portion of the companion diagnostic binding molecule. For example, the companion diagnostic reagent for use in the method herein ns only a portion of a full-length HABP and has a sequence of amino acids set forth in any of SEQ ID NOS:207, 360, 361, 371-394 and 416-418 or a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 9 l %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418.

In examples herein, the companion diagnostic reagent for use in the methods herein contains a G1 domain or sufficient portion f to bind to specifically bind to HA. The HABP ning the G1 domain can be derived from a full-length HABP set forth in any of SEQ ID NOS: 233-236 or the mature form thereof. In some examples, the HABP containing the G1 domain is not the complete sequence of an HABP set forth in any of SEQ ID 3-236 or mature form thereof. It is understood that the portion of an HABP containing a G1 domain is generally a contiguous sequence of amino acids that is generally at least 100 amino acids in length, such as 150, 200, 250, 300, 400, or more amino acids. In some examples, the G1 domain is the only HABP portion ofthe companion diagnostic binding molecule.

For example, the companion diagnostic reagent for use in the method herein contains only a portion of a full-length HABP and has a G1 domain having a sequence of amino acids set forth in any of SEQ ID NOS: 423-426 or a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a ce set forth in any of SEQ ID NOS: 423-426.

In some examples, the companion diagnostic can contain more than one link module, such as two or three link modules. The link modules can be from the same or different HABP. The companion diagnostics can contain link modules that are linked directly or indirectly to form a single polypeptide. In other examples, the companion diagnostics can contain link modules that are set forth as separate polypeptides that are ally linked, such as Via a de bond. Exemplary of an HABP fragment provided for use in the methods herein is the link domain of TSG—6 (TSG—6-LM), or a portion thereof sufficient to bind to HA.

In some examples, the HABP is a multimer containing two or more link modules that are linked directly or indirectly via a multimerization domain to effect the ion of dimer or trimer molecules and the generation of le HA g sites. For example, a companion diagnostic for use in the methods herein is one that is generated by expression of a nucleic acid molecule encoding the link module set forth in any one of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418 or a sequence of amino acids that ts at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any RECTIFIED SHEET (RULE 91) ISA/EP WO 63155 of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418 linked directly or indirectly to a nucleic acid encoding a multimerization domain, such as an Fc portion of an immunoglobulin. Hence, the resulting HABP multimer or LM-multimer contains a first polypeptide set forth in any one of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418 or a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418 linked directly or indirectly to a multimerization domain; and a second polypeptide set forth in any one of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418 or a ce of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any of SEQ ID NOS: 207, 360, 361, 371-394 and 416-418 linked directly or indirectly to a multimerization domain. The ce of the link module in the first and second polypeptide can be the same or different. Exemplary of an HABP multimer provided for use in the methods herein is a er containing two polypeptide chains, whereby each contains the TSGLM, variant thereof or sufficient portion thereof to bind HA linked directly or ctly to a multimerization domain that effects multimerization. For example, provided herein for use in the methods is a TSG LM:Fc molecule (see e.g. SEQ ID NO:212 or 215).

A description of exemplary HABPs ning link domains, including structure and on ption, is ed below. Any of the described HABPs or ns thereof, such as a fragment containing only a link domain or sufficient portion thereof to bind HA, can be used as a companion diagnostic reagent in the methods . It is understood that reference to amino acids, including to a specific sequence set forth as a SEQ ID NO used to describe domain organization of a link domain or other domain are for illustrative purposes and are not meant to limit the scope of the embodiments provided. It is understood that polypeptides and the description of domains thereof are tically derived based on homology analysis and alignments with similar molecules. Thus, the exact locus can vary, and is not necessarily the same for each HABP. Hence, the specific domain, such as specific link domain, can be several amino acids (one, two, three or four) longer or shorter. a. Type A: TSG—6 sub-group Provided herein as a companion diagnostic for use in the methods herein are HABPS that are members of the Type A oup that contain a single link module that binds to hyaluronan. Type A HABPs bind to HA with a minimum chain length of six sugars, hexasaccharide (HA6), or greater. Members of the Type A sub-group that can be used as companion diagnostics in the methods provided herein include, but are not limited to, TSG—6, Stabilin~l, Stabilin-Z, CAB61358 and KIAA0527, link modules thereof, or sufficient portions of a link module that binds HA. i. TSG-6 Exemplary of a Type A sub-group HABP provided for use as a companion diagnostic reagent in the methods ed herein is TSG-6, or a link module thereof, a sufficient portion of a link module to bind to HA, variants thereof or multimers thereof. Tumor necrosis factor-Stimulated Gene—6 (TSG—6, tumor necrosis factor alpha—induced protein 6, TNFAIP6; SEQ ID NO:206) is a ~35 kDa secreted IS glycoprotein composed of a single inal link module and inal CUB domain. Expression of TSG—6 is inducedin many cell types by atory mediators, including cytokines and growths factors. Via its link module, TSG—6 is a potent inhibitor ofpolymorphonuclear leukocyte migration. TSG-6 forms a stable complex with the serine protease inhibitor Inter-alpha—Inhibitor (led) and potentiates the anti-plasmin ty of Iul. TSG—6 also is ant for the formation and remodeling of h pericellular coats and extracellular matrices.

The human TSG—6 transcript (SEQ ID NO:205) is ly translated to form a 277 amino acid precursor peptide (SEQ ID NO:206) containing a 17 amino acid signal sequence at the N—terminus. The mature TSG-6 (set forth in SEQ ID NO:222), therefore, is a 260 amino acid protein containing amino acids 18—277 of SEQ ID N02206 (Lee et a]. (1992) J Cell Biol 5—557). TSG—6 is composed of two main domains, the link module and the CUB . The link module of TSG-6 is variously reported in the literature to be located at amino acids 35-129, 36-128, 36- 129 or 36-132 of SEQ ID N01206 (set forth as SEQ ID NOS: 207, 360, 417 or 418, respectively). It is understood that reference to loci ofa domain can vary by several amino acids due to differences in alignments. Hence, for purposes herein, a TSG ‘LM is one set forth in any ofSEQ ID NOS: 207, 360, 417 or 418 or that varies from RECTIFIED SHEET (RULE 91) ISA/EP 2012/061743 such sequence by one, two or three amino acids. The CUB domain is located at amino acids 135-246 of SEQ ID N02206. Human TSG-6 has two potential N-linked glycans at residues N118 and N258 of SEQ ID NO:206. In addition, residues T259 and T262 of SEQ ID NO:206 are phosphorylated (Molina et al. (2007) Proc Natl Acad Sci USA 99—2204). Human TSG~6 has eight native cysteines which form four disulfide bonds at residues C58-C127, C82-C103, l61 and C188-C210 of preprotein TSG-6 (SEQ ID NO:206).

TSG-6 link module (SEQ ID NO:360) has a relatively small size and a well- characterized structure. Thethree ional structure of the TSG-6 link domain was determined and found to have the same fold as other known link modules, containing two alpha helices and two antiparallel beta sheets arranged around a large hydrophobic core (Kohda et al. (1996) Cell 86:767-775). In addition, the interaction ofthe link module of TSG-6 and HA has been studied revealing that the ic rings of Tyrl 2, Tyr59, Phe70, Tyr78, Trp88 and basic residues Lysl 1, Lys72, Asp77, Arg 81, and G1u86 of the link domain of TSG-6 (SEQ ID N02360) are important for binding to HA (see, e.g., Kahmann et al. (2000) ure 82763-774; y et al. (2001) J Biol Chem 276:22764-22771; Kohda et al. (1996) Cell, 882767-775; Blundell et al. (2003) J Biol Chem 278 :49261-49270; Lesley et al. (2004) JBiol Chem 279:25745-25754; Blundcll et al. (2005) J Biol Chem 280: l 8 1 89-18201). Structural studies also show that there is only a single HA-binding site ned in the link module, which is localized to one region of the molecule based on the structural map dues Lysl 1, Tyrl2, Tyr59, Phe70 and Tyr78 that are most directly implicated in HA binding (see e.g. Mahoney et al. (2001) J Biol Chem 276:22764-22771).

The link module of TSG-6 exhibits binding activity to several glycosaminoglycans. For example, studies have revealed binding of the link module to HA, chondroitin—4-sulphate (C48), Gl-domain of the proteoglycan aggrecan, heparin and the bikunin chain of Ial (see e.g. , Milner er al. (2003) Journal ofCell Science, 116:1863—1873; Mahoney et al. (2005) Journal ofBiological Chemistry, 280:27044-27055). The binding of TSG-6 to heparin and HA is ed by a distinct binding site in the LM of TSG—6. The residues ed in TSGLM binding to hyaluronan are Lysl l, Tyr12, Tyr59, Phe70 and Tyr78, whereby the mutants Kl 1Q, Y12F, Y59F, F70V and Y78F have between 10-and IOO-fold lower RECTIFIED SHEET (RULE 91) ISA/EP HA-binding affinity compared to wildtype; the residues in the TSG LM involved in binding to heparin are LysZO, Ly534, Lys4l , Ly354, Arg56 and Arg84, whereby the mutants K20A, K34A, K41A and K54A exhibit impaired n binding properties; and the residues ed in TSG—6—LM binding to bikunin is overlapping with but not identical to the HA binding site (Mahoney er a1. (2005) Journal ogical Chemistry, 280:27044—27055).

Binding of TSG~6 to hyaluronan is pH dependent, with binding activity exhibited at acidic pH of about or pH 5.6 to 6.4, such as or about pH 5.8 to pH 6.0.

TSG—6 polypeptides, HA binding s thereof, c.g. link modules, , TSG~6 or fragments thereof sufficient to bind to HA provided herein for use as a companion stic in the in, the methods herein can include any of SEQ ID NOS: 206, 207, 222, 360, 417 or 418, or variants thereof such as variants that exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOS: 206, 207, 222, 360, 417 or 418.

Exemplary variants include, for example, species variants, c variants and variants that contain conservative and non—conservative amino acid mutations. Natural allelic variants of human TSG—6 e, for example, TSG-6 containing the amino acid replacement Q144R (SEQ ID NO:407, Nentvw'ch et al. (2002) J Biol Chem 277:15354—15362). TSG—G is highly conserved among species with mouse and human protein being >94% identical. Species ts of TSG—6 or HA binding fragments thereof for use as a companion diagnostic in the methods provided herein also include, but are not limited to, mouse (SEQ ID NO:252), rabbit (SEQ ID N0:253), bovine (SEQ ID N01254), horse (SEQ ID NO:409), chimpanzee (SEQ ID NO:408), dog (SEQ ID ), mouse (SEQ ID NO:4l 1), chicken (SEQ ID NO:412), frog Xenopus laevis (SEQ ID NO:413), zebra fish (SEQ ID NO:414), mature forms thereof or link modules or sufficient ns thereofto bind HA. ts of TSG—6 or HA binding fragments thereof for use in the provided methods include variants with an amino acid modification that is an amino acid replacement (substitution), deletion or insertion. Exemplary ations are amino acid replacements such as an amino acid replacement at any o acid residues 4, 6, 8, 13, 20, 29, 34, 41, 45, 54, 67, 72 or 96 corresponding to residues in the TSG—6 set forth in SEQ ID NO: 360, 417 or 418.7Thereplacement amino acid can be any RECTIFIED SHEET (RULE 91) ISA/EP 2012/061743 other amino acid residue. Exemplary amino acid replacements of a TSG-6 polypeptides or HA binding fragments thereof provided herein for use as a companion diagnostic reagent in the methods provided herein include modified TSG-6 ptides or HA-binding fragments thereof that contain at least one amino acid replacement corresponding to H4K, H4S, E6A, E6K, R8A, Kl3A, K20A, H29K, K34A, K4lA, H45S, K54A, N67L, N67S, K72A, H96K, K34A/K54A or K20A/K34A/K41A corresponding to residues in the TSG-6 set forth in SEQ ID NO: 360, 417 or 418 (see, e.g., Mahoney et al. (2005) JBz'ol Chem 280:27044-27055, Blundell et al. (2007) JBz'ol Chem 282: 12976-12988, Lesley et al. (2004) JBz'ol Chem 279:25745-25754, Kahmann et al. (2000) Structure 15:763-774). It is understood that residues important or otherwise required for the binding of TSG-6 to HA, such as any bed above or known to one of skill in the art, are generally invariant and cannot be changed. Thus, for example, amino acid residues ll, 12, 59, 70, 78 and 81 of SEQ ID NO: 360 in the link module of TSG-6 are generally invariant and are not altered. Further, it is tood that amino acid modifications that result in improper folding or perturbation of the folding of the link module are generally invariant. Thus, for example, a modified TSG-6 provided for use in the methods herein will not n any one or more of the amino acid modifications H4S, H29A, H45A, H45K, R56A, D77A, R84A and D89A of SEQ ID NO:360 (Mahoney et al. (2005) JBz'ol Chem 280:27044-27055, Blundell et al. (2007).]Bz'01 Chem 282: 12976- 12988, Lesley et al. (2004).]Bz'01 Chem 279:25745-25754).

In particular, the modification, for example amino acid replacement or replacements, is one that confers an altered, such as improved, activity ed to a TSG-6 not containing the modification. Such variants include those that contain amino acid modifications that enhance the binding affinity of TSG-6 to HA, increase the specificity of TSG-6 for HA, and/or increase the solubility of TSG-6. For example provided herein for use in the methods herein are TSG-6 variants, HA binding domains, or portions f sufficient to bind to HA that increase the city of TSG-6 for HA by sing the binding of TSG-6 to other glycosaminoglycans, including heparin, chondroitinsulfate, heparan sulfate and dermatan sulfate. Binding to the other aminoglycan that is not onan can be reduced at least l.2-fold, l.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold or more compared to binding of TSGLM not containing the modification. For example, provided herein is a mutant TSGLM containing amino acid replacement(s) at amino acid residues , 34, 41, 54, 56, 72 and/or 84, and in particular at amino acid residues 20, 34, 41, and/or 54 sponding to amino acid residues set forth in SEQ ID NO:206). The replacement amino acid can be to any other amino acid residue, and generally is to a non-basic amino acid residue. For example, amino acid replacement can be to Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Ala (A), Val (V), Ile (I), Leu (L), Met (M), Phe (F), Tyr (Y) or Trp (W). The amino acid replacement or replacements confer decreased binding to heparin. For example, ts that decrease the ability of TSG-6 to bind to heparin are known to one of skill in the art. Such variants are those that e at least one mutation corresponding to K20A, K34A, K41A and K54A, including variants 54A or K20A/K34A/K41A (Mahoney et al. (2005) JBiol Chem 280:27044-27055). ary variants that decrease or reduce g to heparin are variant TSGLM set forth in SEQ ID NO:361 or 416.

Exemplary of a TSG-6 polypeptide provided herein for use in the methods provided herein is a TSG-6 polypeptide that contains at least an HA binding domain, for example, a TSG-6 link module. Thus, provided herein is a TSG-6 link module, or variant thereof, for use in the provided methods. Exemplary of such a polypeptide reagent is one that has a sequence of amino acids set forth in SEQ ID NO: 207, 360, 361, 416, 417 or 418, or has a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 95%, 99% or more sequence identity to any of SEQ ID NOS: 207, 360, 361, 416, 417 or 418. For e, the TSG-6 link module can be modified to alter its specificity, affinity or solubility, as long as it s its ability to bind to HA.

In yet another example, the affinity of the TSG-6 link module is increased by dimerization or multimerization, such as, for example, by fusion to a multimerization domain, such as an EC domain (see Section C3 . Hence, the TSG-6 link module can be modified to produce a multimer containing two or more link modules that are linked directly or indirectly via a multimerization domain to effect the formation of dimer or trimer molecules and the generation of multiple HA binding sites. For example, a ion diagnostic for use in the methods herein is one that is generated by expression of a nucleic acid molecule encoding the link module set forth in any one of SEQ ID NOS: 207, 360, 361, 417 or 418 or a nucleic acid encoding a link module having a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ce ty to a sequence set forth in any of SEQ ID NOS: 207, 360, 361, 417 or 418 linked directly or indirectly to a nucleic acid encoding a multimerization domain, such as an Fc portion of an immunoglobulin. Hence, the resulting TSGLM multimer contains a first polypeptide set forth in any one of SEQ ID NOS: 207, 360, 361, 417 or 418 or a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ce identity to a sequence set forth in any of SEQ ID NOS: 207, 360, 361, 417 or 418 linked directly or indirectly to a erization domain; and a second polypeptide set forth in any one of SEQ ID NOS: 207, 360, 361, 417 or 418 or a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any of SEQ ID NOS: 207, 360, 361, 417 or 418 linked directly or indirectly to a multimerization domain.

Generally, the LM or sufficient portion thereof to effect HA binding is the only TSG- 6 portion of the multimer. For example, provided herein for use in the methods is a TSGLM:Fc molecule (see e.g. SEQ ID NO:212 or 215).

In r example, the TSG-6 link module is linked to a PC domain to increase its solubility (see Section C3 below). ii. Stabilin-l and Stabilin-Z Exemplary of a Type A sub-group HABP provided for use as a companion diagnostic reagent in the s provided herein is Stabilin-1 or Stabilin-2, or a link module thereof, a sufficient portion of a link module to bind to HA, variants thereof or multimers thereof Stabilin-1 (also called STABl, CLEVER-l, 46, FEEL- 1, FEX-l and ; SEQ ID NO:223) and Stabilin-2 (also called STAB2, FEEL-2, CD-44 like precursor FELL2, DKFZp434E0321, FEX2, and hyaluronan receptor for endocytosis/HARE; SEQ ID NO:224) are type I transmembrane members of a family of fasciclin-like hyaluronan (HA) receptor homologs. Both contain seven fasciclin- like adhesion domains, multiple EGF-like repeats, and onan-binding link s. Both Stabilin-1 and Stabilin-2 are expressed on idal endothelium and hages, though each is functionally distinct. Stabilin-1 is involved in two intracellular trafficking pathways: receptor mediated endocytosis and ing; and ing between the endosomal compartment and trans-Golgi network (TGN).

Stabilin-2 acts as a scavenger receptor for HA and AGE-modified proteins.

The precursor sequence of Stabilin-l is set forth in SEQ ID NO:223. The link module of in-1 is located at 2208-2300 of SEQ ID NO:223 and is set forth in SEQ ID NO:371. The precursor sequence of in-2 is set forth in SEQ ID NO:224 and the link module of Stabilin-2 is located at amino acids 290 of SEQ ID NO:224 and is set forth in SEQ ID NO:372.

Stabilin-1 or Stabilin-2 polypeptides, HA binding domains thereof, e.g., Stabilin-LM modules or fragments thereof sufficient to bind to HA provided herein for use as a companion diagnostic in the methods herein include the link module set forth in SEQ ID NO:371 or 372, or variants thereof that exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOS: 371 or 372. The variants include ts that exhibit specific binding to HA. Variants include allelic variants, species variants or other variants containing an amino acid modification (6.g. to increase affinity or specificity to HA). Species variants of stabilin-1 provided for use in the methods herein include, but are not limited to, mouse (SEQ ID NO:255) and bovine (SEQ ID NO:256) and s variants of stabilin-2 provided for use in the methods herein include, but are not limited to, mouse (SEQ ID NO:257) and rat (SEQ ID NO:25 8).

Also provided herein for use as a companion diagnostic in the methods herein is a StablinLM or Stabilin-l-LM multimer that exhibits increased affinity for HA.

For example, a companion diagnostic for use in the methods herein is one that is generated by expression of a nucleic acid molecule encoding the link module set forth in any one of SEQ ID NOS: 371 or 372 or a nucleic acid ng a link module having a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any of SEQ ID NOS: 371 or 372 linked directly or ctly to a nucleic acid encoding a erization domain, such as an Fc portion of an immunoglobulin. Hence, the resulting LM er contains a first polypeptide set forth in any one of SEQ ID NOS: 371 or 372 or a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any of SEQ ID NOS: 371 or 372 linked directly or ctly to a multimerization domain; and a second polypeptide set forth in any one of SEQ ID NOS: 371 or 372 or a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any of SEQ ID NOS: 371 or 372 linked directly or indirectly to a multimerization domain. b. Type B: CD44 sub-group Provided herein as a ion stic reagent for use in the methods herein are HABPs that are members of the Type B sub-group having an HA-binding domain that contains a single link module with N- and C- terminal extensions that binds to hyaluronan. Unlike the HA binding domain of the Type A/TSG-6 sub-group, the flanking sequences of the link domain are essential for the structural integrity of the Type B domain and are required for g to HA. Members of the Type B sub- group of HABPs for use in the methods provided herein include, but are not limited to, CD44 and LYVE-l, or HA binding fragments thereof. i. CD44 A Type B sub-group HABP provided for use in the methods herein is CD44, HA g domains of CD44 or ns thereof sufficient to bind to HA. CD44 is an 80- to 250-kDa Type I transmembrane glycoprotein that binds hyaluronan and a variety of extracellular and cell-surface ligands. CD44 has diverse functions and is involved in attachment, organization and turnovers of the extracellular matrix and mediates the migration of lymphocytes during inflammation. The ability of CD44 to ct with HA is ted by factors, including receptor clustering and changes in glycosylation of the extracellular domain. CD exists in numerous isoforms due to alternative splicing of 10 variant exons, all of which contain the hyaluronan binding domain containing the link module. An exemplary CD44 filll length ce is set forth in SEQ ID NO:227. The hyaluronan binding domain of CD44 is approximately 160 amino acids in length (SEQ ID NO: 341) and contains the link module flanked by N- and C-terminal extensions linked by a de bond (Cys9 and Cys110 of the CD44 HA binding domain set forth in SEQ ID NO: 341). Arg41 and Arg78 are critical for HA binding (corresponding to amino acids Arg22 and Arg59 of the CD44 HA binding domain set forth in SEQ ID NO: 341) and Tyr42 and Tyr79 sponding to amino acids Tyr23 and Tyr60 of the CD44 HA binding domain set forth in SEQ ID NO: 341) are essential for CD44 onal activity. The link domain of CD44 is set forth in SEQ ID NO:375. Thus provided herein for use in the methods herein are fragments of CD44 that retain the ability to bind to HA, for example, a fragment of CD44 that contains a link domain and N— and C-terminal flanking domains or a sufficient portion thereof to effect binding to HA.

Also provided herein for use in the provided methods are variants, ing c variants, species variants and other variants containing an amino acid modification, as long as the variants retain their ability to bind to HA. s variants of CD44 for use in the methods provided herein include, but are not limited to, mouse (SEQ ID NO:259), rat (SEQ ID NO:260), bovine (SEQ ID NO:26l), dog (SEQ ID NO:262), horse (SEQ ID NO:263), hamster (SEQ ID NO:264), baboon (SEQ ID NO:265) and golden hamster (SEQ ID NO:266). Variants of CD44, or HA binding fragments thereof, for use in the provided s include variants that have an amino acid modification and that exhibit an altered, such as improved, activity compared to a CD44 not containing the modification. Such variants include those that n amino acid ations that enhance the binding affinity of CD44 to HA, increase the specificity of CD44 for HA, and/or increase the solubility of CD44. ii. LYVE-l Provided herein for use in the methods provided herein is a Type B sub-group HABP that is LYVE-l HA binding domains of LYVE-l or portions f sufficient to bind to HA. Lymphatic Vessel Endothelial onan (HA) Receptor-l (LYVE- 1, also called CRSBP-l, HAR, and XLKDl; SEQ ID NO:228) is a 60-kDa type I transmembrane glycoprotein that is expressed on both the l and ablumenal surfaces of lymphatic endothelium, and also on hepatic blood sinusoidal endothelia.

LYVE-l ipates in HA internalization for degradation and transport ofHA from tissues into the lumen of lymphatic vessels. LYVE-l-directed HA localization to lymphatic surfaces also affects aspects of the immune response or tumor metastases.

The link module of LYVE-l is located at amino acids 40-129 of SEQ ID NO:228 and is set forth in SEQ ID NO:376. Thus provided herein for use in the methods herein are fragments of LYVE-l that retain the ability to bind to HA, for example, a fragment of LYVE-l that contains a link domain and N— and C-terminal flanking domains or a sufficient portion thereof to effect binding to HA.

Also provided herein for use in the provided methods are variants, including c variants, species variants and other variants containing an amino acid modification, as long as the variants retain their ability to bind to HA. Species ts of LYVE-l include, but are not limited to, mouse (SEQ ID ) and bovine (SEQ ID NO:268). Variants of LYVE-l, or HA binding fragments thereof, for use in the ed methods include variants that have an amino acid modification and that exhibit an altered, such as improved, activity compared to a LYVE-l not containing the modification. Such variants include those that contain amino acid ations that enhance the binding affinity of LYVE-l to HA, increase the specificity of LYVE-l for HA, and/or increase the solubility of LYVE-l. c. Type C: Link Protein sub-group Provided herein for use as a ion diagnostic reagent in the methods herein are HABPs that are members of the Type C sub-group having an HA binding domain that contains an immunoglobulin (Ig) domain, which mediates binding between link protein and other Type C HA binding proteins, and two link modules, both of which are required for binding to HA. The Ig domain and two link modules collectively make up the G1 domain of Type C HABPs. Members of the Type C sub- group of HABPs for use in the methods provided herein include, but are not limited to, HAPLNl/link n, HAPLN2, HAPLN3, HAPLN4, an, versican, brevican, an, and acan, or HA binding fragments thereof i. HAPLN/Link Protein family The Hyaluronan and Proteoglycan Link Protein (HAPLN) family is made up of four secreted proteoglycans that bind hyaluronan and contain one e C2-set domain and two link domains. (1) HAPLNl A Type C sub-group HABP ed herein for use in the methods is HAPLNl HA binding domains of HAPLNl or portions thereof ent to bind to HA. Hyaluronan and Proteoglycan Link Protein 1 (HAPLNl, also called as link protein and CRTLl; SEQ ID NO: 229) contributes to extracellular matrix stability and flexibility by stabilizing ctions ofHA with chondroitin sulfate proteoglycans.

HALPNl contains two link modules (amino acids 159-253 and amino acids 260-350 of SEQ ID NO: 229) that bind to HA and an Ig module (amino acids 53-160 of SEQ ID NO: 229) that binds to the Ig module of the G1 domain of aggrecan. HAPLNl stabilizes associations ofHA with aggrecan by forming a ternary complex ning an HA linear backbone with perpendicularly attached aggrecan and HAPLNl. an and HAPLNl lie parallel to each other, while HA runs between the two HAPLNl link modules and the two an link modules. The complex creates a gel-like substance with resistance to deformation. HAPLNl also stabilizes the interaction ofHA with other chondroitin sulfate proteoglycans, such as versican, neurocan, and brevican, which also have Gl s containing an Ig module and two link modules, similar to aggrecan.

The G1 domain of HAPLNl contains the Ig domain and the 2 link modules.

The Ig domain of the G1 domain of HAPLNl is located at amino acids 53-160 of SEQ ID NO:229. The link modules of the G1 domain of HAPLNl are located at amino acids 159-253 and 259-350 of SEQ ID NO:229 and are set forth in SEQ ID 7 and 378. Thus, provided herein for use in the s herein are fragments of HAPLNl that retain the ability to bind to HA, for example, a fragment of HAPLNl that contains the G1 domain or a sufficient portion thereof to effect binding to HA.

For example, provided herein for use in the methods herein is a HA binding fragment of HAPLNl that contains at least the two link modules.

Typically, for use as a diagnostic for the detection of HA, HAPLNl is provided in ation with another HA binding protein that contains the HA- binding region, such as, for e, the G1 domain of another Type C HABP, such as aggrecan, versican, brevican, neurocan, or phosphacan.

Also provided herein for use in the provided methods are variants, including allelic variants, species variants and other variants ning an amino acid modification, as long as the variants retain their ability to bind to HA. Species variants of HAPLNl include, but are not limited to, bovine (SEQ ID NO:269 and 273), mouse (SEQ ID NO:270), rat (SEQ ID NO:271), chicken (SEQ ID NO:272), horse (SEQ ID NO:274) and pig (SEQ ID NO:275). Variants of HAPLNl, or HA binding fragments thereof, for use in the provided s include variants that have an amino acid modification and that exhibit an altered, such as ed, activity compared to a HAPLNl not containing the modification. Such variants include those that n amino acid modifications that enhance the binding affinity of HAPLNl to HA, increase the specificity of HAPLNl for HA, and/or se the lity of HAPLNl. (2) HAPLN2 Provided herein for use in the methods provided herein is a Type C sub-group HABP that is HAPLN2, HA binding domains of HAPLN2 or portions f sufficient to bind to HA. Hyaluronan and Proteoglycan Link n 2 (HAPLN2; SEQ ID NO: 230), also known as brain link protein 1, is predominantly expressed in brain. The G1 domain of HAPLN2 contains the Ig domain and the 2 link modules.

The Ig domain of the G1 domain of HAPLN2 is located at amino acids 49-149 of SEQ ID NO:230. The link modules of the G1 domain of HAPLN2 are located at amino acids 148-241 and 247-337 of SEQ ID NO:230 and are set forth in SEQ ID NOS:379 and 380.

Thus, ed herein for use in the methods herein are nts of HAPLN2 that retain the ability to bind to HA, for example, a fragment of HAPLN2 that contains the G1 domain or a sufficient portion thereof to effect binding to HA. For example, provided herein for use in the methods herein is a HA binding fragment of HAPLN2 that contains at least the two link modules. Typically, for use as a diagnostic for the detection of HA, HAPLN2 is ed in combination with another HA binding protein that contains the HA-binding region, such as, for example, the G1 domain of another Type C HABP, such as aggrecan, versican, brevican, neurocan, or phosphacan.

Also provided herein for use in the provided methods are variants, including allelic variants, species variants and other variants containing an amino acid modification, as long as the variants retain their ability to bind to HA. Species ts of HAPLN2 include, but are not limited to, mouse (SEQ ID NO:276), rat (SEQ ID NO:277) and bovine (SEQ ID NO:278). Variants of HAPLN2, or HA binding fragments thereof, for use in the provided methods include variants that have an amino acid modification and that exhibit an altered, such as improved, activity compared to a HAPLN2 not ning the modification. Such variants include those that contain amino acid modifications that enhance the binding affinity of HAPLNZ to HA, increase the specificity ofHAPLNI for HA, and/or increase the solubility of HAPLNZ. (3) HAPLN3 A Type C oup HABP provided herein for use in the methods herein is HAPLN3, HA binding domains of HAPLN3 or portions thereof sufficient to bind to HA. Hyaluronan and glycan Link Protein 3, (HAPLN3; SEQ ID N02231), functions in hyaluronic acid binding and cell adhesion. HAPLN3 is upregulated in breast cancer and, thus, may be related to cancer development and metastasis. The G1 m rinmqin nFI—IAPIN’X nnntaine the Ig rlnmain and the 7 link mndnles The Ig domain nf‘ the G1 domain ofHAPLN3 is located at amino acids 62-167 of SEQ ID . The link modules of the G1 domain ofHAPLN3 are located at amino acids 166—260 and 266-357 ofSEQ ID N0:231 and are set forth in SEQ ID NOS:381 and 382.

Thus, provided herein for use in the methods herein are fragments of HAPLN3 that retain the ability to bind to HA, for e, a fiagment ofHAPLN3 that contains the G1 domain or a sufficient portion thereof to effect binding to HA. For example, ed herein for use in the methods herein is a HA binding fragment of HAPLN3 that contains at least the two link modules. Typically, for use as a diagnostic for the detection ofHA, HAPLN3 is provided in combination with another HA binding protein that contains the HA—binding region, such as, for example, the G1 domain of r Type C HABP, such as aggrecan, versican, brevican, neurocan, or acan.

Also provided herein for use in the methods herein are ts, including allelic variants, species variants and other ts containing an amino acid modification, as long as the variants retain their ability to bind to HA. Species variants of HAPLN3 include, but are not limited to, mouse (SEQ ID NO:279), rat (SEQ ID NO:280) and bovine (SEQ ID NO:281). Variants ofHAPLN3, or HA binding fragments thereof, for use in the ed methods include variants that have an amino acid modification and that t an altered, such as improved, activity compared to a HAPLN3 not containing the modification. Such variants e those that n amino acid modifications that enhance the binding affinity of HAPLN3 to RECTIFIED SHEET (RULE 91) ISA/EP HA, increase the specificity of HAPLN3 for HA, and/or increase the solubility of HAPLN3. (4) HAPLN4 ed herein for use in the methods herein is a Type C sub-group HABP that is HAPLN4, HA binding domains ofHAPLN4 or portions thereof sufficient to bind to HA. Hyaluronan and Proteoglycan Link Protein 4, (HAPLN4; SEQ ID NO:232), also known as brain link protein 2, is predominantly expressed in brain.

HAPLN4 participates in the development of the perineuronal matrix. Human and mouse HAPLN4 share 91% amino acid sequence ty. The G1 domain of HAPLN4 contains the Ig domain and the 2 link modules. The Ig domain of the G1 domain ofHAPLN4 is located at amino acids 60-164 of SEQ ID NO:232. The link modules of the G1 domain of HAPLN4 are located at amino acids 163-267 and 273- 364 of SEQ ID NO:232 and are set forth in SEQ ID NOS:383 and 384.

Thus, provided herein for use in the methods herein are fragments of HAPLN4 that retain the y to bind to HA, for example, a nt of HAPLN4 that contains the G1 domain or a sufficient portion thereof to effect binding to HA. For example, provided herein for use in the methods herein is a HA binding fragment of HAPLN4 that contains at least the two link modules. Typically, for use as a stic for the ion of HA, HAPLN4 is provided in combination with another HA binding protein that contains the HA-binding region, such as, for example, the G1 domain of another Type C HABP, such as aggrecan, an, brevican, neurocan, or phosphacan.

Also provided herein for use in the provided methods are variants of , including allelic variants, species variants and other variants containing an amino acid modification, as long as the ts retain their ability to bind to HA.

Species variants of HAPLN4 include, but are not limited to, mouse (SEQ ID NO:282), bovine (SEQ ID ) and rat (SEQ ID NO:284). Variants of HAPLN4, or HA binding fragments thereof, for use in the provided methods e variants that have an amino acid modification and that exhibit an altered, such as improved, activity compared to a HAPLN4 not containing the modification. Such variants include those that contain amino acid modifications that e the binding affinity ofHAPLN4 to HA, increase the specificity of HAPLN4 for HA, and/or increase the lity of HAPLN4. (5) Aggrecan Provided herein for use in the methods herein is a Type C sub-group HABP that is aggrecan, HA binding s of aggrecan or portions thereof sufficient to bind to HA. Aggrecan (SEQ ID NO:233) belongs to the chondroitin sulfate (CS) proteoglycan family, which also includes versican, brevican, neurocan, and phosphacan. Each aggrecan le contains approximately 100 and 30 keratan sulfate and glycosaminoglycan (GAG) side chains, respectively. Aggrecan non- covalently associates with hyaluronan via the link modules and an Ig domain in its N- terminus. It is the most abundant proteoglycan in cartilage, and contributes to the load-bearing capacity of this tissue.

The G1 domain of an is located at amino acids 45-352 of SEQ ID NO:233. The Ig domain of the G1 domain of an is d at amino acids 45- 154 of SEQ ID NO:233 and is set forth in SEQ ID . The link s of the G1 domain of aggrecan are located at amino acids 153-247 and 254-349 of SEQ ID NO:233 and are set forth in SEQ ID NOS:385 and 386. Link modules 3 and 4 are set forth in SEQ ID NOS:387 and 388. Thus, provided herein for use in the methods herein are fragments of aggrecan that retain the ability to bind to HA, for example, a fragment of aggrecan that contains the G1 domain or a sufficient portion f to effect binding to HA.

Also provided herein for use in the provided methods are variants of aggrecan, including allelic variants, species variants and other variants containing an amino acid modification, as long as the ts retain their ability to bind to HA. Species variants of aggrecan include, but are not limited to, pig (SEQ ID NO:285), chicken (SEQ ID ), mouse (SEQ ID NO:287), bovine (SEQ ID NO:288), dog (SEQ ID NO:289), rat (SEQ ID ) and rabbit (SEQ ID NO:29l). Variants of aggrecan, or HA binding fragments f, for use in the provided methods include variants that have an amino acid modification and that exhibit an altered, such as improved, activity compared to an aggrecan not containing the modification. Such variants include those that contain amino acid modifications that enhance the binding affinity of aggrecan to HA, increase the specificity of aggrecan for HA, and/or se the solubility of aggrecan. (6) Brevican Provided herein for use in the methods herein is a Type C sub-group HABP that is brevican, HA binding domains of brevican or portions thereof sufficient to bind to HA. Brevican (SEQ ID NO:234) is a 160 kDa member of the aggrecan/versican proteoglycan family of matrix proteins. It is brain-derived and serves as a linker between hyaluronan and other matrix molecules such as the tenascins and fibulins.

The G1 domain of an is located at amino acids 51-356 of SEQ ID NO:234 and is set forth in SEQ ID NO:424. The Ig domain of the G1 domain of brevican is located at amino acids 51-158 of SEQ ID NO:234. The link modules of the G1 domain of an are d at amino acids 157-251 and 25 8-353 of SEQ ID NO:234 and are set forth in SEQ ID NOS:389 and 390. Thus, provided herein for use in the methods herein are fragments of brevican that retain the ability to bind to HA, for example, a fragment of brevican that contains the G1 domain or a sufficient portion thereof to effect binding to HA. For example, provided herein for use in the methods herein is a HA binding fragment of brevican that contains at least the two link modules.

Also provided herein for use in the provided methods are variants of brevican, including allelic variants, species variants and other variants containing an amino acid modification, as long as the variants retain their y to bind to HA. Species variants of brevican include, but are not limited to, rat (SEQ ID NO:292), mouse (SEQ ID NO:293), bovine (SEQ ID NO:294) and cat (SEQ ID NO:295). Variants of an, or HA binding fragments thereof, for use in the provided methods include variants that have an amino acid modification and that exhibit an altered, such as ed, activity compared to a brevican not containing the ation. Such variants include those that contain amino acid modifications that enhance the binding y of brevican to HA, increase the specificity of brevican for HA, and/or increase the lity of brevican. (7) Versican ed herein for use in the methods herein is a Type C sub-group HABP that is versican, HA binding domains of versican or portions thereof sufficient to bind to HA. Versican (SEQ ID NO:235) is a large extracellular matrix proteoglycan that is present in a variety of tissues. It plays important structural roles, forming loose, hydrated matrices during development and disease. It also interacts directly or indirectly with cells to regulate such physiological processes as cell adhesion, al, proliferation, and motility. The G1 domain of an is located at amino acids 38-349 of SEQ ID NO:235 and is set forth in SEQ ID NO:425. The Ig domain of the G1 domain of versican is located at amino acids 38-151 of SEQ ID NO:235.

The link modules of the G1 domain of versican are located at amino acids 150-244 and 251-346 of SEQ ID NO:235 and are set forth in SEQ ID NOS:39l and 392.

Thus, provided herein for use in the s herein are fragments of versican that retain the y to bind to HA, for example, a fragment of versican that contains the G1 domain or a sufficient portion f to effect binding to HA. For example, provided herein for use in the s herein is a HA binding fragment of versican that contains at least the two link modules.

Also provided herein for use in the provided methods are variants of an, including allelic variants, species variants and other variants containing an amino acid modification, as long as the variants retain their ability to bind to HA. Species variants of versican include, but are not limited to, mouse (SEQ ID NO:296), rat (SEQ ID NO:297), iled e (SEQ ID NO:298), bovine (SEQ ID NO:299) and n (SEQ ID NO:300). Variants of versican, or HA binding fragments thereof, for use in the provided methods include variants that have an amino acid modification and that exhibit an altered, such as improved, activity compared to a versican not containing the modification. Such ts include those that contain amino acid modifications that enhance the binding affinity of versican to HA, increase the specificity of versican for HA, and/or increase the solubility of versican. (8) Neurocan Provided herein for use in the methods herein is a Type C sub-group HABP that is neurocan, HA binding s of neurocan or ns f sufficient to bind to HA. Neurocan, also known as CSPG3 and lDl (SEQ ID NO:236), is a secreted chondroitin sulfate proteoglycan that is primarily expressed in the central nervous system. Human Neurocan is predicted to be cleaved following , resulting in N—terminal (Neurocan-130) and C-terminal (Neurocan-C) fragments.

Neurocan and Neurocan-C are produced by astrocytes and accumulate in the matrix surrounding axonal bundles and neuronal cell bodies. Neurocan-130 is found mainly in the glial cell cytoplasm. Neurocan inhibits neuronal adhesion and neurite outgrth through interactions with a variety of matrix and transmembrane molecules. The G1 domain of an is located at amino acids 53-359 of SEQ ID NO:236 and is set forth in SEQ ID NO:426. The Ig domain of the G1 domain of neurocan is located at amino acids 53-161 of SEQ ID . The link modules of the G1 domain of an are located at amino acids 4 and 261-356 of SEQ ID NO:236 and are set forth in SEQ ID NOS:393 and 394. Thus, provided herein for use in the methods herein are fragments of neurocan that retain the ability to bind to HA, for example, a fragment of neurocan that contains the G1 domain or a sufficient portion thereof to effect g to HA. For example, provided herein for use in the methods herein is a HA g fragment of an that contains at least the two link s.

Also provided herein for use in the provided methods are ts, including allelic variants, species variants and other variants containing an amino acid modification, as long as the ts retain their ability to bind to HA. Species variants of neurocan include, but are not limited to, mouse (SEQ ID NO:30l), rat (SEQ ID NO:302) and chimpanzee (SEQ ID NO:303). Variants of neurocan, or HA binding fragments thereof, for use in the provided methods include variants that have an amino acid modification and that exhibit an altered, such as improved, activity compared to a neurocan not containing the modification. Such variants include those that contain amino acid modifications that enhance the binding y of neurocan to HA, increase the specificity of neurocan for HA, and/or increase the solubility of 116111002111. (9) acan ed herein for use in the method provided herein is a Type C sub-group HABP that is phosphacan, HA binding domains of phosphacan or portions thereof sufficient to bind to HA. Phosphacan (SEQ ID NO:340) a chondroitin sulfate proteoglycan isolated from rat brain that binds to neurons and neural cell-adhesion molecules and modulate cell interactions and other developmental processes in s tissue through heterophilic binding to cell-surface and extracellular matrix molecules, and by competition with ligands of the transmembrane phosphatase.

Phosphacan has 76% identity to the extracellular portion of a human receptor-type protein tyrosine phosphatase (RPTP zeta/beta) and represent an mRNA splicing variant of the larger transmembrane protein.

Also provided herein for use in the methods herein are variants, ing allelic variants, species variants and other variants containing an amino acid modification, as long as the variants retain their ability to bind to HA. Species variants of phosphacan include, but are not limited to rat phosphacan (SEQ ID NO:237). Variants of phosphacan, or HA binding fragments thereof, for use in the provided methods include ts that have an amino acid modification and that exhibit an altered, such as improved, activity compared to a phosphacan not ning the modification. Such variants include those that contain amino acid modifications that enhance the binding affinity of phosphacan to HA, increase the city of phosphacan for HA, and/or increase the solubility of phosphacan. 2. HA Binding Proteins Without Link Modules In some examples, provided herein for use in the s herein are HA binding proteins that do not n link modules. HA binding proteins Without link modules for use in the methods provided herein include, but are not limited to, HABP l/C l QBP, layilin, RHAMM, IuI, CDC37, PHBP, SPACR, SPACRCAN, CD38, IHABP4 and PEP-l , or HA binding fragments thereof. a. HABPl/CIQBP Provided herein for use in the methods herein is a hyaluronan binding protein 1, HA binding domains of HABPl or ns thereof ent to bind to HA.

Hyaluronan binding protein 1 (HABPl; SEQ ID NO:240), also known as C l qBP/C l qR and p32, is a ubiquitous acidic glycoprotein that filnctions in togenesis and as a receptor for proinfiammatory molecules. HABPl binds extracellular onan, vitronectin, complement component Clq, HMW kininogen, and bacterial and viral proteins. Intracellular HABPl binds to molecules ning the Clq globular domain, multiple ms of PKC, mitochondrial Hrk, adrenergic and GABA-A receptors, the mRNA splicing factor ASF/SF2, and the CBF transcription factor.

Also provided herein for use in the methods herein are variants, including allelic variants, species ts and other variants containing an amino acid modification, as long as the variants retain their ability to bind to HA. Variants of HABP 1, or HA binding fragments thereof, for use in the provided methods include variants that have an amino acid modification and that exhibit an altered, such as improved, activity compared to a HABPl not containing the modification. Such ts e those that contain amino acid modifications that enhance the binding affinity of HABPl to HA, se the specificity of HABPl for HA, and/or increase the solubility of HABPl. b. Layilin Provided herein for use in the methods herein is a layilin, HA binding domains of layilin or portions thereof sufficient to bind to HA. Layilin (SEQ ID NOS:238 and 239) is transmembrane protein with homology to C-type lectins and is named after the L-A-Y-I-L-I six amino acid motif in its transmembrane t. Layilin binds specifically to hyaluronan and is found in the ellular matrix of most animal tissues and in body fluids. It thus can modulate cell behavior and functions during tissue remodeling, development, homeostasis, and diseases.

Also provided herein for use in the methods herein are variants, including allelic variants, species variants and other variants containing an amino acid modification, as long as the variants retain their ability to bind to HA. Species variants of layilin include, but are not limited to, mouse (SEQ ID ), Chinese hamster (SEQ ID NO:305) and rat (SEQ ID NO:306). ts of layilin, or HA binding fragments thereof, for use in the provided methods e variants that have an amino acid modification and that exhibit an altered, such as improved, activity ed to a layilin not containing the modification. Such variants include those that contain amino acid modifications that e the binding affinity of layilin to HA, increase the specificity of n for HA, and/or increase the solubility of layilin. c. RHAMM Provided herein for use in the methods herein is a RHAMM, HA binding domains ofRHAMM or portions f sufficient to bind to HA. The receptor for HA-mediated motility (RHAMM; SEQ ID NO:242) is a membrane-associated protein, ranging is size from ~59 to 80 kDa. RHAMM is expressed on most cell types and filnctions to mediate adhesion and cell motility in response to HA binding. Also provided herein for use in the methods herein are ts, including allelic variants, species variants and other variants containing an amino acid modification, as long as the variants retain their ability to bind to HA. s variants ofRHAMM include, but are not limited to, mouse (SEQ ID NO:307) and rat (SEQ ID NO:308). Variants ofRHAMM, or HA binding fragments thereof, for use in the provided methods include variants that have an amino acid modification and that exhibit an altered, such as improved, activity compared to a RHAMM not containing the modification. Such variants e those that contain amino acid modifications that e the binding affinity ofRHAMM to HA, increase the specificity M for HA, and/or increase the solubility of HABPl. (1. Others Other HABPs that bind to HA some of which contain hyaluronan binding domains that can be used in the methods ed herein include, but are not limited to, IOLI (SEQ ID NOS:243-245), CDC37 (SEQ ID NO:250), PHBP (SEQ ID ), SPACR (SEQ ID NO:246), SPACERCAN (SEQ ID NO:247), CD38 (SEQ ID NO:248), IHABP4 (SEQ ID NO:249) and PEP-l (SEQ ID NO:24l), or HA binding domains or portions thereof sufficient to bind to HA. Also provided herein for use in the methods herein are variants, including allelic variants, species ts and other variants containing an amino acid modification, as long as the variants retain their ability to bind to HA. Species ts include, but are not limited to, IOLI from mouse (SEQ ID NOS:309-3 l l) and bovine (SEQ ID NOS:3 12-3 14), CDC37 from Baker’s yeast (SEQ ID NO:326), fruit fly (SEQ ID ), rat (SEQ ID NO:328), mouse (SEQ ID NO:329), fission yeast (SEQ ID NO:330), fruit fly (SEQ ID NO:33 1), chicken (SEQ ID NO:332), bovine (SEQ ID NO:333), a albicans (SEQ ID NO:334). C. elegans (SEQ ID NO:335) and green pufferfish (SEQ ID NO:336), SPACR from chicken (SEQ ID NO:315) and mouse (SEQ ID N03 16), SPACRCAN from mouse (SEQ ID N03 17), rat (SEQ ID NO:318) and n (SEQ ID N03 19), CD38 from mouse (SEQ ID NO:320), rat (SEQ ID NO:321) and rabbit (SEQ ID NO:322), IHABP4 from mouse (SEQ ID NO:324) and chicken (SEQ ID NO:325), and PHBP from mouse (SEQ ID NO:337), rat (SEQ ID NO:338) and bovine (SEQ ID NO:339). Variants of HABPs, or HA binding fragments thereof, for use in the provided methods include variants that have an amino acid modification and that t an altered, such as ed, activity compared to a HABP not ning the modification. Such variants include those that contain amino acid modifications that e the binding affinity of a HABP to HA, increase the Specificity of a HABP for HA, and/or increase the solubility of a HABP, such as an IaI, CDC37, PHBP, SPACR, SPACRCAN, CD38, IHABP4 and PEP-1, or HA binding fi'agments thereof. 3. Modifications ofHA Binding Proteins Modified HABPs are provided herein to improve one or more properties of HABPs for use in the methods provided herein. Such ties include IO modifications increase protein expression in mammalian expression systems, improve biophysical properties such as stability and solubility, improve protein purification and detection and/or increase affinity to HA via zation of the fusion protein. a. Multimers of HABP HABPs provided for use in the s herein can be linked directly or indirectly to a multimerization domain. The presence of a multimerization domain can generate multimers of HABPs or HA binding domains thereof to increase HA binding sites on a molecule. This can result in increased affinity of the HABP for HA. For example, affinity of an HABP multimer can be increased 2-fold, 3-fold, 4- fold, 5—fold, , 7-fold, 8-fold, 9—fold, 10—fold or more compared to an HABP polypeptide not containing a multimerization domain. Affinity of an HABP multimer for HA, when represented as the dissociation constant (Kd), is generally at least less than or less than or 1 x 10‘3 M to 1 x 10'“) M, such as at least less than or less than or 9x 10'9M, 8 x 10'9M, 7x 10'9M, 6x 10'9M, 5 x10‘9M,4x10‘9M,3 x 10'9M, 2x '9M,1x10‘°M, 9x WW, 3 x 10“”M, 7 x IO‘J‘OM, 6 x 10‘10 M, 5 x 10“” M, 4 x 10"°M, 3 x 10“°M, 2 x 10'10 M, 1 x 10"°M or lower Kd.

Provided herein are ers that include an HA binding domain or sufficient n thereof to bind HA of a first HABP and an HA binding domain or sufficient n thereof to bind HA of a second HABP, where the first and second HA—binding domain are linked directly or ctly via a linker to a erization domain. The first and second HA-binding domain can be from the same HABP or from a different HABP. For example, if the PIA-binding domain is the same, then homodimers or homotrimers can be generated. If the HA binding domain is different, RECTIFIED SHEET (RULE 91) ISA/EP then heterodimers or heterotrimers can be generated. For example, HA binding s, such as a link domain or , of HABPS can be ntly-linked, non- covalently-linked or chemically linked to form multimers oftwo or more HA binding domains. The link modules can be linked to form , trimers, or higher multimers. In some ces, multimers can be formed by dimerization oftwo or more HABP polypeptides that each Contain an HA binding domain.

Any portion of an HABP including an HA binding domain can be used as a multimer partner. For example, any of the HABPs described above, or those set forth in any of SEQ ID NOS:206—207, 222—340, 4074i 4 or any portion of an HABP, including an HA binding domain, for example, a link domain or module and variants thereof, including any HA binding domains set forth in any of SEQ ID NOS: 341 and 371—394 can be used to generate chimeric HABP polypeptides, wherein all or part of the HABP polypeptide is linked to a multimerization domain. Typically, at least one, but sometimes both, of the HABP portions is all or a portion of a HABP sufficient to bind HA linked to a multimerization . Examples of HABPs, or portions thereof, for use as multimerization partners are described herein above and are set forth in anyof SEQ ID NOS: 206207, 222-341, 371-394, 407-414, 416418 or 423- 426. In some examples, at least one of the multimer partners is all or patt of the HABP including the HA binding domain. For example, exemplary of multimeric HABP polypeptides is a multimer formed between the HA g domain (3.g. link domain or link module), or portion thereof, of aggrecan, versican, neurocan, brevican, acan, HAPLNI, HAPLN2, HAPLN3, HAPLNI-l, stabilin—l, stabilin-Z, CAB613S8, KIAA0527 or TSG-6 protein. Additionally, a chimeric HABP polypeptide for use in the formation of an HABP multimer can include hybrid HABP polypeptides linked to a erization domain. Exemplary of a multimer provided herein is a multimer, such as a homodimer, generated by erization of the link module (LM) of TSG—6 or ent portion thereof that binds to HA.

Multimerization between two HABP polypeptides can be spontaneous, or can occur due to forced linkage of two or more polypeptides. In one example, ers can be linked by disulfide bonds formed n cysteine residues on different HABP polypeptides or domain or sufficient portions thereof that bind to HA. In another example, multimers can include an HABP polypeptide or domain or sufficient portion RECTIFIED SHEET (RULE 91) ISA/EP —100— thereof to bind to HA joined via covalent or non—covalentinteractions to e moieties fused to the each polypeptide. Such peptides can be peptide linkers (e.g. spacers) or peptides that have the property of ing multimerization. In an additional example, multimers can be formed between two polypeptides through al linkage, such as for example, by using heterobifunctional linkers. i. Peptide Linkers Peptide linkers can be used to produce HABP polypeptide multimers, such as for example a multimer where at least one multimerization partner contains an HA binding domain (ag. , a link domain or module). In one example, peptide linkers can IO be fused to the C—terminal end of a first polypeptide and the N—terrninal end of a second polypeptide. This structure can be repeated multiple times such that at least one, preferably 2, 3, 4, or more polypeptides are linked to one another via peptide linkers at their respective termini. For example, a multimer polypeptide can have a sequence Zl-X-Zz, where 21 and Z; are each a sequence of all or part of an HABP including an HA binding domain and where X is a sequence of a peptide linker. in some instances, Z1 and/or 22 is all of an HABP including an HA binding domain. In other instances, 2; and/or 22 is part of an HABP including an HA binding domain. 21 and 22 are the same or they are different. In another example, the polypeptide has a sequence of z(-X-Z)n, where “n” is any integer, :26. lly 1 or 2.

Typically, the peptide linker is of a sufficient length to allow one or both HA binding domains to bind to a hyaluronan substrate or to permit interaction n the HA binding s (eg. intcraction of two lg modules of the GI HA binding domains of Type C HABPS). Examples of peptide s include, but are not limited to: —Gly—Gly-~ -, GGGGG (SEQ ID NO:342), GGGGS or (GGGGS)n (SEQ ID ), SSSSG or (SSSSG)n (SEQ ID NO:344), GKSSGSGSESKS (SEQ ID NO:345), GGSTSGSGKSSEGKG (SEQ ID NO: 346), GSTSGSGKSSSEGSGSTKG (SEQ ID NO: 347), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 348), EGKSSGSGSESKEF (SEQ ID NO: 349), or AlaAlaProAia 0r (AlaAlaProAla)n (SEQ ID ), where n is I to 6, such as l, 2, 3, or 4. Exemplary linkers include: ( 1) Gly4Ser with NcoI ends (SEQ ID NO: 351) CCATGGGCGG CGGCGGCTCT GCCATGG RECTIFIED SHEET (RULE 91) ISA/EP (2) (Gly4Ser)2 with NcoI ends (SEQ ID NO: 352) CCATGGGCGG CGGCGGCTCT GGCGGCGGCG GCTCTGCCAT GG (3) (Ser4Gly)4 with NcoI ends (SEQ ID NO: 353) CCATGGCCTC GTCGTCGTCG GGCTCGTCGT GCTC GTCGTCGTCG GGCTCGTCGT GCGC CATGG (4) (Ser4Gly)2 with NcoI ends (SEQ ID NO: 354) CCATGGCCTC GTCGTCGTCG TCGT GCGC CATGG Linking es are described, for example, in Huston et al. (1988) PNAS 85:5879-5883, Whitlow et al. (1993) Protein Engineering 6989-995, and Newton et al., (1996) Biochemistry 35:545-553. Other suitable peptide linkers e any of those described in US. Patent Nos. 4,751,180 or 4,935,233, which are hereby orated by reference. A polynucleotide encoding a desired peptide linker can be inserted between, and in the same reading frame as a polynucleotide encoding all or part of an HABP including an HA binding domain, using any suitable conventional technique. In one example, a fusion polypeptide has from two to four HABP polypeptides, including one that is all or part of an HABP polypeptide including an HA g domain, separated by peptide linkers. ii. Heterobifunctional linking agents Linkage of an HABP polypeptide to another HABP polypeptide to create a heteromultimeric fusion polypeptide can be direct or ct. For example, linkage of two or more HABP polypeptides can be achieved by chemical linkage or facilitated by heterobifunctional s, such as any known in the art or provided herein.

Numerous heterobifunctional cross-linking reagents that are used to form covalent bonds between amino groups and thiol groups and to introduce thiol groups into proteins, are known to those of skill in this art (see, e.g., the PIERCE CATALOG, ImmanoTechnology Catalog & Handbook, 1992-1993, which describes the preparation of and use of such reagents and provides a commercial source for such ts; see, also, e.g., Cumber et al. (1992) Bioconjagate Chem. 3:397-401; Thorpe et al. (1987) Cancer Res. 47:5924-5931; Gordon et al. (1987) Proc. Natl. Acad Sci. 84:308-312; Walden et al. (1986) J. Mol. Cell Immunol. 2:191-197; Carlsson et al. (1978) Biochem. J. 173: 723-737; Mahan et al. (1987) Anal. Biochem. 162: 163-170; Wawrzynczak at al. (1992) Br. J Cancer 662361-366; Fattom et a]. (1992) Infection & Immun. 60:584-589). These reagents can be used to form covalent bonds between the inal portion of an HABP polypeptide including an HA g domain and C—tenninus n of another HABP polypeptide including an HA binding domain or between each of those portions and a linker. These reagents include, but are not limited to: N-suceinimidyl-3 -(2-pyridyldithio)propionate (SPDP; de linker); sulfosuccinimidyl 6-[3 -(2-pyridyldithi0)propi0namido]hexanoate (sulfa-LC-SPDP); succinimidyloxycarbonyl-a—methyl benzyl thiosulfate (SMBT, ed disulfate linker); succinimidyl 6—[3-(2~pyridyldithio) propionamido]hexanoate (LC-SPDP); uccinimidyl 4~(N-maleimidomethyl)cyclohexane-l —earb0xylate (sulfo-SMCC); imidyl 3-(2-pyridyldithio)butyrate (SPDB; hindered disulfide bond linker); sulfosuccinimidyl 2-(7-azidomethylcoumarinacetamide) ethyl-l ,3'- dithiopropionate (SAED); sultb-succinimidyl 7-azidomethylcoumarin—3-acetate (SAMCA); sulfosuccinimidyl[alpha-methyl-alpha—(Z~pyridyldithio)toluamido]- ate (sulfo-LC-SMPT); 1,4-di-[3‘-(2'~pyridyldithio)propionamido]butane ' (DPDPB); 4-succinimidyloxycarbonyl-a-methyl—a-(2—pyridylthio)toluene (SMPT, hindered disulfate linker);sulfosuccinimidyl-6—[a-methyl-a-(2-pyrimiyldi- thio)toluamido]hexanoate (sulfo-LC-SMPT); m-maleimidobenzoyl—N—hydroxy— succinimide ester (MBS); m—maleimidobenzoyl-N—hydroxysulfo-succinimide ester (sulfo-MBS); N-succinimidyl(4—iodoacetyl)aminobenzoate (SIAB; thioether linker); uceinimidyl-(4-iodoacetyl)amino benzoate (sulfo-SMB); succinimidyl(p- maleimi-dophenyl)butyrate (SMPB); sulfosuccinimidyl—4-(p-maleimido—phenyl)buty« rate (sulfo-SMPB); azidobenzoyl hydrazide (ABH). These linkers, for example, can be used in combination with peptide linkers, such as those that increase flexibility or lity or that provide for or ate steric hindrance. Any other linkers known to those of skill in the art for linking a polypeptide molecule to another molecule can be employed. General properties are such that the resulting molecule binds to HA.

For in viva diagnostic use of the HABP reagent, generally the linker must be biocompatible for administration to animals, including humans. iii. Polypeptide Multimerization domains Interaction of two or more HABP polypeptides can be facilitated by their linkage, either directly or indirectly, to any moiety or other polypeptide that are RECTIFIED SHEET (RULE 91) ISA/EP themselves able to interact to form a stable structure. For example, separate encoded HABP polypeptide chains can be joined by multimerization, whereby multimerization of the polypeptides is mediated by a multimerization . Typically, the multimerization domain provides for the formation of a stable protein-protein ction between a first HABP polypeptide and a second HABP polypeptide.

HABP polypeptides include, for example, linkage (directly or ctly) of a nucleic acid encoding a HA binding domain (e.g. a link domain or module) of an HABP with a nucleic acid encoding a erization domain. Typically, at least one multimerization partner is a nucleic acid encoding all of part of an HABP including a HA binding domain linked directly or indirectly to a multimerization domain, thus forming a chimeric molecule. Homo- or heteromultimeric polypeptides can be generated from ression of separate HABP polypeptides. The first and second HABP ptides can be the same or different.

Generally, a multimerization domain includes any capable of forming a stable protein-protein interaction. The multimerization s can interact Via an immunoglobulin sequence (e.g. Fc domain; see e.g., International Patent Pub. Nos.

WO 93/10151 and US; US. Pub. No. 2006/0024298; US. Patent No. 5,457,035), leucine zipper (e.g. from nuclear transforming ns fos and jun or the proto-oncogene c-myc or from General Control ofNitrogen (GCN4)), a hydrophobic region, a hydrophilic region, or a fiee thiol which forms an intermolecular disulfide bond between the chimeric les of a homo- or multimer. In addition, a erization domain can include an amino acid sequence comprising a protuberance complementary to an amino acid sequence comprising a hole, such as is described, for example, in US. Patent No. 5, 731,168; International Patent Pub. Nos. WO 98/50431 and ; Ridgway et al. (1996) Protein Engineering, 621. Such a multimerization region can be engineered such that steric interactions not only promote stable interaction, but r promote the formation of heterodimers over homodimers from a mixture of chimeric monomers. Generally, protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory caVities of identical or similar size to the protuberances are optionally created on the interface of the second polypeptide by —104— replacing large amino acid side chains with smaller ones (6.g. , alanine or threonine).

Exemplary multimerization domains are described below.

An HABP polypeptide, such as for example any provided herein, including any HA binding domain (6.g. , a link domain or module) of an HABP, can be joined anywhere, but typically via its N— or C- terminus, to the N— or C- terminus of a multimerization domain to form a chimeric polypeptide The linkage can be direct or indirect via a linker. Also, the chimeric polypeptide can be a fusion protein or can be formed by chemical linkage, such as through covalent or non-covalent interactions.

For example, when preparing a chimeric polypeptide containing a erization domain, nucleic acid encoding all or part of an HABP including an HA binding domain can be operably linked to nucleic acid encoding the multimerization domain sequence, directly or indirectly or optionally via a linker domain. Typically, the construct encodes a ic protein where the C-terminus of the HABP polypeptide is joined to the N—terminus of the multimerization domain. In some instances, a construct can encode a chimeric protein where the N—terminus of the HABP polypeptide is joined to the N— or C-terminus of the multimerization .

A polypeptide multimer contains two chimeric proteins created by linking, directly or indirectly, two of the same or different HABP polypeptides directly or indirectly to a multimerization domain. In some examples, where the multimerization domain is a polypeptide, a gene fiasion encoding the HABP-multimerization domain chimeric polypeptide is inserted into an appropriate expression vector. The ing HABP-multimerization domain chimeric proteins can be expressed in host cells transformed with the inant expression vector, and allowed to assemble into multimers, where the multimerization domains interact to form multivalent polypeptides. Chemical e of erization domains to HABP polypeptides can be ed using heterobifunctional linkers as discussed above.

The resulting chimeric ptides, and multimers formed therefrom, can be purified by any suitable method such as, for example, by ty chromatography over n A or Protein G columns. Where two nucleic acid molecules ng different HABP chimeric polypeptides are transformed into cells, formation of homo- and heterodimers will occur. ions for expression can be adjusted so that heterodimer formation is favored over homodimer formation. -105— (1) Immunoglobulin domain Multimerization domains include those comprising a free thiol moiety e of ng to form an intermolecular disulfide bond with a multimerization domain of an additional amino acid ce. For example, a multimerization domain can include a portion of an immunoglobulin molecule, such as from IgGl, IgGZ, IgG3, lgG4, IgA, IgD, IgM, or lgE. Generally, such a n is an immunoglobulin constant region (Fc). Preparations of fusion proteins ning polypeptides fiised to various portions of antibody—derived polypeptides ding the Fc domain) has been described, see e.g., Ashkenazi et a1. (1991) PNAS 88: 10535; Byrn er al. (1990) Nature, 3442667; and Hollenbaugh and Aruffo, (1992)“Construction of Immunoglobulin Fusion Proteins,” in Current Protocols in Immunology, Ch.10, pp. .19.1-10.19.1 1.

Antibodies bind to specific antigens and contain two identical heavy chains and two identical light chains covalently linked by disulflde bonds. Both the heavy and light chains contain variable regions, which bind the antigen, and constant (C) regions. In each chain, one domain (V) has a variable amino acid sequence depending on the antibody Specificity of the molecule. The other domain (C) has a rather constant sequence common among molecules of the same class. The domains are numbered in ce from the arnino-temiinal end. For example, the IgG light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order VL-CL, referring to the light chain variable domain and the light chain constant domain, respectively. The IgG heavy chain is composed of four immunoglobulin domains linked from the N— to C- terminus in the order VH~CH1—CHZ-CH3, referring to the variable heavy domain, contain heavy domain 1, constant heavy domain 2, and constant heavy domain 3. The resulting antibody molecule is a four chain molecule where each heavy chain is linked to a light chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. Linkage of the heavy chains is mediated by a flexible region of the heavy chain, known as the hinge region.

Fragments of antibody molecules can be ted, such as for e, by enzymatic cleavage. For e, upon protease cleavage by papain, a dimer of the heavy chain constant regions, the Fc domain, is cleaved from the two Fab regions (1‘. e. the portions containing the le regions).

RECTIFIED SHEET (RULE 91) ISA/EP -106— In humans, there are five antibody es classified based on their heavy chains denoted as delta (5), gamma (7 ), mu (u), and alpha (0t) and epsilon (a), giving rise to the IgD, IgG, IgM, IgA, and IgE classes of dies, respectively. The IgA and IgG s contain the subclasses IgA] , IgAZ, IgGl, IgGZ, lgG3, and IgG4.

Sequence differences between immunoglobulin heavy chains cause the various isotypes to differ in, for example, the number ofC domains, the presence of a hinge region, and the number and location of interchain disulfide bonds. For e, IgM and IgE heavy chains contain an extra C domain (C4), that replaces the hinge region.

The F0 regions of lgG, IgD, and IgA pair with each other through their C73, C33, and '10 Cet3 domains, whereas the Fc regions of lgM and IgE dimerize through their Cu4 and C34 domains. IgM and IgA form multimeric structures with ten and four antigen- binding sites, respectively.

HABP immunoglobulin chimeric polypeptides provided herein include a full- length immunoglobulin polypeptide. Alternatively, the immunoglobulin ptide is less than full length, i. e. containing a heavy chain, light chain, Fab, Fabz, Fv, or Fc.

In one example, the HABP globulin chimeric polypeptides are assembled as monomers or hetero-or homo-multimers, and particularly as dimers or tetramers.

Chains or basic units of varying} structures can be utilized to assemble the monomers and - and homo-multimers. For example, an HABP polypeptide can be fused to all or part of an immuneglobulin molecule, including all or part of CH, CL, VH, or VL domain of an immunoglobulin le (see. e.g., US. Patent No. 5,116,964).

Chimeric HABP polypeptides can be readily produced and secreted by mammalian cells transformed with the appropriate nucleic acid molecule. The secreted forms include those where the HABP polypeptide is present in heavy chain dimers; light chain monomers or dimers; and heavy and. light chain heterotetramers where the HABP ptide is fused to one or more light or heavy chains, including heterotetramers where up to and including all four variable region analogues are substituted. In some examples, one or more than one nucleic acid fusion molecule can be transformed into host cells to produce a multimer where the HABP portions of the multimer are the same or different. In some examples, a non~HABP polypeptide light-heavy chain variable-like domain is t, thereby producing a heterobifunctional dy. In some examples, a ic ptide can be made RECTIFIED SHEET (RULE 91) ISA/EP WO 63155 -107~ fused to part of an immunoglobulin molecule lacking hinge disulfides, in which non— covalent or covalent interactions of the two HABP polypeptide ns ate the molecule into a homo~ or dimer. (a) Fc domain Typically, the immunoglobulin portion of an HABP chimeric protein includes the heavy chain of an immunoglobulin polypeptide, most y the constant domains of the heavy chain. Exemplary sequences of heavy chain nt regions for human IgG sub-types are set forth in SEQ ID NOS: 355 (IgGl), SEQ ID NO: 356 (IgGZ), SEQ ID NO: 357 (IgGB), and SEQ ID NO: 358 . For example, for the exemplary heavy chain constant region set forth in SEQ ID NO: 355, the CHI domain ponds to amino acids 1-98, the hinge region corresponds to amino acids 99-] 10, the C142 domain corresponds to amino acids 111-223, and the CH3 domain corresponds to amino acids 224-330.

In one example, an immunoglobulin polypeptide chimeric protein can include the Fc region of an immunoglobulin polypeptide. Typically, such a fusion retains at least a functionally active hinge, CH2 and CH3 domains of the constant region of an immunoglobulin heavy chain. For example, a full-length Fe sequence of IgGl includes amino acids 99-330 of the sequence set forth in SEQ ID NO:355. An exemplary Fe sequence for hIgGl is set forth in SEQ ID NO: 359, and contains almost all of the hinge sequence corresponding to amino acids 100-110 of SEQ LD NO:355, and the complete sequence for the CH2 and CH3 domain as set forth in SEQ ID N01355. Another ary Fe polypeptide is the Fc polypeptide set forth in SEQ ID NO: 212. Another exemplary Fc polypeptide is set forth in PCT application WO 03110151, and is: a mingle chain polypeptide extending from the 1x1-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody (SEQ ID NO:359). The precise site at which the linkage is made is not critical: particular sites are well known and can be selected in order to optimize the biological activity, secretion, or binding characteristics of the HABP polypeptide. For example, other exemplary Fc polypeptide sequences begin at amino acid 0109 or P1 13 of the sequence set forth in SEQ ID NO: 355 (see e.g., U.S. Pub. No. 2006/0024298).

In addition to hIgGl Fc, other PC s also can be ed in the HABP chimeric polypeptides provided herein. For example, where effector functions RECTIFIED SHEET (RULE 91) ISA/EP mediated by Fc/FcyR interactions are to be minimized, fusion with IgG isotypes that poorly recruit complement or effector cells, such as for example, the PC of IgG2 or IgG4, is contemplated. Additionally, the Fc filSlOI‘lS can contain globulin sequences that are substantially encoded by immunoglobulin genes belonging to any of the antibody classes, including, but not limited to IgG (including human sses IgGl, IgG2, IgG3, or IgG4), IgA (including human subclasses IgAl and IgA2), IgD, IgE, and IgM s of antibodies. Further, linkers can be used to covalently link Fc to another polypeptide to generate a PC chimera.

Modified Fc domains also are contemplated herein for use in chimeras with HABP polypeptides. In some examples, the Fc region is modified such that it exhibits altered binding to an FcR so has to result altered (226. more or less) effector fianction than the or filnction of an Fc region of a wild-type immunoglobulin heavy chain.

Thus, a modified Fc domain can have altered affinity, including but not limited to, increased or low or no affinity for the Fc receptor. For e, the different IgG subclasses have ent affinities for the FcyRs, with IgGl and IgG3 typically binding substantially better to the receptors than IgG2 and IgG4. In addition, different FcyRs mediate different effector functions. Fcle, FcyRIIa/c, and Ia are positive regulators of immune complex red activation, characterized by having an intracellular domain that has an immunoreceptor tyrosine-based activation motif (ITAM). FcyRIIb, however, has an immunoreceptor tyrosine-based inhibition motif (ITIM) and is therefore inhibitory. In some instances, an HABP ptide Fc chimeric protein provided herein can be modified to enhance binding to the complement n Clq. Further, an PC can be modified to alter its binding to FcRn, thereby improving the pharmacokinetics of an c chimeric polypeptide.

Thus, altering the affinity of an Fc region for a receptor can modulate the effector functions and/or pharmacokinetic properties associated by the Fc domain. Modified Fc domains are known to one of skill in the art and described in the literature, see 6.g.

US. Patent No. 5,457,035; US. Patent Publication No. US 2006/0024298; and ational Patent Publication No. WC 2005/0638 l 6 for exemplary modifications.

Typically, a polypeptide multimer is a dimer of two ic proteins created by linking, directly or indirectly, two of the same or ent HABP polypeptides to an Fc polypeptide. In some examples, a gene fusion encoding the HABP-Fc chimeric -lO9- protein is inserted into an appropriate expression vector. The resulting HABP-Fc ic proteins can be expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, where hain disulfide bonds form between the Fc moieties to yield divalent HABP polypeptides.

The resulting chimeric polypeptides containing Fc moieties, and multimers formed rom, can be easily purified by affinity tography over Protein A or Protein G columns. For the generation of heterodimers, onal steps for purification can be ary. For example, where two nucleic acids ng different HABP chimeric polypeptides are transformed into cells, the formation of dimers must be biochemically achieved since HABP chimeric les carrying the Fc-domain will be expressed as disulfide-linked homodimers as well.

Thus, mers can be reduced under conditions that favor the disruption of inter- chain disulfides, but do no effect intra-chain disulf1des. Typically, chimeric monomers with different HA-binding domain portions are mixed in equimolar amounts and oxidized to form a mixture of homo- and heterodimers. The components of this mixture are separated by chromatographic techniques. Alternatively, the formation of this type of heterodimer can be biased by genetically ering and expressing HABP filSlOI‘l molecules that contain an HABP polypeptide, followed by the Fc-domain of hIgG, followed by either c-jun or the c-fos leucine zippers (see below). Since the leucine zippers form predominantly heterodimers, they can be used to drive the formation of the heterodimers when desired.

HABP chimeric polypeptides containing Fc regions also can be engineered to include a tag with metal chelates or other e. The tagged domain can be used for rapid purification by metal-chelate tography, and/or by antibodies, to allow for detection of western blots, immunoprecipitation, or activity depletion/blocking in bioassays.

Exemplary HABP-Fc chimeric polypeptides e fusion protein of the TSG-6 link module (TSGLM) and EC. An exemplary TSGLM-Fc is set forth in SEQ ID NO: 212, and encoded by a sequence of nucleotides set forth in SEQ ID NO: 21 I. In addition, HABP-Fc molecules, including for example the exemplary TSG Fc molecules, can optionally contain an epitope tag or a signal for expression and WO 63155 -llO- secretion. For example, the exemplary TSGLM-Fc chimeric polypeptide set forth as SEQ ID NO:2l2 contains human immunoglobulin light chain kappa (K) leader signal peptide sequence (SEQ ID NO: 210), an EC fragment of the human IgG1 heavy chain (SEQ ID NO:204) and a human TSG-6 link module (SEQ ID ). The cDNA sequence encoding the TSGLM-Fc chimeric polypeptide is set forth in SEQ ID NO: 21 l. The DNA encoding human IgG1 heavy chain and human TSG-6 link module regions are connected with a 6 bp AgeI restriction enzyme cleavage site and a 12 bp sequence, GACAAAACTCAC (SEQ ID NO: 208), encoding four additional amino acids (DKTH; SEQ ID NO: 209) (2) Leucine Zipper Another method of preparing HABP polypeptide multimers for use in the methods ed herein involves use of a leucine zipper domain. Leucine zippers are peptides that promote multimerization of the proteins in which they are found.

Typically, leucine zipper is a term used to refer to a repetitive heptad motif containing four to five leucine residues present as a conserved domain in several proteins.

Leucine zippers fold as short, parallel coiled coils, and are ed to be sible for oligomerization of the proteins of which they form a domain. The dimer formed by a leucine zipper domain is stabilized by the heptad repeat, designated (abcdefg)n (see e.g., McLachlan and Stewart (1978) J. M01. Biol. 98293), in which residues a and d are generally hydrophobic residues, with d being a leucine, which lines up on the same face of a helix. Oppositely-charged residues commonly occur at positions g and e. Thus, in a parallel coiled coil formed from two helical leucine zipper domains, the “knobs” formed by the hydrophobic side chains of the first helix are packed into the “holes” formed between the side chains of the second helix.

Exemplary leucine zippers for use as multimerization domains herein are derived from either of two nuclear transforming proteins, fos , that t leucine zipper domains, or the product of the murine proto-oncogene, c-myc. The leucine zipper domain is necessary for biological activity (DNA binding ) in these proteins. The products of the nuclear oncogenesf0s andjun contain leucine zipper domains that preferentially form a heterodimer (O’Shea et al. (1989) Science, 6; Turner and Tijian (1989) Science, 243:1689). For example, the leucine zipper domains of the human transcription factors c-jun and c-f0s have been shown to form stable dirners with a 1:1 stoichiometry (see e.g., Busch and Sassone-Corsi (1990) Trends Genetics, 6:3 6-40; Gentz et al., (1989) e, 243:1695-1699).

Although jun-jun mers also have been shown to form, they are about 1000- foid less stable than jun-foe dimers.

Thus, typically an HABP polypeptide multimer provided herein is generated using ajun-fos combination. Generally, the leucine zipper domain of either c-jun or c—fos is fused in frame at the C-terminus of an HABP of a polypeptide by genetically engineering fusion genes. Exemplary amino acid sequences of c-jun and c-fos leucine zippers are set forth in SEQ ID NOS:362 and 363, respectively. In some instances, a sequence of a e zipper can be modified, such as by the addition of a cysteine residue to allow formation of de bonds, or the addition of a tyrosine residue at the C—terminus to facilitate measurement ofpeptide concentration. Such ary sequences of encoded amino acids of a d c-jun and c-fos leucine zipper are set forth in SEQ ID NOS: 362 and 363, respectively. In addition, the linkage of an HABP polypeptide with a leucine zipper can be direct or can employ a flexible linker domain, such as for example a hinge region of lgG, or other polypeptide linkers of small amino acids such as glycine, serine, threonine, or alanine at various lengths and combinations. In some instances, separation of a leucine zipper from the inus of an encoded polypeptide can be effected by fusion with a sequence encoding a protease cleavage site, such as for example, a thrombin cleavage site, Additionally, the chimeric proteins can be tagged, such as for example, by a 6XHis tag, 'to allow rapid purification by metal chelate chromatography and/or by epitopes to which antibodies are available, such as For example a myc tag, to allow for detection on n blots, immunoprecipitation, or activity depletion/blocking bioassays. r exemplary leucine zipper domain for use as a multimerization domain is derived from a nuclear n that functions as a transcriptional activator of a family of genes involved in the General Control. ofNitrogen (GCN4) metabolism in S. cerevisiae. The protein is able to dimerize and bind promoter ces containing the recognition sequence for GCN4, thereby activating transcription in times of nitrogen deprivation. An exemplary sequence of a GCN4 leucine zipper capable of forming a dimeric complex is set forth in SEQ ID NO: 364. Amino acid substitutions in the a and (1 residues of a synthetic peptide representing the GCN4 leucine zipper RECTIFIED SHEET (RULE 91) ISA/EP -llZ- domain (lie. amino acid substitutions in the sequence set forth as SEQ ID NO:364) have been found to change the oligomerization properties of the leucine zipper domain. For example, when all residues at position a are changed to isoleucine, the leucine zipper still forms a parallel dimer. When, in addition to this change, all e residues at position d also are changed to isoleucine, the resultant peptide spontaneously forms a trimeric parallel coiled coil in solution. An exemplary sequence of such a GNC4 leucine zipper domain capable of forming a trimer is set forth in SEQ ID NO:365. Substituting all amino acids at position d with cine and at position a with leucine results in a peptide that tetramerizes. Such an exemplary sequence of a leucine zipper domain of GCN4 capable of forming tetramers is set forth in SEQ ID NO:366. Peptides containing these substitutions are still referred to as leucine zipper domains since the mechanism of oligomer formation is believed to be the same as that for traditional leucine zipper domains such as the GCN4 described above and set forth in SEQ ID NO:364. (3) n-Protein ction between Subunits Exemplary of r type of multimerization domain for use in modifying a HABP provided for use in the methods herein is one where multimerization is facilitated by protein-protein interactions between different subunit ptides.

Exemplary of such a multimerization domain is derived from the mechanism of cAMP-dependent protein kinase (PKA) with its anchoring domain (AD) ofA kinase anchor proteins (AKAP). Thus, a heteromultimeric HABP polypeptide can be generated by linking (directly or indirectly) a nucleic acid ng an HABP polypeptide, such as a HA-binding domain of an HABP polypeptide, with a nucleic acid encoding an R subunit sequence of PKA (i. e. SEQ ID NO:367). This results in a homodimeric molecule, due to the spontaneous formation of a dimer effected by the R t. In tandem, another HABP ptide fusion can be ted by linking a nucleic acid encoding another HABP polypeptide to a nucleic acid ce encoding an AD sequence ofAKAP (i. e. SEQ ID NO:368). Upon co-expression of the two components, such as following co-transfection of the HABP chimeric components in host cells, the dimeric R subunit provides a g site for binding to the AD sequence, resulting in a heteromultimeric molecule. This binding event can be r stabilized by covalent linkages, such as for example, disulfide bonds. In some examples, a flexible linker residue can be fused n the nucleic acid encoding the HABP polypeptide and the multimerization domain. In another example, fusion of a nucleic acid encoding an HABP polypeptide can be to a nucleic acid encoding an R subunit containing a cysteine residue incorporated adjacent to the amino-terminal end of the R subunit to facilitate covalent linkage (see e.g, SEQ ID NO:369 ). Similarly, fusion of a nucleic acid encoding a r HABP polypeptide can be to a nucleic acid encoding an AD subunit also containing incorporation of cysteine residues to both the amino- and carboxyl-terminal ends ofAD (see e.g., SEQ ID NO:370). iv. Other multimerization domains Other multimerization domains that can be used to multimerize a HABP provided for use in the methods herein are known to those of skill in the art and are any that tate the protein-protein interaction of two or more polypeptides that are separately generated and expressed as HABP fiasions. Examples of other multimerization domains that can be used to provide n-protein interactions between two ic polypeptides include, but are not limited to, the bamase-barstar module (see e.g., Deer et al., (2003) Nat. Biotechnol. 21 :1486-1492); use of particular protein domains (see e.g., Terskikh et al., (1997) Proc Natl Acad Sci USA 94: 1663-1668 and Muller et al., (1998) FEBS Lett. 422:259-264); use ofparticular peptide motifs (see e.g., de Kruif et al., (1996) J. Biol. Chem. 271 :7630-7634 and Muller et al., (1998) FEBS Lett. 432: 45-49); and the use of disulfide s for enhanced stability (de Kruif et al., (1996).]. Biol. Chem. 271:7630-7634 and dl et al., (2000) n Eng. 13 :725-734). b. Mutations to e HA Binding In a further example, provided herein for use in the methods herein are HABPs that are modified, such as by amino acid replacement, to exhibit increased specificity for hyaluronan ed to other GAGs. For example, provided herein is a mutant TSGLM containing amino acid replacement(s) at amino acid residue 20, 34, 41, 54, 56, 72 and/or 84, and in particular at amino acid residue 20, 34, 41, and/or 54 (corresponding to amino acid residues set forth in SEQ ID NO:360). The ement amino acid can be to any other amino acid residue, and generally is to a non-basic amino acid residue. For example, amino acid replacement can be to Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Ala (A), Val (V), Ile (I), Leu (L), Met (M), Phe WO 63155 2012/061743 (F), Tyr (Y) or Trp (W). The amino acid ement or replacements confer decreased binding to heparin. Binding can be reduced at least 12-fold, 1.5»fold, 2- fold, 3-fold, 4-fold, , 6-fold, 7-fold, , 9-fold, 10-fold, ZO-fold, 30-fold, 40- fold, 50-fold, lOO-fold or more compared to binding of TSGLM to heparin not containing the amino acid replacement. Exemplary of a TSGLM mutant for use as a reagent in the method provided herein is K20A/K34A/K41A. Hence, for example, binding to heparin is reduced such that specificity to hyaluronan is increased. The mutant TSGLM can be conjugated directly or indirectly to a multimerization domain to te multimers. For example, exemplary of a reagent for use in the methods herein is TSGLM(K2OA/K34A/K41A)-Fc. c. Modifications of HA Binding Proteins for Detection For use in the diagnostic s provided herein, the HA binding proteins can be modified to contain a detectable n or a moiety to facilitate detection. i. Conjugation to Detectable Proteins or Moieties The HA binding proteins for use in the diagnostic methods provided herein can be modified by conjugation to detectable moieties, including, but not limited to, peptides tags, radiolabels, fluorescent molecules, uminescent molecules, bioluminescent molecules, Fe domains, biotin, enzymes that catalyze a detectable reaction or catalyze formation of a detectable product and proteins that bind a detectable compound. Detectable moieties, including proteins and compounds, or moieties that facilitate detection are known to one of skill in the art. The detectable moieties can be used to facilitate detection and/or ation of the HABP.

In one example, the HA binding protein is modified by ation to a detectable protein or to a protein that s a detectable signal. The detectable protein or n that induces a detectable signal can be selected from among a luciferase, a fluorescent protein, a bioluminescent n, a receptor or transporter protein that binds to and/or transports a contrast agent, chromophore, compound or ligand that can be detected. For example, the detectable protein or protein that induces a detectable signal is a green fluorescent protein (GFP) or a red fluorescent protein (RFP).

Detectable labels can be used in any of the diagnostic methods provided . Exemplary detectable labels include, for example, uminescent RECTIFIED SHEET (RULE 91) ISA/EP moieties, biolumineseent moieties, fluorescent moieties, radionuclides, and metals.

Methods for detecting labels are well known in the art. Such a label can be detected, for e, by visual tion, by fluorescence spectroscopy, by reflectance measurement, by flow cytometry, by X-rays, by a variety of magnetic nce U) methods such as magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). Methods of ion also include any of a variety of tomographic methods including computed tomography (CT), computed axial tomography (CAT), on beam computed tomography (EBCT), high resolution computed tomography (HRCT), hypocyeloidal tomography, positron emission tomography (PET), single-photon emission computed tomography ), spiral computed tomography, and onic tomography.

Exemplary of such ns are enzymes that can catalyze a detectable reaction or catalyze formation of a detectable product, such as, for e, luciferases, such as a click beetle luciferase, a Renillq. luciferase, a firefly luciferase or beta-glucuronidase (GusA). Also exemplary of such proteins are proteins that emit a detectable , including fluorescent proteins, such as a green fluorescent n (GFP) or a red fluorescent protein (RFP). A variety of DNA sequences encoding proteins that can emit a detectable signal or that can catalyze a able reaction, such as luminescent or fluorescent proteins, are known and can be used in the methods provided herein, Exemplary genes encoding emitting proteins include, for example, genes from bacterial lueiferase from Vibrio harveyz‘ (Belas et at, (1982) Science 218:791-793), bacterial rase from fischerii (Foran and Brown, (1988) Nucleic acids Res. 16:777), firefly luciferase (de Wet et al., (1987) Mol. Cell.

Biol. 7:725—737), aequorin from Aequorea Victoria (Prasher et al, (1987) Biochem. 26: 1326—1332), Renilla luciferase from Rem'lla renformis (Lorenz ct a1, (1991) Proc Natl Acad Sci USA 88:4438-4442) and green fluorescent protein from Aequorea Victoria (Prasher et al, (1987) Gene 111:229-233). The [MA and luxB genes of bacterial lueil‘erase can be fused to produce the fusion gene (Fabg), which can be expressed to produce a fully fimctional luciferase protein (Escher et al, (1989) PNAS 86: 6528-6532).

Exemplary detectable proteins that can be conjugated to the HA binding proteins for use in the diagnostic methods provided herein also include proteins that RECTIFIED SHEET (RULE 91) ISA/EP WO 63155 -ll6- can bind a contrasting agent, chromophore, or a compound or ligand that can be detected, such as a transferrin receptor or a ferritin; and reporter proteins, such as E. coli ctosidase, B—glucuronidase, xanthine-guanine phosphoribosyltransferase (gpt), and ne phosphatase. Also exemplary of detectable proteins are proteins that can specifically bind a detectable compound, including, but not d to receptors, metal binding proteins (6.3., siderophores, ferritins, transf‘eifin receptors), ligand binding proteins, and antibodies.

The HABP also can be conjugated to a protein or peptide tag. In one example, the HA binding protein is conjugated to an Fc domain. Protein and peptide tags also include, but are not limited to, s tag (SEQ ID N054), hemagglutinin tag (SEQ ID N0:420), FLAG tag (SEQ ID N055), c—myc tag (SEQ ID NO:419), VSV—G tag (SEQ ID N02421), HSV tag (SEQ ID NO:422) and VS tag (SEQ ID N0:415), chitin binding protein (CBP), maltose binding protein (MBP), and glutathione s-transferase (GST).

Detectable labels can be coupled or conjugated to an HABP through recombinant methods or by chemical methods. For example, conjugation can be ' effected by linked the n, directly or indirectly to a linker such as a peptide linker or a al linker. Linkers can be polypeptide sequences, such as poly-Glycine- sequences of between about 5 and 200 amino acids. Proline residues can be incorporated into a polypeptide linker to prevent the formation of significant secondary structural elements, 1'. e. the linker. An example of a , a-helix/B—sheet, by flexible linker is a polypeptide that includes a glycine chain with an ediate proline. In other examples, a chemical linker is used to connect synthetically or recombinantly produced binding and labeling domain subsequences. Such flexible linkers are known to s of skill in the art. For example, poly(ethylene glycol) linkers are ble from Shearwater Polymers, Inc. ille, Ala. These s optionally have amide es, sulfliydryl linkages, or heterofunctional linkages. 4. Selection ofHA Binding Proteins for stic Use An HA binding protein suitable for use as a diagnostic agent can be selected based on one or more desired properties or activities, including, but not limited to, specificity or y for HA, solubility, peptide stability, homogeneity, ease of expression and purification, minimum batch to batch variations in the expressed RECTIFIED SHEET (RULE 91) ISA/EP 2012/061743 -ll7- peptide, and low sample variability in HA binding and detection. In some es, a single polypeptide stic agent is contemplated over a stic with multiple polypeptide components. For example, a link module that binds to HA in the absence of a complete link protein. The y of an HABP provided herein to bind to hyaluronan can be assessed by methods well known in the art including, but not limited to ELISA-based assays, competitive binding assays with HA, heparin and other glycosaminoglycans, such as chondroitin sulfates A or C, heparan sulfates or dermatan sulfates. Exemplary assays for assessing HA binding activity are provided herein in n D and in the Examples.

D. ASSAYS AND CLASSIFICATION The methods ed herein are based on assaying the expression or levels of hyaluronan (HA) in a sample or samples, such as a tissue sample or body fluid sample. The methods herein are based on g methods using a hyaluronan binding protein companion diagnostic (HABP, such as a TSGLM, multimer or variant thereof) for assessing, ting, determining, quantifying and/or otherwise ically detecting hyaluronan expression or levels in a sample. The assays can be performed in vitro or in viva. By comparisons to a control or reference sample or fications based on a predetermined level, such values can be used for diagnosis or prognosis of a hyaluronan-associated disease or condition, to predict responsiveness of a subject having a hyaluronan-associated disease or condition to a hyaluoman-degrading enzyme therapy, and/or to monitor or predict efficacy of treatment of a subject having a hyaluronan-associated disease or condition that has been treated with a hyaluronan-degrading enzyme therapy. For example, as described herein, it is found that HA levels and extent specifically are associated with responsiveness to treatment with a hyaluronan-degrading , such as a hyaluomidase or modified hyaluronidase (e.g. PEGylated hyaluronidase such as PEGPHZO).

In any of the above examples, the hyaluronan-associated es or conditions are diseases and conditions in which hyaluronan levels are elevated as cause, consequence or otherwise observed in the disease or condition. Exemplary hyaluronan-associated diseases or conditions, include, but are not limited to, ones that are associated with high interstitial fluid pressure, a cancer and in particular a 2012/061743 hyaluronan rich , edema, disc pressure, an inflammatory disease, and other es associated with hyaluronan. In some cases, hyaluronan-associated diseases and conditions are associated with increased interstitial fluid pressure, decreased vascular volume, and/or sed water content in a tissue, such as a tumor. In ular, hyaluronan-associated diseases and conditions, include, but are not limited to, hyaluronan-rich cancers, for example, , including solid tumors such as late- stage cancers, metastatic cancers, undifferentiated cancers, n cancer, in situ carcinoma (ISC), squamous cell carcinoma (SCC), te cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer and other cancers.

In one example, based on the levels or expression of hyaluronan, a patient or subject can be selected for treatment with an anti-hyaluronan agent (e.g. a hyaluronan- degrading enzyme). For example, a sample from a subject can be contacted with a hyaluronan-binding protein (HABP) companion diagnostic, such as TSGLM, a multimer or variant thereof, and the binding of the HABP to the sample can be detected in order to determine the amount of hyaluronan in the sample. Based on ermined selection or classification ia as described herein, a patient can be diagnosed with a hyaluronan-associated disease or condition, and hence selected for treatment of the disease or condition. Also, based on the predetermined ion or classification criteria as described herein, the methods herein can be used for prognosis of the t. Depending on the course of the disease or condition, the dose, treatment schedule and/or dosing regime of the therapeutic agent (e.g. a hyaluronan-degrading enzyme) can be optimized and adjusted accordingly. In particular examples herein, based on the predetermined selection or classification criteria as described herein, a patient or subject can be selected for ent that is predicted to be responsive to treatment with an anti-hyaluronan agent, for example a hyaluronan-degrading , such as a hyaluronidase or modified hyaluronidase (e.g. a PEGylated hyaluronidase such as PEGPHZO). Hence, the method can be used to predict the efficacy of treatment by an anti-hyaluronan agent, for example a hyaluronan-degrading enzyme.

In examples of methods herein, the efficacy of the treatment by an anti- onan agent, for example a hyaluronan-degrading enzyme, can be determined by monitoring the expression or levels of hyaluronan over the course of treatment.

WO 63155 Hence, the method is a post-treatment method of monitoring disease status and/or resolution, which information can be used to alter the course oftreatment of a subject depending on individualized status information. For example, a sample from a subject can be contacted with a hyaluronan—binding protein (HABP) companion diagnostic, for e a TSG—6—LM, a multimer or variant thereof, and the binding of the HABP to the sample. can be detected in order to ine the amount of hyaluronan in the sample. The expression or level of onan in the sample can be compared to a reference or control sample in order to assess ences in hyaluronan levels or expression. For example, elevated or accumulated hyaluronan levels in a diseased subject compared to a healthy or normal subject is indicative of a hyaluronan-associated disease or condition (e. 9. tumor or cancer) and the extent of the hyaluronan expression or levels correlates to disease aggressiveness. In such methods, the control or reference sample is a sample from a healthy subject, is a baseline sample from the t prior to treatment with an anti-hyaluronan agent (e. g. a hyaluronan-degrading enzyme) (pm-treatment reference) or is a sample from a t rior to the last dose of an anti—hyaluronan agent ag a onan-dc rading enzyme . bor example, tor momtormg patlent se, e assay can be run a the initiation of therapy to establish ne (or predetermined) levels of hyaluronan in a sample (e.g. tissue or body fluid). The same sample (6.g. tissue or body fluid) is then sampled using the same assay and the levels of hyaluronan compared to the baseline or predetermined levels. 1. Assays For Measuring Hyaluronan It is within the level of one of skill in the art to assess, quantify, determine and/or detect hyaluronan levels in a sample using an HABP companion diagnostic, such as TSG—6-LM, multimer (e.g. TSG-6LM-Fc) or variant thereof, as described herein. Assays include in vitro or in viva assays. Exemplary binding assays that can be used to assess, evaluate, ine, quantify and/or otherwise specifically detect hyaluronan expression or levels in a sample include, but are not limited to, solid phase binding assays (ag. enzyme linked immunosorbent assay )), radioimmunoassay (RIA), immunoradiometric assay, fluorescence assay, chemiluminescent assay, bioluminescent assay, western blot and histochemistry methods, such as immunohistochemistry (IHC) or pseudo innnunohistocltemiStry RECTIFIED SHEET (RULE 91) ISA/EP using a tibody binding agent. In solid phase g assay methods, such as ELISA methods, for example, the assay can be a sandwich format or a competitive inhibition format. In other examples, in vivo imaging methods can be used. a. Histochemical and Immunohistochemical Methods The methods of assessing hyaluronan accumulation are based on the ability of an HABP companion diagnostic to bind to HA in a sample, for example a tissue or cell sample, such that the amount of the HABP companion diagnostic that binds correlates with amount of HA in the sample. Any HABP companion diagnostic provided herein can be used to detect HA using tissue staining methods known to one of skill in the art, including but not limited. to, cytochemical or histochemical methods, such as immunohistochemistry (IHC) or histochemistry using a non- antibody binding agent (e.g. pseudo inununohistochemistry). Such histochemical s permit quantitative or semi-quantitative detection of the amount of HABP that binds to HA in a sample, such as a tumor tissue sample. In such methods, atissue l5 sample can be contacted with an HABP reagent provided herein, and in particular one that is detectably d or capable of detection, under conditions that permit binding to tissue- or cell-associated HA.

A sample for use in the s provided herein as determined by hemistry can be any biological sample that can be ed for its HA levels, such asa tissue or cellular sample. For example, a tissue sample can be solid tissue, including a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate, or cells. In some examples, the tissue sample is tissue or cells obtained from a solid tumor, such as primary and metastatic tumors, ing but not limited to, breast, colon, , lung, stomach, ovary, cervix, uterus, , bladder, te, thyroid and lung cancer tumors. In particular examples, the sample is a tissue sample from a cancer that is a late-stage cancer, a metastatic cancer, undifferentiated cancer, ovarian cancer, in situ carcinoma (ISC), squamous cell oma (SCC), prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer. In other examples, the tissue sample ns cells from primary or cultured cells or cell lines.

Cells may be have various states of differentiation, and may be normal, pre—cancerous or cancerous, may be fresh tissues, diespersed cells, immature cells, including stem RECTIFIED SHEET (RULE 91) ISA/EP -l2l- cells, cells of intemiediate maturity and fully matured cells. lly, the cells selected for use in the methods ed herein are cancer cells.

When the tumor is a solid tumor, isolation of tumor cells is typically achieved by surgical biopsy. Biopsy techniques that can be used to harvest tumor cells from a subject include, but are not limited to, needle biopsy, CT-guided needle , aspiration biopsy, endoscopic biopsy, bronchoscopic , bronchial lavage, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy, skin biopsy, bone marrow biopsy, and the Loop Electrosurgical Excision ure (LEEP). Typically, a non-necrotic, sterile biopsy or specimen is obtained that is greater than 100 mg, but which can be smaller, such as less than 100 mg, 50 mg or less, 10 mg or less or 5 mg or less; or , such as more than 100 mg, 200 mg or more, or 500 mg or more, 1 gm or more, 2 gm or more, 3 gm or more, 4 gm or more or 5 gm or more. The sample size to be extracted for the assay can depend on a number of factors including, but not limited to, the number of assays to be performed, the health of the tissue sample, the type of cancer, and the condition of the subject. The tumor tissue is placed in a sterile vessel, such as a sterile tube or culture plate, and can be ally immersed in an appropriate medium.

Tissue obtained from the patient after biopsy is often fixed, y by formalin (forrnaldehyde) or glutaraldehyde, for example, or by alcohol immersion.

For histochemical methods, the tumor sample can be sed using known ques, such as ation and embedding the tumor tissue in a paraffin wax or other solid supports known to those of skill in the art (see Plenat et al., (2001) Ann Pathol January 21(1):29~47), slicing the tissue into sections le for staining, and processing the sections for staining according to the histochemical staining method selected, including l of solid supports for embedding by c solvents, for example, and rehydration of preserved tissue. Thus, samples for use in the methods herein can contain compounds that are not naturally present in a tissue or cellular sample, including for example, preservatives, anticoagulants, buffers, fixatives, nutrients and antibiotics.

In exemplary methods to select a subject for treatment with a hyaluronan— degrading enzyme, harvesting of the tumor tissue is generally performed prior to treatment of the subject with a hyaluronan-degrading enzyme. In exemplary methods RECTIFIED SHEET (RULE 91) ISA/EP WO 63155 —122- of monitoring therapy of a tumor with a hyaluronan-degrading enzyme, ting of the tumor tissue from the subject can be performed before, during or after the subject has received one or more treatments with a hyaluronan—degrading enzyme.

Assays for use in the methods ed herein are those in which HA present in the sample is detected using histochemistry or immunohistochemistry. hemistry (HC) is a staining method based on enzymatic reactions using a binding partner, such as an antibody (e.g. monoclonal or polyclonal antibodies) or other binding partner, to detect cells or specific proteins such as tissue ns; or biomarkers, for example, HA. For example, histochemistry assays for use in the methods herein include those where an HABP is used as a binding partner to detect HA associated with cells or tissues. Typically, histochemistry protocols include detection systems that make the presence of the s visible, to either the human eye or an automated g system, for qualitative or quantitative analyses. In a direct HC assay, binding is determined directly upon binding of the binding partner (a. g. first antibody) to the tissue or ker due to the use of a labeled reagent. In an indirect HC assay, a secondary antibody or second binding partner is necessary to detect the g of the first binding partner, as it is not labeled.

In such methods, generally a slide-mounted tissue sample is stained with a labeled binding reagent (e.g. labeled HABP) using Common histochemistry techniques. Thus, in exemplary HC methods provided herein, the HABP companion diagnostics are modified to contain a moiety capable of being detected (as described in Section 3C above). In some examples, the HABP ion diagnostics are conjugated to small les, e.g, biotin, that are detected via a labeled binding partner or antibody. In some examples, the IHC method is based on staining with an HABP protein that is detected by tic staining with horseradish dase.

For example, the HABP can be biotinylated and ed with avidin or streptavidin conjugated to detectable n, such as streptavidin-horseradish peroxidase (see Example 6 below). In other es, the HABP companion diagnostics are conjugated to detectable proteins which permit direct detection, such as, for example, HABP companion diagnostics conjugated to a fluorescent protein, bioluminescent protein or enzyme. Various enzymatic staining methods are known in the art for detecting a protein of interest. For example, enzymatic interactions can be visualized RECTIFIED SHEET (RULE 91) ISA/EP -l23- using ent enzymes such as peroxidase, alkaline atase, or different chromogens such as DAB, AEC or Fast Red. In other examples, the HABP companion diagnostics are conjugated to peptides or proteins that can be detected via a labeled binding partner or dy.

In other examples, HA is detected by HC methods using a HABP companion diagnostic provided herein where the HABP companion diagnostics are detected by labeled secondary reagents, such as labeled antibodies that ize one or more epitopes of the HABPs, HABP link domains, or HA binding fragments thereof. In other examples, HABP companion diagnostics are detected using an anti-HABP antibody. For detecting a HABP, any anti-HABP antibody can be used so long as it binds to the HABP, HABP link domain, or HA binding fragment thereof used to detect HA. For example, for detecting TSG-6 or a TSGLM, an anti-TSG-6 link module monoclonal dy can be used, such as antibodies designated A38 and Q75 (see, Lesley et al. (2002) J Biol Chem 277:26600-26608). The anti-HABP antibodies can be labeled for detection or can be detected with a ary antibody that binds the first antibody. The selection of an appropriate anti-HABP antibody is within the level of one of skill in the art.

The resulting stained specimens are each imaged using a system for viewing the detectable signal and acquiring an image, such as a digital image of the staining.

Methods for image acquisition are well known to one of skill in the art. For example, once the sample has been stained, any l or non-optical g device can be used to detect the stain or biomarker label, such as, for example, upright or inverted l microscopes, ng al copes, cameras, scanning or tunneling electron microscopes, canning probe microscopes and imaging infrared detectors. In some examples, the image can be captured digitally. The obtained images can then be used for quantitatively or semi-quantitatively determining the amount ofHA in the sample. Various automated sample processing, scanning and analysis systems suitable for use with immunohistochemistry are available in the art. Such systems can include automated staining and microscopic scanning, computerized image analysis, serial section comparison (to control for variation in the orientation and size of a sample), digital report tion, and archiving and tracking of samples (such as slides on which tissue sections are placed). ar imaging systems are 2012/061743 —124— commercially available that combine tional light microscopes with digital image processing s to perform quantitative analysis on cells and tissues, including immunostained samples. See, e.g., the CAS-200 system (Becton, Dickinson & Co.). In particular, detection can be made manually or by image processing techniques involving computer processors and software. Using such software, for example, the images can be configured, calibrated, standardized and/or validated based on factors including, for example, stain quality or stain intensity, using procedures known to one of skill in the art (see e.g. published US. patent Appl. No.

US20100136549).

The image can be tatively or semi-quantitatively analyzed and scored based on staining intensity of the . Quantitative or semi-quantitative histochemistry refers to method of scanning and scoring samples that have undergone histochemistry, to identify and quantitate the presence of a specified biomarker, such as an antigen or other protein (e.g. HA). Quantitative or semi-quantitative methods can employ imaging software to detect staining densities or amount of staining or methods of detecting staining by the human eye, where a trained operator ranks results numerically. For e, images can be quantitatively analyzed using a pixel count algorithms (e.g. Aperio Spectrum Software, Automated QUantitatative Analysis platform (AQUA® platform), and other standard methods that measure or quantitate or semi-quantitate the degree of staining; see e.g. US. Patent No. 8,023,714; US.

Patent No. 7,257,268; US. Pat No. 7,219,016; US. Patent No. 7,646,905; hed US. Pat. Appl. Nos. US20100136549 and 20110111435; Camp et al. (2002) Nature Medicine, 8:1323-1327; Bacus, et al. (1997) Analyt Quant Cytol , 19:316-328).

A ratio of strong positive stain (such as brown stain) to the sum of total stained area can be calculated and scored.

Using histochemical, such as immunohistochemical or pseudo immunohistochemical methods, the amount ofHA ed is quantified and given as a percentage ofHA positive pixels and/or a score. For example, the amount ofHA ed in the sample can be quantified as a percentage ofHA ve pixels. In some examples, the amount ofHA present in a sample is quantified as the percentage of area stained, e.g, the percentage ofHA positive . For example, a sample can have at least or about at least or about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, WO 63155 %, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more HA positive pixels as compared to the total staining area.

In some examples, a score is given to the sample that is a numerical representation of the intensity or amount of the histochemical staining of the sample, and represents the amount of target biomarker (e.g. in the sample.

, HA) present Optical density or percentage area values can be given a scaled score, for example on an integer scale, for example, 0-10, 0-5, or 0-3. In particular examples, the amount of hyaluronan in a sample is classified on a scale of 0-3, 6.g. 0, HA+1, HA+2, and HA+3.

The amount ofHA present is relative to the percentage ofHA pixels, that is, low percentages ofHA pixels indicates a low level of HA whereas high percentages of HA pixels indicate high levels of HA. Scores can correlated with percentages ofHA ve , such that the percentage area that is d is scored as 0, HA+1, HA+2, and HA+3, representing no staining, less than 10% staining, 10-25% staining or more than 25% staining respectively. For example, if the ratio (6.g. strong pixel stain to total stained area) is more than 25% the tumor tissue is scored as HA+3, if the ratio is 10-25% of strong positive stain to total stain the tumor tissue is scored as HA+2, if the ratio less than 10% of strong positive stain to total stain the tumor tissue is scored as HA+1, and if the ratio of strong positive stain to total stain is 0 the tumor tissue is scored as 0. A score of 0 or HA+1 indicates low levels ofHA in the tested sample, whereas a score of HA+2 or HA+3 indicates higher levels ofHA in the tested samples. b. Solid Phase Binding Assays The methods of assessing hyaluronan accumulation are based on the ability of an HABP companion diagnostic to bind to HA in a sample such that the amount of the HABP ion diagnostic that binds correlates with amount ofHA in the sample.

In ular solid-phase g assays can be used. Exemplary of binding assays that can be used to assess, evaluate, determine, fy and/or otherwise specifically detect hyaluronan expression or levels in a sample include, but are not limited to, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluroassay, chemiluminescent assay, bioluminescent assay.

For example, a HABP companion diagnostic provided herein can detect HA using any —126- binding assay known to one of skill in the art, including but not limited to, enzyme- linked immunosorbent assay ) or other similar immunoassay, including a sandwich ELISA or competitive ELISA assay. Exemplary methods provided herein include ELISA based methods for quantitative or semi-quantitative detection of the amount ofHABP that binds to HA in a sample, such as a tumor tissue sample or fluid sample from a subject having a tumor or suspected of having a tumor. The use of solid phase binding assays can be used when HA is detected in a bodily fluid.

As described herein, patients that t high levels ofhyaluronan production in the tumor tissue also exhibit high levels of hyaluronan in blood. Accordingly, the methods provided herein encompass s of predicting the responsiveness of a t to treatment with a hyaluronan—degrading enzyme, to select ts for treatment with a hyaluronan-degrading enzyme, or to monitor treatment with a hyaluronan degrading enzyme, including assessing the accumulation ofhyaluronan in a fluid sample from a patient having a tumor or a patient suspected of having a tumor.

Fluid samples for is of HA production in an HA-associated disease, such as cancer, include but are not limited to serum, urine, plasma, cerebrOSpinal fluid, and lymph. The subject can have or be suspected of having a cancer, such as a primary and metastatic tumors, in breast, colon, rectum, lung, stomach, ovary, cervix, uterus, testes, bladder, prostate, thyroid, lung cancer. In particular examples, the cancer is a late—stage cancer, a metastatic cancer, undifferentiated cancer, ovarian , in situ carcinoma (ISC), squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer, non—small cell lung cancer, breast cancer, or colon cancer.

In exemplary s to t the responsiveness of subject to treatment with a hyaluronan—degrading enzyme or to select subjects for treatment with a hyaluronan-degrading enzyme, collection of a fluid sample from a subject is lly med prior to treatment of the subject with a hyaluronan-degrading . In exemplary methods ofmonitoring therapy of a tumor with a hyaluronan—degrading enzyme, collection of the fluid sample from a subject can be performed before, during or after the subject has received one or more treatments with a hyaiuronan-degrading ‘ enzyme. Harvesting of the fluid sample also can be performed before, during, or after the subject has undergone one or more rounds of anti—cancer y, such as ion and/or chemotherapy treatment.

RECTIFIED SHEET (RULE 91) ISA/EP -l27- The fluid sample then can be assessed for the presence or amount ofHA using a solid-phase binding assay. Solid-phase binding assays can detect a substrate (e.g.

HA) in a fluid sample by binding of the substrate to a binding agent that is fixed or immobilized to a solid e. A substrate specific antibody or binding protein (e.g. an HABP provided ), coupled to detectable label (e.g. an enzyme), is applied and allowed to bind to the substrate. Presence of the antibody or bound protein is then detected and quantitated. Detection and quantitation methods include, but are not limited to, colorimetric, fluorescent, luminescent or radioactive methods. The choice of detection method is dependent on the detectable label used. In some examples, a colorimetric reaction employing the enzyme coupled to the antibody. For example, enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. The amount of substrate present in the sample is tional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy. The concentration ofHA in a sample can be calculated by interpolating the data to a standard curve. The amount of HA can be expressed as a concentration of fluid sample.

In an ary method, an HABP reagent that is generally unlabeled is first immobilized to a solid support (e.g. coated to wells of a microtiter plate), followed by tion with a fluid sample ning HA (6.g. serum or plasma) to e HA.

After washing the fluid sample with an appropriate buffer, bound HA can be detected.

In some examples to detect the bound HA, a second HABP that is the same or different than the immobilized HABP and that is labeled (labeled HABP), such as a ylated HABP, is used to bind to the HA on the plate. Following removal of the d labeled HABP, the bound d HABP is detected using a detection reagent. For example, biotin can be detected using an avidin detection reagent. In some examples, the HABP bound to the plate is different from the HABP used for detection. In other examples, the HABP bound to the plate and the HABP for detection are the same. In other examples to detect the bound HA, bound HA is detected by addition of HABP and subsequent addition of an anti-HABP antibody.

For example, for detecting TSG-6 or a TSGLM, an anti-TSG-6 link module onal antibody can be used, such as antibodies designated A38 and Q75 (see, Lesley et al. (2002) J Biol Chem 600-26608). The anti-HABP antibodies can -l28- be labeled for detection or can be detected with a secondary dy that binds the first antibody. In yet other examples to detect the bound HA, bound HA is directly detected with an anti-HA antibody. Anti-HA antibodies are well known to one of skill in the art, and include, for example, a sheep anti-hyaluronic acid polyclonal antibody (e.g., Abcam #53842 or #93321). c. In vivo Imaging Assays In some es herein, the amount ofHA is detected using in viva imaging s. In such s, the HABP, such as a TSGLM, multimer (e.g. TSG- 6LLM-Fc) or t thereof, is ated to a detectable moiety or moiety that is capable of detection by an g . Exemplary imaging methods include, but are not limited to, fluorescence imaging, X-rays, magnetic resonance methods, such as magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS), and tomographic s, including computed tomography (CT), computed axial tomography (CAT), electron beam computed tomography (EBCT), high resolution ed aphy (HRCT), hypocycloidal tomography, positron emission tomography (PET), -photon emission computed tomography (SPECT), spiral computed tomography and ultrasonic tomography. For example, for fluorescence imaging, fluorescent signals can be analyzed using a fluorescent microscope or fluorescence stereomicroscope. Also, a low light imaging camera also can be used.

In particular, the HABP, such as a TSGLM, multimer (e.g. TSG-6LLM-Fc) or variant thereof, is labeled or conjugated with a moiety that provides a signal or induces a signal that is detectable in viva, when imaged, such as, but not d to, magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), positron emission tomography (PET), scintigraphy, gamma camera, a [3+ detector, a y detector, cence imaging and inescence imaging.

Exemplary imaging/monitoring methods include any of a variety magnetic resonance methods such as magnetic resonance imaging (MRI) and ic resonance spectroscopy (MRS), and also include any of a variety of tomographic methods including computed tomography (CT), computed axial tomography (CAT), electron beam computed tomography (EBCT), high resolution computed tomography (HRCT), hypocycloidal tomography, positron emission tomography (PET), gamma rays (after —129- annihilation of a positron and an electron in PET scanning), single—photon emission computed tomography (SPECT), spiral computed tomography and ultrasonic tomography. Other exemplary g s include low-light g, X-rays, ultrasound signal, fluorescence absorption and bioluminescence. In on, the proteins can be labeled with light-emitting or other electromagnetic spectrum-emitting compounds, such as fluorescent compounds or molecules. ion can be effected by detecting emitted light or other emitted electromagnetic radiation.

Detectable labels include reagents with directly detectable elements (e.g. radiolabels) and reagents with indirectly detectable elements (e.g. a on product).

Section C.3.c also describes detectable labels. Examples of able labels include radioisotopes, bioluminescent nds, chemiluminescent nds, fluorescent compounds, metal chelates and enzymes. A detectable label can be incorporated into - an HABP by chemical or recombinant methods, Labels appropriate for X~ray imaging are known in the art, and e, for example, h (111), Gold (III), Lanthanum (III) or Lead (II); a radioactive ion, such as 67Copper, 67Gallium, 68Gallium, 1 l 11ndium, IIBIndium, 123Iodine, l2SIodine, 13lIodine, 197Mercury, 203Mercury, 186Rhenium, I88Rhenium, 97Rubidium, 103Rubidium, 99Technetium or 90Yttrium; a nuclear magnetic spin~ resonance isotope, such as Cobalt (II), COpper (II), Chromium (III), Dysprosium (III), Erbium (III), Gadolinium (III), m (III), Iron (11), Iron (III), Manganese (II), Neodymium (III), Nickel (11), Samarium (III), Terbium (III), Vanadium (H) or ium (III); or ine or fluorescein.

Contrast agents are used for magnetic resonance imaging. Exemplary contrast agents include iron, gold, gadolinium and gallium. Labels appropriate for magnetic resonance imaging are known in the art, and include, for example, fluorine, gadolinium chelates, metals and metal oxides, such as for e, iron, gallium, gold, gadolinium, magnesium, 1H, 19F, 13C and 15N labeled compounds. Use of es in contrast agents is known in the art. Labels appropriate for tomographic imaging methods are known in the art, and include, for example, B-emitters such as “C, l3’N, 15O or 64Cu or (b) y—emitters such as 1231- Other exemplary radionuclides that can, be used, for example, as tracers for PET include 55Co, 67Ga, 68Ga, 60Cu(II), 67Cu(Il), 99Tc, 57Ni, 52Fe and 18F. The reagent, such as TS6-6 or the FC portion RECTIFIED SHEET (RULE 91) ISA/EP ~130- thereof can be conjugated to a le label and/or the protein can include a radiolabel in its constituent molecules.

An exemplary list of radionuclides useful for the g methods provided herein includes, for example, on, llFluorine, ‘3Carbon, 13Nitrogen, ”Nitrogen, ”Oxygen, ”Flourine, 19Flourine, 24Sodium, phate, 42Potassium, 5IChromiurn, 55Iron, 59Iron, 57Cobalt, lt, 64Copper, “Gallium, “Gallium, ”Selenium, 8‘Krypton, 82Rubidium, 89Strontium, 92Strontium, 90Yttirum, 99Technetium, 103Palladium, méRuthenium, lllIndium, 117Lutetium,123lodine, 12slodine, 13Ilodine, on, 137Cesium, 153Samarium, 153Gadolinium, 165Dysprosium, ‘66Holmium, ‘69Ytterbium, 177Le:utium186Rhe1'liurri, lssRhenium, ‘921ridium, lgsGold,2°1Thallimn, 21'Astatine, muth and 213Bismuth. One of skill in the art can alter the ters used in different g methods (MRI, for example) in order to visualize different radionuclides/metals.

Fluorescent labels also can be used. These include fluorescent proteins, ' fluorescent probes or fluorescent substrate. For example, fluorescent ns can include, but are not limited to, fluorescent proteins such as green cent protein (GFP) or homologs thereof or RFP; fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green®, rhodamine and derivatives (e. g., Texas red and tetramethyl rhodamine isothiocyanate (TRITC)), biotin, phycoerythrin, AMCA, Alexa Fluor®, Li—COR®, cynyese or DyLight® Fluors); thomato, mCherry, InPlum, e, TagRFP, mKateZ, TurboRFP and TurboFP635 (Katushka). The fluorescent reagent can be chosen based on user desired excitation and emission spectra. Fluorescent substrates also can be used that result in cent cleavage products.

The in vivo imaging methods can be used in the diagnosis of HA—associated tumors or cancers. Such a technique permits diagnosis without the use of biopsy. In vivo imaging methods based on the extent or level of binding of an HABP to a tumor also can. be used for prognoses to cancer patients. The in vivo imaging methods also can be used to detect metastatic cancers in other parts ofthe body or circulating tumor cells (CTCs). It is Within the level of one of skill in the art to ascertain ound levels of hyaluronan in tissues other than tumors. Hyaluronan-expressing tumors will have higher levels of signal than background tissues. In some examples, threshold RECTIFIED SHEET (RULE 91) ISA/EP -l3l- criteria can be determined by comparisons to signal detected in normal or healthy subjects. 2. Classification of Subjects Once the amount of hyaluronan in the sample is determined, the amount can be compared to a control or threshold level. The control or threshold level is generally a predetermined old level or amount that is indicative ofa hyaluronan-associated disease or condition (e. g. a tumor or cancer). Such level or amount can be empirically determined by one skilled in the art. It is understood that the particular predetermined selection or classification criteria for; the methods herein are dependent on the particular assay that is used to detect hyaluronan and the particular sample that is being tested. It is within the level of one of skill in the art to determine if an assay is compatible with g a particular sample. Generally, in vitro solid phase assays are used for testing body fluid samples. Solid phase assays such as histochemistry or immunohistochemistry are generally used for g tissue samples. It also is understood that in methods involving comparisons to a predetermined level or amount or to a control or reference sample that the references are made with the same type of sample and using the same assay and HABP reagent {including the same detectable moiety and detecting method).

For example, the predetermined old level can be determined based on the level or amount of the marker in a nce or control sample, such as the median or mean level or amount of the marker in a population of subjects, in order to assess differences in levels or expression. In one e, the predetermined threshold level can represent the mean or median level or amount of hyaluronan in a sample from a healthy subject or a subject known tohave a hyaluronan-associated e or condition (e. g. a tumor or cancer). In one embodiment, the predetermined level or amount of hyaluronan from a normal tissue or bodily fluid sample is the mean level or amount observed in normal s (e.g., all normal samples analyzed). In r embodiment, the level or amount of hyaluronan from a normal tissue or bodily fluid sample is the median value for the level or amount observed in normal samples. The predetermined threshold level also can be based on the level or amount of hyaluronan in a cell line or other control sample (ag. tumor cell line). As described below, these predetermined values can be determined by comparison or dge of HA levels in RECTIFIED SHEET (RULE 91) ISA/EP 2012/061743 a corresponding normal sample as determined by the same assay of detection and using the same HABP t.

The reference or control sample can be r tissue, cell or body fluid, such as a normal tissue, cell or body fluid, for example, a tissue, cell or body fluid that is analogous to the sample being tested, but isolated fiom a different subject. The control or reference subject can be a subject or a population of subjects that is normal (i.e. does not have a disease or condition), a subject that has a disease but does not have the type of disease or ion that the subject being tested has or is Suspected of having, for example, a subject that does not have a hyaluronan-associated disease or condition (9. g. a tumor or cancer), or an analogous tissue from another subject that has a similar disease or condition, but whose disease is not as severe and/0r expresses relatively less hyaluronan. For example, when the cell, tissue or fluid being tested is a subject or a population of subjects having a , the level or amount of the marker can be compared to the level or amount of the marker in a tissue, cell or fluid from a subject having a less severe , such as an early stage, differentiated or other type of cancer. In another example, a control or reference sample is a fluid, tissue, extract (a.g. cellular or nuclear extract), nucleic acid or peptide preparation, cell line, biopsy, rd or other sample, with a known amount or relative amount of hyaluronan, such as a sample, for example a tumor cell line, known to express relatively low levels ofHA, such as exemplary tumor cell lines that express low levels of HA, for example, the HCT 116 cell line, the HT29 cell line, the NCI H460 cell line, the DU145 cell line, the Capan-l cell line, and tumors from tumor models generated using such cell lines.

In any method herein, the 1evel(s) of onan in samples from subjects suspected or known to have a hyaluronan—associated disease or condition (e.g. , cancer) can be determined rently with the determination of 1evel(s) of onan in reference or normal tissues. Alternatively, the levels of hyaluronan in samples from subjects suspected or known to have a hyaluronan-associated disease or condition (e. g. cancer) can be compared to the 1evel(s) of hyaluronan previously determined in normal tissue or bodily fluid. Thus, the level of onan in normal or healthy samples or other reference s employed in any detection, comparison, determination, or evaluation can be a level or amount determined prior to any RECTIFIED SHEET (RULE 91) ISA/EP ~133- detection, determination, or evaluation ofthe level or amount of hyaluronan in a sample from a human patient.

The level or amount of hyaluronan is determined and/or scored and compared to predetermined phenotypes ofhyaluronan associated with disease. It is within the level of one of skill in the art to determine the threshold level for disease diagnosis depending on the particular disease, the assay being used for ion of the hyaluronan and/or the HABP detection reagent being used. It is within the level of one of skill in the art to determine the threshold level of the hyaluronan for classifying responsiveness to treatment with an anti-hyaluronan agent (6.g. a onan- degrading enzyme). Exemplary methods for stratification of tumor samples or bodily fluid samples for diagnosis, prognosis or selection of subjects for treatment are provided herein.

It is tood that the particular change, e. g. increase in or decrease of hyaluronan is dependent on the assay used." In an ELISA, the fold increase or decrease in ance at a particular wavelength or in quantity of n (e. g. as determined by using a standard curve) can be expressed relative to a control. In a PCR assay, such as RT-PCR, expression levels can be compared to control expression levels (e. g. expressed as fold change) using methods known to those in the art, such as using standards.

In particular examples of the methods herein, a subject is selected as a candidate for therapy with an anti-hyaluronan agent ifthe amount of hyaluronan is determined to be elevated in the sample. For example, elevated or lated hyaluronan levels in a ed subject compared to a healthy or normal subject is indicative of a hyaluronan-associated e or condition (e.g. tumor or cancer). The ' hyaluronan can be elevated 05—fold, , , 3—fold, 4-fold, 5~fold, 6—fold, 7~ fold, 8-fold, 9-fold, lO-fold or more. Thus, in examples of the methods herein, when the amount of onan in a sample from a subject is being tested, detection of the marker can be determining that the amount ofHA in the sample (e.g. cancerous cell, tissue or fluid) from the t is elevated compared to a predetermined level or amount or control . In one example, the subject is determined to have a hyalruonan—associated disease or condition if the amount ofHA in the tissue,» cell or fluid is elevated at or about 05-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- RECTIFIED SHEET (RULE 91) ISA/EP - 134.. fold, , 9—fold, 10-fold, 11-fold, 12-fold, 13-fold, l4~fold, 15—fold, 20—fold, or more, compared to the predetermined level or amount or control sample.

A subject can be ed as a candidate for therapy with an anti-hyaluronan agent (6.g. a onan-degrading enzyme) based on the level or amount of hyaluronan in a sample (e. g. a bodily fluid or other fluid) from the subject. HA greater than 0.010 ug/mL, 0.015 pg/mL, and generally greater than 0.02 ug/mL, 0.03 ng/mL, 0.04 pg/mL, 0.05 pg/mL, 0.06 pg/mL or higher correlates to the presence of a tumor or cancer. Using such methods, in exemplary methods provided herein, a subject can be selected for treatment with a an anti-hyaluronan agent (e. g. hyaluronan-degrading enzyme) if the concentration ofHA in the fluid , such as a serum sample, ns HA levels greater than 0.010 pg/mL, 0.015 ug/mL, and generally r than 0.02 ng/mL, 0.03 ng/mL, 0.04 ng/mL, 0.05 ug/mL, 0.06 pg/mL or higher.

A t can be selected as a candidate for therapy with ananti—hyaluronan agent (e.g. a hyaluronan-degrading enzyme) based on the level or amount of hyaluronan in a cell or tissue sample. In such an example, if the level is indicative of e, then the patient is diagnosed With a hyaluronan-associated disease or condition. For example, using immunohistochemistry methods of tumor tissues a score of HA+2 or HA+3 can be determinative of disease. For example, a percentage of staining ofHA over total tumoral area of greater than 10%, such as 10 to 25%, or greater than 25% is indicative of disease. In the methods herein, a subject is selected for treatment with an anti-hyaluronan agent (e.g. a hyaluronan~degrading enzyme) if the scaled score of the sample is an HA+2 or HA+3 sample. For example, a high score, e. g. indicates the subject has a HA-rich tumor indicative of the presence of a , HA”, tumor that would benefit from treatment with an anti-hyaluronan agent (e.g. a hyaluronan—degrading enzyme) and thus is a candidate for therapy with an anti- onan agent (e.g. a hyaluronan-degrading enzyme). In other examples, a t can be selected for ent with an anti-hyaluronan agent (e.g. a hyaluronan- degrading enzyme) based on the percentage of staining, for example, if the degree of HA staining is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more ofthe total staining area, and generally at least 25% or marqi RECTIFIED SHEET (RULE 91) ISA/EP Efficacy of treatment with an anti—hyaluronan agent (e. g. a hyaluronan- degrading enzyme) or responsiveness to treatment also can be monitored by comparing the level or amount ofhyaluronan in a t over time. Changes in the level or amount of hyaluronan can be used to optimize dosing or scheduling of treatment with an yaluronan agent (e.g. a hyaluronan-degrading enzyme). In the method, the level of HA expression in samples, in particular as assessed in tumor tissues (e.g. via immunohistochemistry or other similar method), from treated subjects are compared to a predetermined level ofHA expression. For purposes of monitoring ent after administration of a hyaluronan—degrading enzyme, in particular one with an extended half-life (e. g. PEGPHZO), the sample that is monitored is not a bodily fluid in which ic levels ofenzyme can be present.

For purposes of monitoring ent, the predetermined level ofIIA can be from a normal or healthy subject, a baseline HA value prior to treatment, the prior measured HA level in the same subject at an r time after treatment, or a classification or stratification ofHA levels known to be associated with disease progression or regression. For example, if the hyaluronan level is about the same as or below (or decreased) as compared reference or control sample, the treatment is likely efficacious and the treatment can be continued or tinued or altered. For example, the predetermined level of HA can be HA levels from a normal or healthy tissue sample, and if the level ofHA measured in the t after treatment is higher than the normal HA levels, then ent is d or continued. For example, the predetermined level of HA can be an HA level as determined from a baseline HA value prior to treatment, and the course oftreatment determined accordingly. For example, if the level ofHA is the same as baseline HA, then treatment is continued or resumed; if the level ofHA is higher than baseline HA then treatment is continued or resumed or treatment is accelerated or increased (eg. by increasing the dosage of hyaluronan- degrading enzyme or increasing the dose le in a dosage regimen ; if the level ofHA is less than baseline HA then treatment is continued or resumed, terminated or is reduced or decreased (e.g. by decreasing the dosage of hyaluronan-degrading . enzyme or decreasing the dose schedule in a dosage regimen cycle). In a further example, the predetermined level of HA can be an HA level as determined in a prior measurement in an earlier course of ent of the same subject. For e, if RECTIFIED SHEET (RULE 91) ISA/EP the level of HA is the same as the earlier measured HA, then treatment is continued or d; if the level of HA is biwer than the earlier measured HA then treatment is continued or resumed or treatment is accelerated or increased (6. g. by increasing the dosage of hyaluronan~degrading enzyme or increasing the dose schedule in a dosage U1 regimen ; ifthe level ofHA is less than the earlier measured IIA then treatment is continued or resumed, ated or is reduced or decreased (ag. by decreasing the dosage of hyaluronan-degrading enzyme or decreasing the dose schedule in a dosage regimen cycle).

In the monitoring s or methods of determining efficacy of treatment, ll) the particular therapy can be altered during the course of treatment to maximize individual response. Dosing and scheduling of treatment can be modified in response to changing levels. Combination therapy using other anti-cancer agents also can be employed in such treatment methods. It is within the level of the skill of the treating physician to determine the exact course of ent. For example, the treatrnent can be altered, such that the dosing amount, schedule (2.g freqency of administration), or regime is adjusted accordingly, such as discontinued, decreased or made less frequent, or combined with another treatment for the disease or ion. On. the other hand, if the hyaluronan level is above a compared reference or l sample, the patient is likely not responding to the treatment. In such instances, the particular nature and type of anti-hyaluronan agent (e. g. onan-degrading enzyme) or combination therapy can be modified or changed. In other instances, the dosing, amount, le and/or regime can be adjusted accordingly, such as increased or made more Frequent. .

It is within the level of the treating physician to determine the exact course of ent.

For purposes of monitoring efficacy of treatment, predetermined levels or amounts of hyaluronan can be empirically determined, whereby the level or amount indicates that the treatment is working. These predetermined values can be ined by comparison or dge of HA levels in a ponding normal sample or samples of disease subjects as determined by the same assay of detection and using the same HABP reagent. For example, high levels of HA as assessed by immunohistochemistiy methods using a quantitative score scheme (cg. HA“) or percentage of tumor staining for hyaluronan of greater than 25% ate to the RECTIFIED SHEET (RULE 91) ISA/EP -l37- existence of malignant disease across a range of cancer types, and te that a patient is not ding to treatment. In another example, HA levels in bodily fluid such as plasma of greater than 0.015 ug/mL, and generally greater than 0.02 ug/mL, such as 0.03 pg/mL, 0.04 pg/mL, 0.05 ug/mL or 0.06 ug/mL HA, is associated with advanced disease stage. On the other hand, a subject is likely responding to treatment if the scaled score of the sample is less than an HA+2 or HA+3 or the percentage ofHA staining is less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 7% (11-1ch A en‘hjpnf ie manly rpcpmnrling m trnafmpnt arm: ”A law] in bodily fluid such as plasma is less than 0.03 ug/mL, 0.02 ug/mL, 0.01 ug/mL or less.

In the methods herein, the comparison to a predetermined level or to levels of a control or reference sample can be determined by any method known of skill in the art. For example, the comparison of the level of hyaluronan with a reference, control or predetermined level can be done by an automated system, such as software program or intelligence system that is part of, or compatible with, the equipment (e.g. computer platform) on which the assay is carried out. Alternatively, this comparison can be done by a physician or other trained or experienced professional or technician.

E. ENT OF SELECTED SUBJECTS WITH AN ANTI- HYALURONAN AGENT The s provided herein include methods of treating a tumor-bearing t with an anti-hyaluronan agent, for example a hyaluronan~degrading enzyme, where the subject has been selected for treatment based on level of HA ed in the tumor. The methods of treatment also include methods for assessing effects of ent with an anti—hyaluronan agent, for example a hyaluronan-degrading enzyme, such as efficacy of treatment, such as for example, tumor inhibition or regression, or side effects of treatment, such as for example, musculoskeletal side effects. Combination therapies with one or more additional anti-cancer agents or one more agents to treat one or more side effects of y with an anti-hyaluronan agent (2. g. a hyaluronan-degrading enzyme) also are provided. 1. Anti-Hyaluronan Agent yaluronan agents include agents that inhibit onan synthesis or degrade onan. Anti-hyaluronan , such as hyaluronan degrading enzymes, can be used to treat a hyaluronan~associated disease or condition, including tumors RECTIFIED SHEET (RULE 91) ISA/EP -l38- and cancers or inflammatory diseases or conditions. For example, HA lation, such as by altered hyaluronan metabolism, distribution and fianction is associated with arthritis, immune and inflammatory ers, pulmonary and vascular es and cancer (Morohashi et al. (2006) Biochem. Biophys. Res. C0mm., 345:1454-1459).

Such diseases can be treated by inhibiting HA synthesis or degrading HA (see e.g. shi 2006; US. published application No. 20100003238 and International published PCT Appl. No WC 2009/128917). In some es, such treatments that reduce hyaluronan levels on cells and tissues can be associated with adverse side effects, such as musculoskeletal side s. Hence, treatment with an anti- hyaluronan-agent can r include treatment with a corticosteroid to rate or reduce such side effects. 21. Agents that Inhibit Hyaluronan Synthesis HA can be synthesized by three enzymes that are the products of three related ian genes identified as HA synthases, designated has-I has-2 and has-3.

Different cell types express different HAS enzymes and expression of HAS mRNAs is correlated with HA biosynthesis. It is known that silencing HAS genes in tumor cells inhibits tumor grth and asis. An yaluronan agent includes any agent that inhibits, reduces or downregulates the expression or level of an HA synthase. Such agents are known to one of skill in the art or can be fied.

For example, downregulation of a HAS can be accomplished by providing oligonucleotides that specifically hybridize or otherwise interact with one or more nucleic acid molecules encoding an HAS. For example, anti-hyaluronan agents that inhibit hyaluronan synthesis include antisense or sense molecules against an has gene.

Such antisense or sense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded or otherwise rendered inoperable. In other examples, post-transcriptional gene silencing (PTGS), RNAi, ribozymes and DNAzymes can be employed. It is within the level of one skill in the art to generate such constructs based on the sequence of HASl (set forth in SEQ ID NO:219), HAS2 (set forth in SEQ ID NO:220) or HAS3 (set forth in SEQ ID NO:22l). It is understood in the art that the sequence of an nse or sense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.

WO 63155 Moreover, an oligonucleotide can hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g. a loop structure or hairpin structure). Generally, the antisense or sense compounds have at least 70% sequence mentarity to a target region within the target nucleic acid, for example, 75% to 100% complementarity, such as 75%, 80%, 85%, 90%, 95% or 100%. Exemplary sense or nse molecules are known in the art (see e.g.

Chao et al. (2005) J. Biol. Chem, 280:27513-27522; Simpson et al. (2002) J. Biol.

Chem, 277: 10050-10057; Simpson et al. (2002) Am. JPath., 161 :849; a et al. (1999) J. Biol. Chem, 274:21893-21899; Edward et al. (2010) British JDermatology, 162: 1224-1232; Udabage et al. (2005) Cancer Res., 65:6139; and published US.

Patent ation No. US20070286856).

Another exemplary anti-hyaluronan agent that is an HA synthesis inhibitor is 4-Methylumbelliferone (4-MU; 7-hydroxymethylcoumarin) or a derivative f. 4-MU acts by reducing the UDP-GlcUA precursor pool that is required for HA synthesis. For example, in mammalian cells, HA is synthesized by HAS using UDP- glucuronic acid (UGA) and UDP-N—acetyl-D-glucosamine precursors. 4-MU interferes with the process by which UGA is generated, thereby depleting the intracellular pool ofUGA and resulting in inhibition ofHA synthesis. 4-MU is known to have mor activity (see e.g. war et al. (2010) Cancer Res., 70:2613-23; Nakazawa et al. (2006) Cancer Chemother. Pharmacol, 57: 165-170; Morohashi et al. (2006) Biochem Biophys. Res. Comm, 3451459). Oral administration of 4-MU at 600 d reduces metastases by 64% in the B16 melanoma model (Yoshihara et al. (2005) FEBS Lett., 579:2722-6). The structure of 4-MU is set forth below. Also, derivatives of 4-MU exhibit anti-cancer activity, in ular 6,7-dihydrozymethyl coumarin and 5,7-dihydroxymethyl coumarin (see e.g. Morohashi et al. (2006) Biochem Biophys. Res. Comm, 3451459). 4-Methylumbelliferone (4-MU; C10H803) HOmyO O Further exemplary anti-hyaluronan agents are tyrosine kinase inhibitors, such as Leflunomide (Arava), genistein or erbstatin. Leflunomide also is a pyrimidine —140— synthesis tor. Leflunomide is a known drug for the treatment of Rheumatoid arthritis (RA), and also is effective in treating the rejection of afts as well as xenografts. HA is known to directly or indirectly bute to RA (see e.g.

Stuhlmeier (2005) Jlmmun01., 76-73 82). Tyrosine kinase inhibitors inhibit HAS] gene expression (Stuhlmeier 2005).

In one example, leflunomide, or derivatives thereof, lly are available as tablets containing 1-100 mg of active drug, for e, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg of drug. For the treatment of a hyaluronan-associated disease and conditions, for example Rheumatoid arthritis or a tumor or cancer, it is administered at 10 to 500 mg per day, typically 100 mg per day. The dosage can be ued as needed for treatment of the disease or conditions, or can be tapered or reduced to successively lower doses. For example, for treatment of Rheumatoid arthristis, leflunomide can be administered at an initial loading dose of 100 mg per day for three days and then administered at a continued dose of 20 mg/day. b. Hyaluronan-degrading s Hyaluronan is an essential component of the extracellular matrix and a major constituent of the titial barrier. By catalyzing the hydrolysis of hyaluronan, hyaluronan-degrading enzymes lower the Viscosity of hyaluronan, thereby increasing tissue permeability and increasing the absorption rate of fluids administered parenterally. As such, hyaluronan-degrading enzymes, such as hyaluronidases, have been used, for example, as spreading or dispersing agents in conjunction with other , drugs and proteins to enhance their dispersion and delivery.

Hyaluronan degrading s act to e hyaluronan by cleaVing hyaluronan polymers, which are composed of repeating harides units, D- glucuronic acid (Gch) and N—acetyl-D-glucosamine (GlcNAc), linked together Via alternating B-l—>4 and B-l—>3 glycosidic bonds. Hyaluronan chains can reach about ,000 disaccharide repeats or more in length and rs of hyaluronan can range in size from about 5,000 to 20,000,000 Da in viva. Accordingly, hyaluronan degrading enzymes for the uses and methods provided include any enzyme having the ability to catalyze the cleavage of a hyaluronan disaccharide chain or polymer. In some examples the hyaluronan degrading enzyme cleaves the B-l—>4 glycosidic bond in the hyaluronan chain or polymer. In other examples, the hyaluronan degrading —141— enzyme catalyze the cleavage of the B-l—>3 glycosidic bond in the hyaluronan chain or polymer.

Hence, hyaluronan degrading enzymes, such as hyaluronidases, are a family of enzymes that e 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, hyaluronan degrading enzymes lower the viscosity of hyaluronic acid, thereby increasing tissue permeability. As such, hyaluronan ing s, such as 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. Hyaluronan-degrading s also are used as an adjuvant to increase the absorption and dispersion of other injected drugs, for hypodermoclysis (subcutaneous fluid stration), and as an adjunct in subcutaneous urography for improving resorption of radiopaque agents. Hyaluronan-degrading enzymes, for example, hyaluronidase can be used in applications of ophthalmic procedures, for example, peribulbar and non’s block in local anesthesia prior to ophthalmic surgery.

Hyaluronidase also can be used in other eutic and cosmetic uses, for example, by promoting akinesia in cosmetic surgery, such as blepharoplasties and face lifts.

Various forms of onan degrading enzymes, including hyaluronidases have been prepared and approved for eutic use in subjects, including humans.

The provided itions and methods can be used, Via these and other therapeutic uses, to treat hyaluronan-associated diseases and conditions. For example, animal- derived hyaluronidase preparations include Vitrase (ISTA Pharmaceuticals), a purified ovine testicular hyaluronidase, ase (Amphastar Pharmaceuticals), a bovine testicular hyaluronidase and Hydase (Prima Pharm Inc.), a bovine testicular hyaluronidase. It is understood that any animal-derived hyaluronidase preparation can be used in the methods and uses provided herein (see, e. g., US. Patent Nos. 564, 2,488,565, 2,676,139, 2,795,529, 815, 2,808,362, 5,747,027 and ,827,721 and Intemation PCT Application No. WO2005/l l8799). Hylenex (Halozyme Therapeutics) is a human recombinant onidase produced by genetically engineered Chinese Hamster Ovary (CHO) cells containing nucleic acid encoding soluble forms of PH20, designated rHuPH20. —142— Exemplary of onan degrading enzymes in the compositions and methods ed herein are soluble hyaluronidases. Other exemplary onan degrading enzymes include, but are not limited to particular chondroitinases and lyases that have the ability to cleave hyaluronan.

As described below, hyaluronan-degrading enzymes exist in membrane-bound or soluble forms that are secreted from cells. For purposes herein, soluble hyaluronan-degrading s are provided for use in the methods, uses, compositions or combinations herein. Thus, where hyaluronan-degrading enzymes include a ylphosphatidylinositol (GPI) anchor and/or are otherwise membrane- anchored or insoluble, such hyaluronan-degrading s are provided herein in soluble form by truncation or on of the GPI anchor to render the enzyme secreted and soluble. Thus, hyaluronan-degrading enzymes include truncated variants, e.g. truncated to remove all or a portion of a GPI anchor. Hyaluronandegrading enzymes provide herein also e allelic or species variants or other variants, of a soluble hyaluronan-degrading enzyme. For example, hyaluronan degrading enzymes can contain one or more variations in its primary ce, such as amino acid substitutions, additions and/or deletions. A variant of a hyaluronan- degrading enzyme generally exhibits at least or about 60 %, 70 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or more sequence identity compared to the hyaluronan-degrading enzyme not containing the variation. Any variation can be included in the hyaluronan degrading enzyme for the purposes herein provided the enzyme retains hyaluronidase activity, such as at least or about 5 %, lO %, l5 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 % or more of the activity of a hyaluronan degrading enzyme not containing the variation (as measured by in vitro and/or in viva assays well known in the art and described herein).

Where the s and uses provided herein describe the use of a soluble onidase, accordingly any hyaluronan degrading enzyme, generally a soluble hyaluronan degrading enzyme, can be used. It is understood that any onidase can be used in the methods and uses ed herein (see, e. g., US. Patent No. 7,767,429 and US. Publication Nos. US20040268425 and US20100143457).

WO 63155 —143— i. onidases Hyaluronidases are members of a large family of hyaluronan degrading enzymes. There are three general classes of hyaluronidases: mammalian-type onidases, bacterial hyaluronidases and hyaluronidases from leeches, other parasites and crustaceans. Such enzymes can be used in the compositions, ations and methods provided herein. (1) Mammalian-type hyaluronidases Mammalian-type hyaluronidases (EC 3.2. l .35) are -N—acetyl- hexosaminidases that hydrolyze the B-l—>4 glycosidic bond of hyaluronan into various oligosaccharide lengths such as tetrasaccharides and ccharides. These enzymes have both hydrolytic and transglycosidase activities, and can e 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:lO, 11 and 64 and BH55 (US. Pat. Nos. 5,747,027 and 5,827,721), nucleic acid molecules set forth in SEQ ID NOS: 190-192), sheep (Ovis aries) (SEQ ID NO: 26, 27, 63 and 65, nucleic acid molecules set forth in SEQ ID NOS:66 and 193-194), yellow jacket wasp (SEQ ID NOS: l2 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: l7- l9, 32), pig (SEQ ID NOS:20-2l), rat (SEQ ID NOS:22-24, 31), rabbit (SEQ ID NO:25), orangutan (SEQ ID NO:28), lgus monkey (SEQ ID NO:29), guinea pig (SEQ ID NO:30), chimpanzee (SEQ ID NO: 101), rhesus monkey (SEQ ID NO:lO2), and human hyaluronidases (SEQ ID NOS:l-2, 36-39). Exemplary of hyaluronidases in the compositions, combinations and methods provided herein are soluble hyaluronidases.

Mammalian hyaluronidases can be r subdivided into those that are neutral active, predominantly found in testes extracts, and acid , 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 , 63 and 65), bovine (SEQ ID NO:ll and 64) and human (SEQ ID NO: 1). Human PH20 (also known as SPAMl or sperm surface protein PH20), is generally attached to the plasma membrane via a glycosylphosphatidyl inositol (GPI) WO 63155 —144— anchor. It is naturally involved in sperm-egg adhesion and aids penetration by sperm of the layer of cumulus cells by digesting hyaluronic acid.

Besides human PH20 (also termed SPAMl), five hyaluronidase-like genes have been identified in the human genome, HYALl, HYAL2, HYAL3, HYAL4 and HYALPl. HYALPl is a pseudogene, and HYAL3 (SEQ ID N038) has not been shown to possess enzyme ty toward any known substrates. HYAL4 (precursor ptide set forth in SEQ ID NO:39) is a chondroitinase and exhibits little activity towards hyaluronan. HYALl (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 HYALl and HYAL2 (precursor polypeptide set forth in SEQ ID NO:37) generally lack catalytic activity at l pH (z'.e. pH 7). For example, HYALl has little catalytic activity in vitro over pH 4.5 (Frost et al. (1997) Anal. Biochem. 251263- 269). HYAL2 is an ctive enzyme with a very low specific activity in vitro. The hyaluronidase-like enzymes also can be characterized by those which are generally attached to the plasma membrane via a ylphosphatidyl inositol (GPI) anchor such as human HYAL2 and human PH20 (Danilkovitch-Miagkova et al. (2003) Proc Natl Acad Sci USA 100(8):45 80-5), and those which are generally soluble such as human HYALl (Frost et al. (1997) Biochem s Res Commun. 236(1): 10-5). (21) PH20 PH20, like other mammalian onidases, is an endo-B-N—acetyl- hexosaminidase that hydrolyzes the [Bl—>4 glycosidic bond of hyaluronic acid into various oligosaccharide lengths such as tetrasaccharides and hexasaccharides. It has both hydrolytic and transglycosidase activities and can degrade hyaluronic acid and chondroitin es, such as C4-S and C6-S. PH20 is naturally involved in sperm- egg adhesion and aids penetration by sperm of the layer of s cells by digesting hyaluronic acid. PH20 is located on the sperm surface, and in the lysosome-derived acrosome, where it is bound to the inner acrosomal membrane. Plasma membrane PH20 has hyaluronidase ty only at neutral pH, while inner acrosomal membrane PH20 has activity at both neutral and acid pH. In addition to being a hyaluronidase, PH20 is reported to be a receptor for HA-induced cell signaling, and a receptor for the zona pellucida surrounding the oocyte. —145— Exemplary PH20 proteins e, but are not limited to, human (precursor polypeptide set forth in SEQ ID NO:1, mature polypeptide set forth in SEQ ID NO: 2), chimpanzee (SEQ ID NO: 101), Rhesus monkey (SEQ ID NO: 102) bovine (SEQ ID NOS: 11 and 64), rabbit (SEQ ID NO: 25), ovine PH20 (SEQ ID NOS: 27, 63 and 65), Cynomolgus monkey (SEQ ID NO: 29), guinea pig (SEQ ID NO: 30), rat (SEQ ID NO: 31) and mouse (SEQ ID NO: 32) PH20 polypeptides.

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 h to the respective carboxy termini due to the absence of a GPI anchor in the bovine polypeptide (see e.g., Frost GI (2007) Expert Opin. Drug. Deliv. 4: 427-440). In fact, clear GPI s are not predicted in many other PH20 species besides humans. Thus, PH20 ptides produced from ovine and bovine naturally 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. (2001) Biol Reprod. 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®).

The human 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 ylphosphatidylinositol (GPI) anchor attachment signal sequence at the C- terminus (amino acid residue positions 491-509). The mature PH20 ore, is a 474 amino acid polypeptide set forth in SEQ ID NO:2. Following transport of the sor polypeptide to the ER and removal of the signal peptide, the inal GPI-attachment signal e is cleaved to facilitate covalent attachment of a GPI anchor to the newly-formed C-terminal amino acid at the amino acid on corresponding to position 490 of the precursor polypeptide set forth in SEQ ID NO: 1.

Thus, a 474 amino acid GPI-anchored mature polypeptide with an amino acid sequence set forth in SEQ ID NO:2 is produced.

Human PH20 exhibits hyaluronidase activity at neutral and acid pH. In one aspect, human PH20 is the prototypical neutral-active hyaluronidase that is generally -l46- locked to the plasma membrane via a GPI anchor. In r aspect, PH20 is expressed on the inner acrosomal membrane where it has hyaluronidase activity at neutral and acid pH. PHZO contains two catalytic sites at distinct regions of the polypeptide: the Peptide 1 and e 3 s (Cherr et al. (2001) Matrix Biology :515-525). Evidence indicates that the Peptide 1 region of PH20, which corresponds to amino acid positions 7 of the mature polypeptide Set forth in SEQ ID N02 and positions 142-172 of the precursor polypeptide set forth in SEQ ID NO: 1 is required for enzyme activity at neutral pH. Amino acids at ons 111 and 113 (corresponding to the mature PH20 polypeptide set forth in SEQ ID NO:2) within this region are reported to be important for activity, as mutagenesis by amino acid replacement results in PH20 polypeptides with 3 % hyaluronidase activity or undetectable hyaluronidase activity, respectively, compared to the wild-type PI—I20 (Arming et al., (1997) Eur. J. Biochem. 247:810-814).

The Peptide 3 region, which corresponds to amino acid positions 2 of the mature ptide set forth in SEQ ID NO:2, and positions 277-297 of the precursor polypeptide set forth in SEQ ID NO: 1, is reported to be important for enzyme activity at acidic pH. Within this region, amino acids at positions 249 and 252 of the mature PH20 polypeptide are reported to be essential for activity as mutagenesis of either results in a polypeptide ially devoid of activity (Arming et 01., (1997) Eur. J. Biochem. 0-814).

In addition to the catalytic sites, PHZO also ns a hyaluronan-binding site.

Experimental evidence indicate that this site is located in the Peptide 2 , which corresponds to amino acid positions 205-235 ofthe precursor polypeptide set forth in SEQ ID NO: 1 and positions 170-200 of the mature polypeptide set forth in SEQ ID N022. This region is highly conserved among hyaluronidases and is similar to the heparin binding motif. Mutation of the arginine residue at position 176 (corresponding to the mature PH20 ptide set forth in SEQ ID NO:2) to a glycine results in a polypeptide with only about 1 % of the hyaluronidase activity of the Wild type polypeptide (Arming er al., (1997) Eur. J. Biochem. 247:810-814).

There are seven potential glycosylation sites, including ed glycosylation sites, in human PHZO at N82, N166, N235, N254, N368, N393, S490 of the polypeptide exemplified in SEQ 10 NO: 1. Because amino acids 36 to 464 of SEQ ID RECTIFIED SHEET (RULE 91) ISA/EP 2012/061743 -147— NO:1 is reported to contain the minimally active human PH20 hyaluronidase , the glycosylation site 8-490 is not required for proper onidase activity. There are six disulfide bonds in human PHZO. Two disulfide bonds between the cysteine residues C60 and C35] and between C224 and C238 of the polypeptide exemplified in SEQ ID NO: 1 (corresponding to residues C25 and C316, and C189 and C203 of the mature polypeptide set forth in SEQ ID N022, respectively). A further four di sulfide bonds are formed between the cysteine residues C376 and C387; between C381 and C435; between C437 and C443; and between C458 and C464 of the polypeptide exemplified in SEQ ID NO: 1 (corresponding to residues C341 and C352; n C346 and C400; between C402 and C408; and between C423 and C429 of the mature polypeptide set forth in SEQ ID N02, respectively). (2) Other hyaluronidasés Bacterial hyaluronidases (EC 4.2.2.1 or EC .1) degrade hyaluronan and, to various extents, chondroitin sulfates and dennatan sulfates. Hyaluronan lyases isolated from bacteria differ from hyaluronidases (from other sources, e.g., hyaluronoglucosaminidases, EC 3.2.1.35) by their mode of action. They are endo-fi- N~acetylhexosaminidases that catalyze an elimination reaction, rather than hydrolysis, of the -glycosidic linkage between N-acetyl—beta-D—giucosamine and D- glucuronic acid residues in hyaluronan, yielding 3—(4-deoxy-B-D—gluc—4-enuronosyl)— N-acetyl-D-glucosamine tetra— and hexasaccharides, and disaccharide end products.

The reaction results in the formation of accharides with unsaturated hexuronic acid residues at their nonreducing ends.

Exemplary onidases from bacteria for use in the compositions, combinations and methods provided include, but are not limited to, hyaluronan degrading enzymes in microorganisms, including strains ofArthrobacter, Bdellovibrio, Clostridz'um, Micrococcus, ococcus, Peptococcus, Propiom‘bacterium, Bact-eroz'des, and Streptomyces. Particular examples of such strains and enzymes include, but are not limited to Arthrobacter Sp. n FB24 (SEQ ID NO:67)), Bdellovibrz'o bacteriovorus (SEQ ID NO:68), Propz‘onibaclerium acnes (SEQ ID N069), Streptococcus qgalactiae ((SEQ ID NO:70); lSRSZl (SEQ ID ; serotype Ia (SEQ ID NO:72); serotype III (SEQ ID ), Staphylococcus aureus (strain COL (SEQ ID NO:74); strain MRSA252 (SEQ ID RECTIFIED SHEET (RULE 91) ISA/EP NOS:75 and 76); strain MSSA476 (SEQ ID NO:77); strain NCTC 8325 (SEQ ID NO:78); strain bovine RF122 (SEQ ID NOS:79 and 80); strain USA300 (SEQ ID NO:8l)), Streptococcus pneumoniae ((SEQ ID NO:82); strain ATCC 5 / R6 (SEQ ID NO:83); serotype 2, strain D39 / NCTC 7466 (SEQ ID NO:84)), Streptococcus pyogenes (serotype Ml (SEQ ID NO:85); pe M2, strain MGASlO270 (SEQ ID NO:86); serotype M4, strain MGASlO750 (SEQ ID NO:87); serotype M6 (SEQ ID NO:88); serotype M12, strain MGAS2096 (SEQ ID NOS:89 and 90); serotype Ml2, strain MGAS9429 (SEQ ID NO:9l); serotype M28 (SEQ ID NO:92)), Streptococcus suz's (SEQ ID NOS:93-95); Vibrz’ofischerz’ (strain ATCC 700601/ ESl l4 (SEQ ID NO:96)), and the Streptomyces hyaluronolytz'cus hyaluronidase enzyme, which is specific for hyaluronic acid and does not cleave chondroitin or chondroitin sulfate (Ohya, T. and Kaneko, Y. (1970) Biochim. Biophys.

Acta 7). Hyaluronidases from s, other parasites, and crustaceans (EC 3.2.1.36) are endo-B-glucuronidases that generate tetra- and ccharide end- products. These enzymes catalyze hydrolysis of l—>3-linkages between [3-D- glucuronate and N—acetyl-D-glucosamine residues in hyaluronate. Exemplary hyaluronidases from leeches e, but are not d to, onidase from nidae (e.g., Hirudo medicinalz's), Erpobdellidae (e.g., Nephelopsz's obscura and Erpobdella punctata,), Glossiphoniidae (e.g., Desserobdella pz'cta, Helobdella stagnalz's, Glosslphonz'a complanata, Placobdella ornata and Theromyzon sp.) and Haemopidae (Haemopis marmorata) (Hovingh et al. (1999) Comp Biochem l B Biochem Mol Biol. l24(3):3 . An exemplary hyaluronidase from bacteria that has the same mechanism of action as the leech hyaluronidase is that from the cyanobacteria, Synechococcus sp. (strain RCC307, SEQ ID NO:97). (3) Other hyaluronan degrading enzymes In addition to the hyaluronidase family, other hyaluronan degrading enzymes can be used in the compositions, combinations and methods provided. For example, enzymes, including particular chondroitinases and lyases, that have the ability to cleave hyaluronan can be employed. Exemplary chondroitinases that can degrade hyaluronan e, but are not limited to, chondroitin ABC lyase (also known as chondroitinase ABC), chondroitin AC lyase (also known as chondroitin sulfate lyase or chondroitin e eliminase) and chondroitin C lyase. Methods for production —149— and purification of such enzymes for use in the compositions, combinations, and s provided are known in the art (e.g., US. Patent No. 569; Yamagata, et al. (1968) J. Biol. Chem. 243(7):l523-l535; Yang et al. (1985) J. Biol. Chem. 160(30):1849-1857). oitin ABC lyase contains two enzymes, chondroitin-sulfate-ABC endolyase (EC 4.2.2.20) and chondroitin-sulfate-ABC exolyase (EC 4.2.2.21) (Hamai et al. (1997) JBiol Chem. 272(l4):9l23-30), which degrade a variety of glycosaminoglycans of the oitin-sulfate- and dermatan-sulfate type.

Chondroitin e, chondroitin-sulfate proteoglycan and dermatan sulfate are the preferred substrates for chondroitin-sulfate-ABC endolyase, but the enzyme also can act on onan at a lower rate. Chondroitin-sulfate-ABC endolyase degrades a variety of glycosaminoglycans of the chondroitin-sulfate- and dermatan-sulfate type, producing a mixture of A4-unsaturated oligosaccharides of different sizes that are ultimately degraded to A4-unsaturated tetra- and disaccharides. Chondroitin-sulfate- ABC exolyase has the same substrate specificity but removes disaccharide residues from the non-reducing ends of both polymeric oitin sulfates and their oligosaccharide fragments produced by chondroitin-sulfate-ABC endolyase (Hamai, A. et al. (1997) J. Biol. Chem. 23-9130). Exemplary chondroitin-sulfate-ABC ases and chondroitin-sulfate-ABC exolyases include, but are not limited to, those from Proteus vulgaris and Pedobacter nus (the Proteus is chondroitin-sulfate-ABC endolyase is set forth in SEQ ID NO: 98 (Sato et al. (1994) Appl. Microbiol. Biotechnol. 4l(l):39-46). oitin AC lyase (EC 4.2.2.5) is active on chondroitin sulfates A and C, chondroitin and hyaluronic acid, but is not active on dermatan sulfate (chondroitin sulfate B). Exemplary chondroitinase AC enzymes from the bacteria include, but are not limited to, those from Pedobacter heparinus and Victivallis vadensis, set forth in SEQ ID NOS:99 and 100, respectively, and Arthrobacter aurescens (Tkalec et al. (2000) Applied and Environmental Microbiology 66(1):29-35; Ernst et al. (1995) Critical Reviews in Biochemistry and Molecular Biology 30(5):387-444).

Chondroitinase C cleaves chondroitin e C producing tetrasaccharide plus an unsaturated 6-sulfated disaccharide (delta Di-6S). It also cleaves hyaluronic acid producing unsaturated non-sulfated disaccharide (delta Di-OS). Exemplary WO 63155 -l50- oitinase C enzymes from the bacteria include, but are not limited to, those from ococcus and Flavobacterz'um (Hibi et al. (1989) FEMS-Microbiol—Lett. 48(2): 121-4; Michelacci et al. (1976) J. Biol. Chem. 251:1 154-8; Tsuda et al. (1999) Eur. J. Biochem. 262:127-133). ii. Soluble hyaluronan degrading enzymes Provided in the compositions, ations, uses and methods herein are soluble hyaluronan degrading s, ing soluble hyaluronidases. Soluble hyaluronan ing enzymes include any hyaluronan degrading enzymes that are secreted from cells (e.g. CHO cell) upon expression and exist in soluble form. Such enzymes e, but are not limited to, soluble hyaluronidases, including non-human soluble hyaluronidases, including non-human animal soluble hyaluronidases, ial soluble hyaluronidases and human hyaluronidases, Hyall, bovine PH20 and ovine PH20, allelic variants thereof and other variants thereof. For example, included among soluble hyaluronan degrading s are any hyaluronan degrading enzymes that have been modified to be soluble. For example, hyaluronan ing enzymes that contain a GPI anchor can be made soluble by truncation of and removal of all or a portion of the GPI . In one example, the human hyaluronidase 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 hyaluronan degrading enzymes also include neutral active and acid active hyaluronidases. Depending on factors, such as, but not limited to, the desired level of actiVity of the enzyme following administration and/or site of administration, neutral active and acid active hyaluronidases can be selected. In a particular example, the hyaluronan degrading enzyme for use in the compositions, combinations and methods herein is a soluble l active hyaluronidase.

Exemplary of a soluble hyaluronidase is PH20 from any species, such as any set forth in any of SEQ ID NOS: 1, 2, ll, 25, 27, 29-32, 63-65 and 101-102, or truncated forms thereof lacking all or a portion of the C-terminal GPI anchor, so long as the hyaluronidase is soluble (secreted upon expression) and retains hyaluronidase actiVity. Also included among soluble hyaluronidases are allelic variants or other ts of any of SEQ ID NOS:l, 2, ll, 25, 27, 29-32, 63-65 and 101-102, or truncated forms thereof. Allelic variants and other variants are known to one of skill in the art, and e polypeptides having 60 %, 70 %, 80 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %., 97 %, 98 %, 99 % or more sequence ty to any of SEQ ID NOS: 1, 2, 11, 25, 27, 29-32, 63-65 and 101-102, or truncated forms thereof. Amino acid variants include conservative and non-conservative mutations. It is understood that residues that are important or otherwise required for the activity of a hyaluronidase, such as any described above or known to skill in the art, are generally ant and cannot be changed. These include, for example, active site residues.

Thus, for example, amino acid es 111, 113 and 176 (corresponding to residues in the mature PH20 polypeptide set forth in SEQ ID NO:2) of a human PH20 polypeptide, or soluble form thereof, are generally invariant and are not altered.

Other residues that confer glycosylation and formation of disulfide bonds required for proper folding also can be ant.

In some instances, the soluble hyaluronan degrading enzyme is normally GPI- ed (such as, for example, human PH20) and is rendered soluble by truncation at the C-terminus. Such truncation can remove all of the GPI anchor attachment signal sequence, or can remove only some of the GPI anchor attachment signal sequence.

The resulting polypeptide, however, is soluble. In instances where the soluble hyaluronan degrading enzyme retains a portion of the GPI anchor attachment signal sequence, 1, 2, 3, 4, 5, 6, 7 or more amino acid residues in the GPI-anchor attachment signal ce can be retained, provided the polypeptide is soluble. Polypeptides containing one or more amino acids of the GPI anchor are termed extended soluble hyaluronan degrading enzymes. One of skill in the art can determine whether a polypeptide is GPI-anchored using s well known in the art. Such methods include, but are not limited to, using known algorithms to predict the presence and location of the GPI-anchor attachment signal sequence and oa-site, and performing solubility analyses before and after digestion with phosphatidylinositol-specific phospholipase C (PI-PLC) or D (PI-PLD).

Extended soluble hyaluronan degrading enzymes can be produced by making C-terminal truncations to any naturally GPI-anchored hyaluronan ing enzyme such that the ing polypeptide is soluble and ns one or more amino acid es from the chor attachment signal sequence (see, e.g., U.S. Published Pat. Appl. No. US20100143457). Exemplary extended e hyaluronan degrading WO 63155 enzymes that are C-terminally truncated but retain a portion of the GPI anchor attachment signal sequence include, but are not limited to, extended soluble PH20 (esPH20) polypeptides of primate , such as, for example, human and chimpanzee esPH20 polypeptides. For e, the esPH20 polypeptides can be made by C-terminal truncation of any of the mature or sor polypeptides set forth in SEQ ID NOS: 1, 2 or 101, or allelic or other variation thereof, including active fragment thereof, wherein the resulting ptide is soluble and retains one or more amino acid residues from the GPI-anchor attachment signal ce. 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 ty to any of SEQ ID NOS: 1 or 2. The esPH20 polypeptides provided herein can be C-terminally truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids compared to the Wild type polypeptide, such as a polypeptide with a sequence set forth in SEQ ID NOS: 1, 2 or 101, provided the resulting esPH20 polypeptide is soluble and retains 1 or more amino acid residues from the GPI-anchor attachment signal sequence.

Typically, for use in the compositions, combinations and methods , a soluble human hylauronan degrading enzyme, such as a soluble human PH20, is used.

Although hylauronan degrading enzymes, such as 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 d immunogenicity. Such modifications are Within the level of one of skill in the art and can e, for example, removal and/or replacement of one or more antigenic epitopes on the molecule.

Hyaluronan degrading enzymes, including onidases (e.g., PH20), used in the methods herein can be recombinantly produced or can be purified or partially- purified from natural sources, such as, for e, from testes extracts. Methods for -l53- production of recombinant proteins, including recombinant hyaluronan degrading enzymes, are provided elsewhere herein and are well known in the art. (1) e Human PH20 Exemplary of a soluble hyaluronidase is soluble human PH20. Soluble forms of inant human PH20 have been ed and can be used in the compositions, combinations and methods described . The production of such soluble forms of PH20 is described in US. Published Patent Application Nos.

US20040268425; US20050260186, US20060104968, US20lOOl43457 and International PCT application No. WO20091 1 1066. For example, soluble PH20 polypeptides, include C-terminally truncated variant polypeptides that e a ce of amino acids in SEQ ID NO:1, or have at least 91 %, 92 %, 93 %, 94 %, 95 %, 95 %, 97 %, 98 % sequence identity to a sequence of amino acids included in SEQ ID NO:1, retain hyaluronidase activity and are soluble. Included among these polypeptides are soluble PH20 polypeptides that completely lack all or a portion of the GPI-anchor ment signal sequence.

Also included are extended soluble PH20 (esPH20) polypeptides that contain at least one amino acid of the GPI anchor. Thus, instead of having a chor covalently attached to the C-terminus of the protein in the ER and being anchored to the extracellular leaflet of the plasma membrane, these polypeptides are secreted and are soluble. C-terminally truncated PH20 ptides can be C-terminally truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, l2, l3, l4, l5, l6, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more amino acids compared to the fill length wild type ptide, such as a full length wild type polypeptide with a sequence set forth in SEQ ID NOS:l or 2, or allelic or species variants or other variants thereof.

For example, soluble forms include, but are not limited to, C-terminal ted polypeptides of human PH20 set forth in SEQ ID NO:1 having a C-terminal amino acid residue 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 and 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 ofthe sequence of amino acids set forth in SEQ ID NO:1, or polypeptides that exhibit at least 85% identity thereto. Soluble forms of human PH20 generally include those that contain amino acids 36-464 set forth in SEQ ID N021. For example, when expressed in mammalian cells, the 35 amino acid N- WO 63155 —154— 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 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 and 483 of SEQ ID N021. Table 3 provides non-limiting examples of exemplary C- terminally truncated PH20 polypeptides, including C-terminally truncated soluble PH20 polypeptides. In Table 3 below, the length (in amino acids) of the precursor and mature polypeptides, and the sequence identifier (SEQ ID NO) in which exemplary amino acid sequences of the precursor and mature polypeptides of the C- terminally truncated PH20 ns are set forth, are ed. The Wild-type PH20 lO polypeptide also is ed in Table 3 for comparison. In particular, exemplary of soluble hyaluronidases are soluble human PH20 ptides 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.

Table 3. Exemlar C-terminall truncated PH20 ol nuetides Precursor Mature (amino Mature (amino acids) SEQ ID NO acids) SEQ ID NO 117 440 161 -l55- Table 3. Exemlar inall truncated PH20 ol nuetides (amino acids) SEQ ID NO acids) SEQ ID NO For example, exemplary C-terminally truncated PH20 polypeptides that exhibit hyaluronidase activity, are secreted from cells and are e include any of the mature forms of a truncated human PH20 set forth in Table 3, or variants thereof that exhibit hyaluronidase activity. For example, the PH20 or truncated form thereof contains the sequence of amino acids set forth in any of SEQ ID NOS: 4-9, 47, 48, 150-170 and 183-189 or a ce of amino acids that exhibits at least 85% sequence identity to any of SEQ ID NOS: 4-9, 47, 48, 150-170 and 9. For example, the PH20 polypeptide can exhibit at least 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 4-9, 47, 48, 150-170 and 183-189.

Generally soluble forms of PH20 are produced using protein expression systems that tate correct N-glycosylation to ensure the polypeptide retains activity, since ylation is important for the catalytic activity and stability of hyaluronidases. Such cells e, for e Chinese Hamster Ovary (CHO) cells (e.g. DG44 CHO cells). (2) rHuPH20 Recombinant soluble forms of human PH20 have been generated and can be used in the compositions, combinations and methods provided herein. The tion of such soluble forms of recombinant human PH20 are described, for example, in US. Published Patent Application Nos. US20040268425; US 20050260186; US20060104968; US20100143457; and International PCT Appl. No.

W02009l l 1066. Exemplary of such polypeptides are those generated by expression of a nucleic acid molecule encoding amino acids l-482 (set forth in SEQ ID NO:3).

Such an exemplary nucleic acid molecule is set forth in SEQ ID NO:49. Post -l56- translational processing s the 35 amino acid signal sequence, leaving a 447 amino acid e recombinant human PH20 (SEQ ID NO:4). As produced in the culture medium there is heterogeneity at the C-terminus such that the product, designated rHuPH20, includes a mixture of species that can include any one or more of SEQ ID NOS. 4-9 in s abundance. Typically, rHuPH20 is produced in cells that facilitate correct N-glycosylation to retain activity, such as CHO cells (6.g. DG44 CHO cells). iii. Glycosylation of hyaluronan degrading enzymes Glycosylation, ing N- and O-linked glycosylation, of some hyaluronan degrading enzymes, including hyaluronidases, can be important for their catalytic activity and stability. While altering the type of glycan modifying a rotein can have dramatic affects on a n's antigenicity, structural folding, solubility, and stability, most enzymes are not thought to require glycosylation for optimal enzyme activity. For some hyaluronidases, removal ofN-linked glycosylation can result in near complete inactivation of the hyaluronidase activity. Thus, for such hyaluronidases, the presence ofN-linked glycans is critical for generating an active enzyme. ed 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 es that fall Within -Asn-Xaa-Thr/Ser- sequences (Where Xaa is not Pro). Glycosylation at an aa-Cys-site has been reported for coagulation protein C. In some instances, a hyaluronan degrading enzyme, such as a onidase, can contain both N-glycosidic and O-glycosidic linkages. For example, PH20 has O-linked oligosaccharides as well as N-linked oligosaccharides. There are seven potential ed glycosylation sites at N82, N166, N235, N254, N368, N393, N490 of human PH20 exemplified in SEQ ID NO: 1. Amino acid residues N82, N166 and N254 are occupied by complex type glycans Whereas amino acid residues N368 and N393 are occupied by high mannose type glycans. Amino acid residue N235 is occupied by approximately 80 % high mannose type glycans and 20 % complex type glycans. As noted above, ed glycosylation at N490 is not required for hyaluronidase activity. -l57- In some examples, the onan degrading enzymes for use in the compositions, combinations and/or methods provided are glycosylated at one or all of the glycosylation sites. For example, for human PH20, or a soluble form thereof, 2, 3, 4, 5, or 6 of the N-glycosylation sites corresponding to amino acids N82, N166, N235, N254, N368, and N393 of SEQ ID NO: 1 are glycosylated. In some examples the hyaluronan ing enzymes are glycosylated at one or more native glycosylation sites. In other examples, the hyaluronan degrading enzymes are modified at one or more non-native glycosylation sites to confer glycosylation of the ptide at one or more additional site. In such examples, attachment of additional sugar es can enhance the pharmacokinetic properties of the molecule, such as improved half- life and/or improved activity.

In other examples, the hyaluronan ing enzymes for use in the compositions, combinations and/or s ed herein are partially deglycosylated (or N-partially glycosylated polypeptides). For example, partially deglycosylated soluble PH20 polypeptides that retain all or a portion of the onidase activity of a fially glycosylated hyaluronidase can be used in the compositions, combinations and/or methods provided herein. Exemplary partially deglycosylated hyalurodinases include soluble forms of a partially deglycosylated PH20 polypeptides from any species, such as any set forth in any of SEQ ID NOS: 1, 2, ll, 25, 27, 29-32, 63, 65, and 101-102, or allelic variants, truncated variants, or other variants thereof. Such 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 identity to any of SEQ ID NOS: 1, 2, ll, 25, 27, 29-32, 63, 65, and 101-102, or truncated forms thereof The partially deglycosylated hyaluronidases provided herein also include hybrid, fusion and chimeric partially deglycosylated onidases, and partially deglycosylated hyaluronidase conjugates.

Glycosidases, or glycoside hydrolases, are enzymes that catalyze the ysis of the glycosidic linkage to generate two smaller sugars. The major types ofN-glycans in vertebrates include high mannose glycans, hybrid s and complex glycans. There are l glycosidases that result in only partial protein deglycosylation, including: EndoFl, which s high mannose and hybrid type glycans; EndoF2, which cleaves biantennary complex type glycans; EndoF3, which -l58- cleaves biantennary and more branched complex glycans; and EndoH, which cleaves high mannose and hybrid type glycans. ent of a hyaluronan degrading enzyme, such as a soluble hyaluronidase, such as a soluble PH20, with one or all of these glycosidases can result in only partial deglycosylation and, therefore, retention of hyaluronidase activity.

Partially deglycosylated onan degrading enzymes, such as partially deglycosylated soluble hyaluronidases, can be produced by digestion with one or more glycosidases, generally a glycosidase that does not remove all N-glycans but only lly deglycosylates the protein. For example, treatment of PH20 (e.g. a recombinant PH20 ated rHuPH20) with one or all of the above glycosidases (e.g. EndoFl, EndoF2 and/or EndoF3) results in l deglycosylation. These partially deglycosylated PH20 polypeptides can exhibit hyaluronidase enzymatic activity that is comparable to the fully glycosylated polypeptides. In contrast, treatment of PH20 with PNGaseF, a glycosidase that cleaves all N-glycans, results in complete removal of all ans and thereby renders PH20 enzymatically inactive.

Thus, gh all N-linked glycosylation sites (such as, for example, those at amino acids N82, Nl66, N235, N254, N368, and N393 of human PH20, exemplified in SEQ ID NO: 1) can be glycosylated, treatment with one or more glycosidases can render the extent of glycosylation reduced compared to a hyaluronidase that is not digested with one or more glycosidases.

The lly deglycosylated hyaluronan degrading s, including partially deglycosylated soluble PH20 polypeptides, can have 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 % or 80 % of the level of glycosylation of a fially glycosylated polypeptide. In one example, 1, 2, 3, 4, 5 or 6 of the N-glycosylation sites ponding to amino acids N82, N166, N235, N254, N368, and N393 of SEQ ID NO:1 are partially deglycosylated, such that they no longer contain high mannose or complex type glycans, but rather contain at least an N-acetylglucosamine moiety. In some es, 1, 2 or 3 of the N-glycosylation sites corresponding to amino acids N82, N166 and N254 of SEQ ID NO:1 are deglycosylated, that is, they do not contain a sugar moiety. In other examples, 3, 4, 5, or 6 of the N-glycosylation sites ponding to amino acids N82, N166, N235, N254, N368, and N393 of SEQ ID NO:1 are glycosylated. Glycosylated amino acid residues lly contain an N- -lS9- acetylglucosamine moiety. lly, the partially deglyclosylated hyaluronan degrading enzymes, including partially deglycosylated soluble PH20 polypeptides, exhibit hyaluronidase activity that is 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 110 %, 120 %, 130 %, 140 %, 150 %, 200 %, 300 %, 400 %, 500 %, 1000 % or more of the hyaluronidase activity exhibited by the fully ylated polypeptide. iv. Modified (Polymer-Conjugated) hyaluronan degrading enzymes In one example, the provided itions and combinations contain onan ing enzymes, in particular soluble hyaluronidases, that have been modified by conjugation to one or more polymeric le (polymer), typically to increase the half-life of the hyaluronan degrading enzyme, for example, to promote prolonged/sustained ent s in a subject.

Covalent or other stable attachment gation) of polymeric molecules, such as polyethylene glycol (PEGylation moiety (PEG)), to the hyaluronan degrading enzymes, such as hyaluronidases, impart beneficial ties to the resulting onan degrading enzyme-polymer composition. Such properties include improved biocompatibility, extension of protein (and enzymatic activity) ife in the blood, cells and/or in other tissues Within a subject, effective shielding of the protein from proteases and hydrolysis, improved biodistribution, enhanced pharmacokinetics and/or pharmacodynamics, and increased water solubility.

Hence, in particular examples herein, the hyaluronan ing enzyme is conjugated to a polymer. Exemplary of polymers are such as polyols (i. e. poly-OH), polyamines (i. e. poly-NH2) and polycarboxyl acids (1'. e. poly-COOH), and further heteropolymers z'.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups. Examples of suitable polymeric molecules include polymeric molecules selected from among polyalkylene oxides (PAO), such as polyalkylene s (PAG), including polypropylene glycols (PEG), methoxypolyethylene s (mPEG) and polypropylene glycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG) branched polyethylene glycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-maleic 2012/061743 acid anhydride, dextrans including carboxymethyl-dextrans, heparin, gous albumin, celluloses, including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of chitosan, starches such as hydroxyethyl- starches and hydroxypropyl-starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, , alginic acid hydrolysates and bio-polymers.

In particular, the polymer is a polyethylene glycol (PEG). Suitable ric molecules for attachment to the hyaluronan degrading enzymes, including onidases, include, but are not limited to, hylene glycol (PEG) and PEG derivatives such as methoxy-polycthylene glycols (mPEG), PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched PEGs, and polyethylene oxide (PEO) (see e.g. Roberts et a1, , Advanced Drug Delivery Review (2002) 54: 459-476; Harris and Zalipsky, S (eds) "Poly(ethylene glycol), Chemistry and Biological Applications" ACS Symposium Series 680, 1997; Mehvar et al., J.

Pharm. Pharmaceut. Sci. , 3(1): 125-136, 2000; , (2003) Nature Reviews Drug Discovery 21214-221; and Tsubery, (2004) JBiol. Chem 279(37):381 18—24). The polymeric molecule can be of a molecular weight typically ranging from about 3 kDa to about 60 kDa. In some embodiments the polymeric molecule that is conjugated to a protein, such as rHuPH20, has a molecular weight of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more than 60 kDa.

Various methods of modifying polypeptides by covalently attaching gating) a PEG or PEG derivative (i.e. “PEGylation”) are known in the art (see e.g., US. 2006/0104968; U.S. 5,672,662; US. 6,737,505; and US. 2004/0235734).

Such ques aredescribed elsewhere herein. 2. Pharmaceutical Compositions and Formulations Provided herein are pharmaceutical compositions of anti—hyaluronan agents, for example, a hyaluronan—degrading enzyme or modified form thereof (2.g. a PEGylated hyaluronan—degrading enzymes, such as PEGylatcd hyaluronidascs), for use in the treatment methods ed. Also provided herein are pharmaceutical compositions ning a seCond agent that is used to treat a disease or disorder associated with a onan-associated disease or ion, such as cancer.

RECTIFIED SHEET (RULE 91) ISA/EP ~161- Exemplary of such agents include, but are not limited to, anti-cancer agents including drugs, polypeptides, nucleic acids, antibodies, peptides, small molecules, gene therapy vector, viruses and other therapeutics. Anti-hyaluronan agents, for example, a hyaluronan—degrading enzyme or modified form f (ag. a PEGylated hyaluronan-degrading enzymes, such as ted hyaluronidases or PEGPHZO), can be co-formulated or co—administered with pharmaceutical formulations of such second agents to enhance their delivery to desired sites or tissues within the body associated with excess or lated hyaluronan.

Pharmaceutically acceptable compositions are prepared in View of approvals for a regulatory agency or other agency prepared in accordance with generally recognized acopeia for use in animals and in humans. The compounds can be formulated into any suitable pharmaceutical preparations for any of oral and enous administration such as solutions, suspensions, powders, or sustained release ations. 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). The formulation should suit the mode of administration.

In one example, pharmaceutical preparation can be in liquid foun, for example, ons, syrups or suspensions. 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 ed by conventional means with ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, ose 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 e before use.

Pharmaceutical compositions can e carriers such as a diluent, adjuvant, excipient, or vehicle with which the compositions (e. g. corticosteroid or anti- hyaluronan agent, such as a PEGylated hyaiuronan-degrading enzymes) are administered. Examples of suitable pharmaceutical carriers are described in RECTIFIED SHEET (RULE 91) ISA/EP -l62- "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound or agent, generally in purified form or lly purified form, together with a le 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 eum, , vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a typical carrier. 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 ient: a t such as lactose, sucrose, dicalcium ate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acacia, gelatin, 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, e, 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. For e, suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. A composition, if desired, also can contain other minor amounts of xic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility ers, and other such agents, such as for e, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

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

Examples of aqueous vehicles include Sodium de Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable , cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fiangistatic concentrations can be added to parenteral preparations packaged in multiple-dose containers, which e phenols or s, mercurials, -l63- benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium de and benzethonium chloride. Isotonic agents include sodium de and dextrose. Buffers include ate and citrate.

Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and sing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone.

Emulsifying agents include Polysorbate 80 (TWEEN 80). A sequestering or chelating agent of metal ions include EDTA. Pharmaceutical carriers also include ethyl l, polyethylene glycol and propylene glycol for water miscible es and sodium hydroxide, hloric acid, citric acid or lactic acid for pH adjustment.

Injectables can be prepared in tional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Preparations for intraprostatic stration include sterile solutions ready for ion, 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 e just prior to use, sterile emulsions. The solutions can be either aqueous or nonaqueous. 3. Dosages and Administration Typically, the dose of an anti-hyaluronan agent, for example, a hyaluronan- degrading enzyme, is one that also achieves a therapeutic effect in the treatment of a hyaluronan associated disease or condition, such ascancer. Hence, itious of an anti—hyaluronan agent, for example a hyaluronan-degrading enzyme, are included in an amount sufficient to exert a therapeutically useful effect. The composition 2.5 containing the active agent can include a pharmaceutically acceptable carrier; The compositions of an anti-hyaluronan agent also can include a second therapeutic agent.

Therapeutically effective concentrations of an anti-hyaluronan agent, for example a hyaluronan-degrading enzyme, can be determined empirically by testing the compounds in known in vitro and in vivo systems, such as the assays ed herein. For example, the concentration of an anti—hyaluronan agent, such as a hyaluronan—degrading enzyme or modified form thereof (e.g a PEGylated hyaluronan- degrading enzyme, such as PEGylated hyaluronidase) s on absorption, RECTIFIED SHEET (RULE 91) ISA/EP inactivation and excretion rates, the ochemical characteristics, 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 e dosage and duration of treatment is a function of the tissue being treated, the disease or condition being treated, the route of administration, the patient or subject and the particular anti- hyaluronan agent and can be determined cally using known testing protocols or by extrapolation from in vivo or in vitro test data and/or can be determined from known dosing s of the particular agent. The amount of an anti-hyaluronan agent, for example a hyaluronan-degrading enzyme or modified form f (6.g. a PEGylated hyaluronan-degrading enzyme, such as a PEGylated onidase), to be administered for the treatment of a disease or condition, for e a hyaluronan- associated disease or condition such as an HA-rich tumor, can be determined by standard clinical techniques. In addition, in vitro assays and animal models can be employed to help identify optimal dosage ranges. The precise dosage, which can be determined cally, can depend on the particular enzyme, the route of administration, the type of disease to be treated and the seriousness of the disease.

For example, methods of using anti-hyaluronan agents, such as hyaluronan- degrading enzymes or modified forms thereof (e.g. PEGylated forms) for treatment of hyaluronan-associated diseases and ions are well known in the art (see e.g. U.S. published application No. 20100003238 and International published PCT Appl. No.

WC 2009/128917). Thus, dosages of an anti-hyaluronan agent, such as a hyaluronan- degrading enzyme for example a onidase, can be chosen based on standard dosing regimes for that agent under a given route of stration.

Examples of effective amounts of an anti-hyaluronan agent for treatment of a hyaluronan-associated disease or condition is a dose ranging from 0.01 ug to 100 g per kg of body weight. For example, an effective amount of an anti-hyaluronan agent is a dose g from 0.01 ug to 100 mg per kg ofbody weight, such as 0.01 ug to 1 mg per kg ofbody weight, 1 ug to 100 ug per kg ofbody weight, 1 ug to 10 ug per kg ofbody weight or 0.01 mg to 100 mg per kg of body weight. For example, effective amounts include at least or about at least or about or 0.01 ug, 0.05, 0.1, 0.5, 1, 2, 3, 4, , 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 ug/kg body weight Other examples of -165— effective amounts include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 g/kg body weight. For example, an anti- hyaluronan agent, such as a onan-degrading enzyme for example a hyaluronidase (e.g. a PEGylated onidase such as a PEGPH20), can be administered at or about 0.1 pg/kg to 1 rug/kg, for example 0.5 pg’kg to 100 pg/kg, 0.75 pg/kg to 15 pg/kg, 0.75 pg/kg to 7.5 pg/kg or 1.0 pg/kg to 3.0 pg/kg. In other examples, an anti-hyaluronan agent such as a onan—degrading enzyme for example a hyaluornidase (ag, a PEGylated hyaluronidase such as a PEGPH20), can be stered at or 1 mg/kg to 500 mg/kg, for example, 100 mg/kg to 400 rug/kg, such as 200 mg’kg. For example, compositions contain 0.5 mg to 100 grams of anti- hyaluronan agent, for example, 20 pg to 1 mg, such as 100 pg to 0.5mg or can contain 1 mg to 1 gram, such as 5 mg to 500 mg.

For example, agents and treatments for treatment of hyaluronan-associated diseases and conditions, such as anti-cancer agents, are well known in the art (see e. g.

U.S. published application No. 20100003238 and Intematioual hed PCT Appl.

No. W0 2009/128917). Thus, dosages ofa hyaluronanedegrading enzyme, for e a hyaluronidase, or other second agents in a composition can be chosen based on rd dosing regimes for that agent under a given route of administration.

Examples of effective amounts ofa hyaluronan-degrading enzyme is a dose ranging from 0.0] pg to 100 g per kg ofbody weight. For example, an effective amount of a hyaluronan-degrading enzyme is a dose ranging from 0.01 pg to 100 mg per kg of body weight, such as 0.01 pg to 1 mg per kg of body weight, 1 pg to l00 pg per kg of body weight, 1 pg to 10 pg per kg ofbody weight or 0.01 mg to 100 mg per kg of body weight. For example, effective amounts include at least or about at least or about or 0.01 pg, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 pg/kg body weight Other examples of effective amounts include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5,6, 7, 8, 9, 10, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 g/kg body weight. FUl' example, a liyalut'un‘cui-degradiug enzyme for example a hyaluronidase (ag. a PEGylated hyaluronidase such as a 0), can be RECTIFIED SHEET (RULE 91) ISA/EP -l66- administered at or about 0.1 rig/kg to 1 mg/kg, for example 0.5 ug/kg to 100 lag/kg, 0.75 pg/kg to 15 rig/kg, 0.75 gig/kg to 7.5 pig/kg or 1.0 rig/kg to 3.0 rig/kg. In other examples, a hyaluronan-degrading enzyme for example a hyaluornidase (e.g. a PEGylated hyaluronidase such as a PEGPHZO), can be administered at or 1 mg/kg to 500 mg/kg, for e, 100 mg/kg to 400 mg/kg, such as 200 mg/kg. Generally, itions contain 0.5 mg to 100 grams of a hyaluronan-degrading enzyme, for ‘ example, 20 pg to 1 mg, such as 100 pg to 0.5mg or can contain 1 mg to 1 gram, such as 5 mg to 500 mg.

The dose or compositions can be for single dosage administration or for multiple dosage administration. The dose or composition can be administered in a single stration once, several times a week, twice weekly, every 15 days, 16 days, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, once monthly, several times a year or yearly. In other examples, the dose or composition an be divided up and administered once, several times a week, twice weekly, every 15 days, 16 days, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, once monthly, several times a year or yearly. Hyaluronan-degrading enzyme compositions can be formulated as liquid compositions or can be lyophilized. The compositions also can be formulated as a tablet or capsule. .

Provided below is description of dosages and dosage regimines of exemplary hyaluronan—degrading enzymes ated to a polymer (e.g. PEGylated) for use in the methods herein. The hyaluronan—degrading enzymes can be used alone in a single agent therapy or in combination with other agents for use in treating an ociated disease or ion, such as cancer. As discussed elsewhere herein, in particular examples ofthe methods and uses herein, the agents can be administered in combination with a corticosteroid in order to ameliorate a side-effect associated with treatment of the anti-hyaluronan—agent. a. stration of a PEGylated hyaluronan-degrading enzyme A hyaluronan-degrading enzyme, such as a PEGylated hyaluronan-degrading enzyme (e.g. a hyaluronidase), can be administered systemically, for e, intravenously (IV), intramus’cularly, or by any another systemic route. In particular examples, lower doses can be given locally. For e, local stration of a RECTIFIED SHEET (RULE 91) ISA/EP hyaluronan-degrading enzyme, such as a PEGylated hyaluronan-degrading enzyme for example a PEGylated hyaluronidase (e.g. PH20) includes intratumoral stration, arterial injection (6.g. hepatic artery), intraperitoneal administration, intravesical administration and other local routes used for cancer therapy that can increase local action at a lower absolute dose.

Exemplary dosage range is at or about 0.3 kg to 320,000 Units/kg, such as 10 Units/kg to 0 Units/kg of a PEGylated hyaluronidase, or a functionally equivalent amount of another PEGylated hyaluronan-degrading enzyme. It is tood herein that a unit of actiVity is normalized to a standard actiVity, for example, an actiVity as ed in a microturbidity assay assaying hyaluronidase actiVity. A PEGylated soluble hyaluronidase can exhibit lower actiVity per mg of total protein, z'.e. exhibits a lower specific activity, compared to a native soluble hyaluronidase not so conjugated. For example, an exemplary rHuPH20 preparation exhibits a specific actiVity of 120,000 Units/mg, while a PEGylated form ofrHuPH20 exhibits a specific actiVity of at or about 32,000 Units/mg. lly, a PEGylated form of a hyaluronan-degrading , such as a hyaluronidase for example rHuPH20, exhibits a specific activity within the range of between at or about 18,000 and at or about 45,000 U/mg. In one example, the PEG-hyaluronan-degrading enzyme can be provided as a stock solution for example, at 3.5 mg/mL at 0 U/mL (~32,000 U/mg), with a PEG to protein molar ratio between 5:1 and 9:1, for example, 7: 1, or can be provided in a less concentrated form. For purposes herein, dosages can be with reference to Units.

For example, PEGylated hyaluronan-degrading enzyme, such as a hyaluronidase, for e PEGPH20, can be administered enously twice , once weekly or once every 21 days. Typically, the PEGylated onandegrading enzyme is administered twice weekly. The cycle of administration can be for a defined period, generally for 3 weeks or 4 weeks. The cycle of administration can be repeated in a dosage regime for more than one month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 , 9 months, 10 months, 11 , 1 year or more. Generally, the cycle of administration is repeated at the discretion of a treating physician, and can depend on factors such as remission of the disease or condition, severity of the disease or condition, adverse events and other factors. In other examples, in subsequent cycles of administration, the hyaluronan-degrading enzyme can be administered less frequently. For example, in a first cycle the onan-degrading enzyme is administered twice weekly for four weeks, and in subsequent cycles of administration the hyaluronan-degrading enzyme is administered once weekly or once every two weeks, once every 3 weeks (e.g. once every 21 days) or once every 4 weeks. As described herein, the dose or dosing regime of corticosteroid is dependent on the dosing regime of onan-degrading .

While dosages can vary ing on the disease and patient, the hyaluronan- degrading enzyme, such as a PEGylated hyaluronidase, is generally administered in an amount that is or is about in the range of from 0.01 ug/kg, such as 0.0005 mg/kg (0.5 ug/kg) to 10 mg/kg (320,000 U/kg), for example, 0.02 mg/kg to 1.5 mg/kg, for example, 0.05 mg/kg. The PEGylated hyaluronidase can be administered, for example, at a dosage of at or about 0.0005 mg/kg (of the t), 0.0006 mg/kg, 0.0007 mg/kg, 0.0008 mg/kg, 0.0009 mg/kg, 0.001 mg/kg, 0.0016 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.016 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg.kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, or more is administered, to an average adult human subject, typically weighing about 70 kg to 75 kg. In particular examples, the hyaluronan- degrading enzyme is administered in lower amounts such as less than 20 ug/kg, for example 0.01 ug/kg to 15 ug/kg, 0.05 ug/kg to 10 ug/kg 0.75 ug/kg to 7.5 ug/kg or 1.0 ug/kg to 3.0 ug/kg.

A hyaluronan-degrading enzyme, such as a PEGylated hyaluronidase (e.g.

PH20), provided herein, for example, PEGPH20, can be administered at or about 1 Unit/kg to 800,000 Units/kg, such as 10 to 800,000 Units/kg, 10 to 0 Units/kg, to 700,000 Units/kg, 10 to 650,000 Units/kg, 10 to 600,000 kg, 10 to 550,000 Units/kg, 10 to 500,000 Units/kg, 10 to 450,000 Units/kg, 10 to 400,000 Units/kg, 10 to 350,000 Units/kg, 10 to 320,000 Units/kg, 10 to 300,000 Units/kg, 10 to 280,000 Units/kg, 10 to 260,000 Units/kg, 10 to 240,000 Units/kg, 10 to 220,000 Units/kg, 10 to 200,000 Units/kg, 10 to 180,000 Units/kg, 10 to 160,000 Units/kg, 10 to 140,000 Units/kg, 10 to 120,000 Units/kg, 10 to 100,000 kg, 10 to 80,000 Units/kg, 10 to 70,000 Units/kg, 10 to 60,000 Units/kg, 10 to 50,000 Units/kg, 10 to 40,000 Units/kg, 10 to 30,000 Units/kg, 10 to 20,000 kg, 10 to 15,000 Units/kg, to 12,800 Units/kg, 10 to 10,000 Units/kg, 10 to 9,000 Units/kg, 10 to 8,000 Units/kg, 10 to 7,000 Units/kg, 10 to 6,000 Units/kg, 10 to 5,000 Units/kg, 10 to 4,000 Units/kg, 10 to 3,000 kg, 10 to 2,000 Units/kg, 10 to 1,000 Units/kg, 10 to 900 Units/kg, 10 to 800 Units/kg, 10 to 700 Units/kg, 10 to 500 Units/kg, 10 to 400 Units/kg, 10 to 300 Units/kg, 10 to 200 Units/kg, 10 to 100 Units/kg, 16 to 600,000 Units/kg, 16 to 500,000 Units/kg, 16 to 400,000 Units/kg, 16 to 0 Units/kg, 16 to 320,000 Units/kg, 16 to 160,000 Units/kg, 16 to 80,000 Units/kg, 16 to 40,000 Units/kg, 16 to 20,000 Units/kg, 16 to 16,000 Units/kg, 16 to 12,800 Units/kg, 16 to ,000 Units/kg, 16 to 5,000 Units/kg, 16 to 4,000 Units/kg, 16 to 3,000 Units/kg, 16 to 2,000 Units/kg, 16 to 1,000 kg, 16 to 900 Units/kg, 16 to 800 Units/kg, 16 to 700 Units/kg, 16 to 500 Units/kg, 16 to 400 kg, 16 to 300 Units/kg, 16 to 200 Units/kg, 16 to 100 Units/kg, 160 to 12,800 kg, 160 to 8,000 Units/kg, 160 to 6,000 Units/kg, 160 to 4,000 Units/kg, 160 to 2,000 Units/kg, 160 to 1,000 Units/kg, 160 to 500 kg, 500 to 5000 Units/kg, 1000 to 100,000 kg or 1000 to ,000 Units/kg, of the mass of the t to Whom it is stered. In some examples, a hyaluronan-degrading enzyme, such as a PEGylated hyaluronidase (e.g.

PH20), provided herein, for example, PEGPH20, can be administered at or about 1 Unit/kg to 1000 Units/kg, 1 Units/kg to 500 Units/kg or 10 Units/kg to 50 Units/kg.

Generally, Where the specific actiVity of the PEGylated hyaluronidase is or is about 18,000 U/mg to 45,000 U/mg, generally at or about 1 kg (U/kg), 2 U/kg, 3 U/kg, 4 U/kg, 5 U/kg, 6 U/kg, 7 U/kg, 8 U/kg, 8 U/kg 10 U/kg, 16 U/kg, 32 U/kg, 64 U/kg, 100 U/kg, 200 U/kg, 300 U/kg, 400 U/kg, 500 U/kg, 600 U/kg, 700 U/kg, 800 U/kg, 900 U/kg, 1,000 U/kg, 2,000 U/kg, 3,000 U/kg, 4,000 U/kg, 5,000 U/kg, 6,000 U/kg, 7,000 U/kg, 8,000 U/kg, 9,000 U/kg, 10,000 U/kg, 12,800 U/kg, 20,000 U/kg, 32,000 U/kg, 40,000 U/kg, 50,000 U/kg, 60,000 U/kg, 70,000 U/kg, 80,000 U/kg, 90,000 U/kg, 100,000 U/kg, 0 U/kg, 0 U/kg, 160,000 U/kg, 180,000 U/kg, 200,000 U/kg, 220,000 U/kg, 240,000 U/kg, 260,000 U/kg, 280,000 U/kg, 300,000 U/kg, 320,000 U/kg, 350,000 U/kg, 400,000 U/kg, 450,000 U/kg, 500,000 U/kg, 550,000 U/kg, 600,000 U/kg, 650,000 U/kg, 700,000 U/kg, 750,000 U/kg, 800,000 U/kg or more, per mass of the subject, is administered.

In some aspects, the ted hyaluronan-degrading enzyme is formulated and dosed to maintain at least 3 U/mL of the PEGylated hyaluronidase in the plasma (see e.g. published US. Patent App. No. US20100003238 and published International Patent App. No. WO2009128917). For example, the PEGylated e hyaluronidase is ated for ic adminstration in a sufficient amount to maintain at least or about 3 U/mL in the plasma, generally 3 U/mL - 12 U/mL or more, for example, from at least or about or at a level of 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL, 9 U/mL, 10 U/mL, 11 U/mL, 12 U/mL, 13 U/mL, 14 U/mL, 15 U/mL, 16 U/mL, 17 U/mL, 18 U/mL, 19 U/mL, 20 U/mL, 25 U/mL, 30 U/mL, 35 U/mL, 40 U/mL, 45 U/mL, 50 U/mL or more. Generally, for purposes herein to maintain at least 3 U/mL of the hyaluronidase in plasma, at or about 0.02 mg/kg (of the subject), 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg.kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg or more is administered. Generally, where the specific actiVity of the modified hyaluronidase is or is about 20,000 U/mg to 60,000 U/mg, generally at or about ,000 U/mg, 60,000 U; 70,000 U; 80,000 U; 90,000 U; 100,000 U; 200,000 U; 300,000 U; 400,000 U; 0 U; 600,000 U; 700,000 U; 800,000 U; 900,000 U; 1,000,000 U; 1,500,000 U; 2,000,000 U; 2,500,000 U; 3,000,000 U; 3,500,000 U; 4,000,000 U or more is administered. To maintain such levels, administration can be daily, several times a week, twice weekly, weekly or monthly.

It is within the level of one of skill in the art to determine the s of PEGylated hyaluron degrading enzyme, for example, PEGylated PH20, to maintain at least 3 U/mL of the hyaluronidase in the blood. The level of hyaluronidase in the blood can be monitored over time in order to ensure that a sufficient amount of the -l7l- hyaluronidase is present in the blood. Any assay known to one of skill in the art to measure the hyaluronidase in the plasma can be performed. For example, a microturbidity assay or enzymatic assay described in the Examples herein can be performed on protein in plasma. It is undersood that plasma normally contains hyaluronidase enzymes. Such plasma hyaluronidase enzymes lly have activity at an acidic pH (US. Patent No. 7,105,330). Hence, before ent of with a modified enzyme, the plasma levels of onidase should be determined and used as a baseline. Subsequent measurements of plasma hyaluronidase levels after ent can be compared to the levels before treatments. Alternatively, the assay can be performed under pH conditions that suppress endogenous lysosomal hyaluronidase activity in plasma, which normally exhibits activity at acidic pH. Thus, where the d soluble hyaluronidase is active at neutral pH (e.g. human PH20), only the level of the ed neutral-active soluble hyaluronidase is measured.

In other examples, the PEGylated hyaluronan-degrading enzyme is formulated and administered at a lower dose, which is found herein to have therapeutic effects to treat a hyaluronan-associated disease or conditions absent a detectable level of hyaluronidase maintained in the blood. For example, the PEGylated soluble onidase is administered in an amount that is less than 20 ug/kg, for example 0.01 ug/kg to 15 ug/kg, 0.05 ug/kg to 10 ug/kg 0.75 ug/kg to 7.5 ug/kg or 1.0 ug/kg to 3.0 ug/kg, such as at or about 0.01 ug/kg (ofthe t), 0.02 ug/kg, 0.03 ug/kg, 0.04 ug/kg, 0.05 ug/kg, 1.0 ug/kg, 1.5 ug/kg, 2.0 ug/kg, 2.5 ug/kg, 3.0 ug/kg, 3.5 ug/kg, 4.0 ug/kg, 4.5 ug/kg, 5.0 ug/kg, 5.5 ug/kg, 6.0 ug/kg, 7.0 ug/kg, 7.5 ug/kg, 8.0 ug/kg, 9.0 ug/kg, 10.0 ug/kg, 12.5 ug/kg or 15 ug/kg. Generally, where the specific activity of the modified hyaluronidase is or is about 20,000 U/mg to 60,000 U/mg, generally at or about 35,000 U/mg, 200 Units to 50,000 (U) is administered, such as 200 U, 300 U; 400 U; 500 U; 600 U; 700 U; 800 U; 900 U; 1,000 U; 1250 U; 1500 U; 2000 U; 3000 U; 4000 U; 5,000 U; 6,000 U; 7,000 U; 8,000 U; 9,000 U; ,000 U; 20,000 U; 30,000 U; 40,000 U; or 50,000 U is administered. To maintain such levels, administration can be daily, several times a week, twice weekly, weekly or monthly.

Typically, volumes of injections or ons of PEGylated hyaluronidase contemplated herein are from at or about 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 ~172- mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40 mL, 50 mL or more. The PEGylated hyaluronan-degrading enzyme, such as a PEGylated hyaluronidase can be ed 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 a fiinctionally equivalent amount) or can be provided in a more concentrated form, for example at or about 1000 U/mL, 2000 Units/mL, 3000 U/mL, 4000 U/mL, 5000 U/mL, 6000 U/mL, 7000 U/mL, 8000 U/mL, 9000 U/mL, ,000 U/mL, 11,000 U/rnL, 12,000 U/mL, or 12,800 U/mL, for use ly or for dilution to the effective concentration prior to use. The volume of PEGylated hyaluronan-degrading enzyme, such as PEGylated hyaluronidase, administered is a function of the dosage required, but can be varied depending on the concentration of a hyaluronan—degrading enzyme, such as soluble hyaluronidase, stock formulation available. For example, it is plated herein that the PEGylated hyaluronan- degrading enzyme, such as PEGylated hyaluronidase, is not administered in volumes than about 50 mL, and typically is stered in a volume of 5-30 mL, , greater lly in a volume that is not greater than about 10 mL. The PEGylated hyaluronan—degrading enzyme, such as a PEGylated hyaluronidase, can be provided as a liquid or lyophilized formulation. Lyophilized formulations are ideal for storage of large unit doses of PEGylated hyaluronan-degrading enzymes. 4. Combination Treatments Anti-hyaluronan agents, such as a hyaluronan-degrading enzymes or modified form thereof (e.g. a PEGylated hyaluronan-degrading enzyme or PEGylated hyaluronaidase such as PEGPHZO) can be administered in a combination treatment, for example, for the treatment of a onan-associated disease or condition, such as cancer. Compositions of an yaluronan agent can be mulated or co- administered together with, prior to, intermittently with, or subsequent to, other therapeutic or pharmacologic agents or treatments, such as procedures, for example, agents or treatments to treat a hyaluronan associated disease or condition, for example ouan—associated cancers. Such agents include, but are not limited to, other ics, anti-cancer agents, small molecule compounds, dispersing agents, anesthetics, vasoconstrictors and surgery, and combinations thereof. Such other agents and ents that are available for the treatment of a disease or condition, RECTIFIED SHEET (RULE 91) ISA/EP -l73- including all those exemplified herein, are known to one of skill in the art or can be empirically determined.

A preparation of a second agent or agents or treatment or treatments can be administered at once, or can be divided into a number of smaller doses to be administered at intervals of time. Selected agent / treatment 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 tion and patient’s tolerability. Generally, dosing regimes for second agents/treatments herein are known to one of skill in the art.

In one example, an anti-hyaluronan agent, for example a hyaluronan- degrading enzyme or modified form thereof conjugated to a polymer (e.g. a PEGylated hyaluronan-degrading enzyme, such as PEGylated hyaluronidase), is stered with a second agent or treatment for treating the e or condition. In one example, an anti-hyaluronan agent, for e a onan-degrading enzyme or a modified form thereof conjugated to a polymer (6.g. a ted onan- degrading enzyme) and second agent or treatment can be co-formulated and administered together. In another example, an anti-hyaluronan agent, for e a hyaluronan-degrading enzyme or modified form thereof conjugated to a polymer (e.g. a PEGylated onan-degrading enzyme, such as PEGylated hyaluronidase) is administered subsequently, intermittently or simultaneously with the second agent or treatment preparation. Generally, an yaluronan agent, for example a hyaluronan-degrading enzyme (6.g. a PEGylated hyaluronan-degrading enzyme) is administered prior to administration of the second agent or treatment preparation to permit the agent to reduce the level or amount of tissue-or cell-associated hyaluronan.

For example, a onan-degrading enzyme, for example a PEGylated hyaluronan- degrading enzyme, is administered prior to a second agent or treatment to permit the enzyme to reduce or degrade the hyaluronic acid in a cell, tissue or fluid of the subject, such as, for example, the interstitial space, extracellular matrix, tumor tissue, blood or other tissue. For example, an anti-hyaluronan agent, such as a hyaluronan- ing enzyme or modified form thereof conjugated to a polymer (e.g. a ted hyaluronan-degrading enzyme, such as soluble hyaluronidase) can be WO 63155 4174- administered 0.5 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 s, 6 minutes, 7 s, 8 minutes, 9 s, 10 s, 20 minutes, 30 minutes, 1 hour or more prior to administration of the second agent preparation. In some examples, an anti-hyaluronan agent, for example a hyaluronan-degrading enzyme or modified form thereof conjugated to a polymer (e.g. a PEGylated hyaluronan-degrading enzyme) is administered together with the second agent preparation. As will be appreciated by those of skill in the art, the desired proximity of co-administration depends in significant part in the effective half lives of the agents in the particular tissue setting, and the particular disease being treated, and can be readily optimized by g the effects of administering the agents at varying times in suitable models, such as in suitable animal . In some situations, the Optimal timing of stration of the anti~hyaluronan agent, for example a hyaluronan—degrading enzyme or modified form f conjugated to a polymer (2. g. a PEGylated hyaluronan—degrading enzyme, such as a PEGylated hyaluronidase) will exceed 60 minutes.

For example, an anti—hyaluronan agent, for example a hyaluronan-degrading enzyme, can be. administered in conjunction with anti-cancer agents (see. p g ITS.

Publication No. US2010-000323 8). The anticancer agent(s) or treatment(s) for use in combination with a hyaluronan-degrading enzyme include, but are not limited to, surgery, radiation, drugs, herapeutics, polypeptides, antibodies, peptides, small molecules or gene therapy vectors, viruses or DNA.

In other es, the methods of treatment provided herein include s ofadministering one or more additional anti—hyaluronan agents for therapy in addition to a hyaluronan—degrading enzyme. Anti-hyaluronan agents include any agent that reduces or eliminates the accumulation or HA in a tumor. Such agents include, but are not limited to, the hyaluronan—degrading enzymes described herein and also agents that inhibit synthesis of HA. For example, anti-hyaluronan agents that inhibit onan synthesis include antisense or sense molecules against an has gene. Such nse or sense inhibition is lly based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded or otherwise rendered inoperable. In other examples, post-transcriptional gene silencing (PTGS), RNAi, ribozymes and DNAzymes can be employed. it is within the level of one skill in the artgto generate such constructs RECTIFIED SHEET (RULE 91) ISA/EP based on the sequence of HAS1 (set forth in SEQ ID NO: 195), HAS2 (set forth in SEQ ID ) or HAS3 (set forth in SEQ ID NO:197 or 198). It is understood in the art that the sequence of an antisense or sense compound need not be 100% mentary to that of its target nucleic acid to be specifically hybridizable. An oligonucleotide may hybridize over one or more ts such that intervening or adjacent segments are not involved in the hybridization event (e.g. a loop structure or n structure). Generally, the antisense or sense nds have at least 70% sequence mentarity to a target region within the target nucleic acid, for example, 75% to 100% complementarity, such as 75%, 80%, 85%, 90%, 95% or 100%. Exemplary sense or antisense molecules are known in the art (see e.g. Chao et al. (2005) J. Biol. Chem. 280:27513-27522; Simpson et al. (2002) J. Biol. Chem. 277:10050-10057; Simpson et al. (2002) Am. JPath. 161 :849; Nishida et al. (1999) J.

Biol. Chem. 274:21893-21899; Edward et al. (2010) British JDermatology 162: 1224- 1232; Udabage et al. (2005) Cancer Res. 65:6139; and published US. Patent application No. 0286856). Another exemplary anti-hyaluronan agent that is an HA synthesis inhibitor is 4-Methylumbelliferone (4-MU; 7-hydroxy methylcoumarin) or a derivative thereof. 4-MU acts by reducing the UDP-GlcUA precursor pool that is required for HA synthesis. Further exemplary anti-hyaluronan agents are tyrosine kinase inhibitors, such as Leflunomide (Arava), genistein or erbstatin.

In some examples, a osteroid can be administered to ameliorate side effects or adverse events of a hyaluronan-degrading enzyme in the combination therapy (see e.g. US. Patent Application No. 13/135,817). In some examples, the glucocorticoid is selected from among cortisones, dexamethasones, hydrocortisones, methylprednisolones, prednisolones and prednisones. In a particular example, the glucocorticoid is dexamethasone. Typically, the corticosteroid is stered orally, although any method of administration of the corticosteroid is contemplated.

Typically, the glucocorticoid is stered at an amount between at or about 0.4 and 20 mgs, for example, at or about 0.4 mgs, 0.5 mgs, 0.6 mgs, 0.7 mgs, 0.75 mgs, 0.8 mgs, 0.9 mgs, 1 mg, 2 mgs, 3 mgs, 4 mgs, 5 mgs, 6 mgs, 7 mgs, 8 mgs, 9 mgs, 10 mgs, 11 mgs, 12 mgs, 13 mgs, 14 mgs, 15 mgs, 16 mgs, 17 mgs, 18 mgs, 19 mgs or mgs per dose. -l76- F. METHODS OF PRODUCING NUCLEIC ACIDS AND ENCODED POLYPEPTIDES OF HYALURONAN BINDING PROTEINS AND HYALURONAN-DEGRADING ENZYMES Polypeptides of a hyaluronan binding protein for use in the compositions and methods provided or a hyaluronan-degrading enzyme, such as a soluble onidase, for treatment 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 d genes can be used. Any method available in the art can be used to obtain a full length (i.e., encompassing the entire coding region) cDNA or genomic DNA clone encoding a hyaluronan binding protein or a hyaluronidase, such as from a cell or tissue source.

Modified or variant hyaluronan binding proteins or hyaluronidases, can be ered from a wildtype polypeptide, such as by site-directed mutagenesis.

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 ization screening, antibody-based screening and activity-based screening.

Methods for amplification of nucleic acids can be used to isolate c acid molecules encoding a d 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 ations, cell extracts, tissue extracts, fluid samples (e.g. blood, serum, saliva), s from healthy and/or ed ts 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 ce. Nucleic acid molecules generated by amplification can be sequenced and ed to encode a d polypeptide.

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 , for example, a protein -l77- expression vector or a vector designed for the cation of the core protein coding DNA sequences. Furthermore, onal nucleotide sequences specifying fianctional DNA elements can be operatively linked to a ptide-encoding nucleic acid molecule. Examples of such sequences e, but are not limited to, promoter sequences designed to facilitate intracellular protein expression, and secretion sequences, for example heterologous signal sequences, designed to tate protein secretion. Such sequences are known to those of skill in the art. onal 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.

In addition, tags or other moieties can be added, for example, to aid in detection or affinity purification of the polypeptide. For example, onal nucleotide es sequences such as sequences of bases ying 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 ng a His tag (e.g., 6xHis, HHHHHH; SEQ ID NO:54) or Flag Tag (DYKDDDDK; SEQ ID NO:55).

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, ds or modified viruses, but the vector system must be compatible with the host cell used. Such s 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 H224 expression vector ified herein. The insertion into a cloning vector can, for example, be accomplished by ng the DNA fragment into a cloning vector which has complementary cohesive termini. Insertion can be effected using TOPO cloning vectors (Invitrogen, ad, CA). If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA 2012/061743 -l78- molecules can be tically 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 lymeric tailing. Recombinant molecules can be uced into host cells via, for example, transformation, ection, infection, electroporation and sonoporation, so that many copies of the gene sequence are generated.

In specific embodiments, transformation of host cells with recombinant DNA molecules that incorporate the isolated protein gene, cDNA, or synthesized DNA ce enables generation of le copies of the gene. Thus, the gene can be ed 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 For recombinant sion of one or more of the desired proteins, such as any hyaluronan binding protein or hyaluronan-degrading enzyme described herein, the nucleic acid ning all or a portion of the nucleotide ce encoding the protein can be inserted into an appropriate sion vector, z'.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.

Also provided are vectors that contain a nucleic acid encoding the enzyme.

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

Prokaryotic and eukaryotic cells, including endothelial cells, containing the vectors are provided. 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 n is expressed by the cell, and ring the expressed protein. For es herein, for example, the enzyme can be secreted into the medium.

Provided are vectors that contain a sequence of nucleotides that encodes the hyaluronan-degrading enzyme ptide, in some examples a soluble hyaluronidase polypeptide, coupled to the native or heterologous signal sequence, as well as multiple copies f. The vectors can be selected for expression of the enzyme n in the cell or such that the enzyme protein is expressed as a secreted protein.

A variety of host-vector systems can be used to express the protein coding sequence. These include but are not limited to ian cell systems infected with virus (e.g. vaccinia virus, adenovirus and other viruses); insect cell systems ed 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 ths and specif1cities.

Depending on the host-vector system used, any one of a number of suitable transcription and ation ts can be used.

Any methods known to those of skill in the art for the insertion ofDNA fragments into a vector can be used to construct expression s containing a chimeric gene containing appropriate transcriptional/translational control signals and protein coding sequences. These methods can include in vitro recombinant DNA and synthetic ques and in viva recombinants (genetic recombination). Expression of nucleic acid sequences encoding protein, or s, derivatives, fragments or gs 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 embodiment, 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 (Bemoist and Chambon, Nature 290:304-310 (1981)), the promoter contained in the 3 ’ long terminal repeat of Rous a 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 [3-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:5543) or the tac promoter r et al., Proc. Natl.

Acad. Sci. USA 80:21-25 (1983)); see also Gilbert and Villa-Komaroff “Useful Proteins from Recombinant Bacteria” Scientific American 242:74-94 (1980)); plant expression vectors containing the nopaline tase promoter (Herrera-Estrella et al., Nature 303:209-213 (1984)) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., Nucleic Acids Res. 92871 (1981)), and the promoter of the photosynthetic enzyme ribulose sphate carboxylase (Herrera-Estrella et al., Nature 310:115-120 (1984)); promoter elements from yeast and other filngi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase er, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene l region which is active in pancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Omitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, Hepatology 515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan et al. , Nature 315: 1 15-122 ), globulin gene control region which is active in id 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 (Pinkert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoprotein gene control region which is active in liver auf et al., Mol. Cell. Biol. 5:1639-1648 (1985); Hammer et al., e 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 ing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al., Science 234:1372-1378 (1986)).

In a specific embodiment, a vector is used that contains a promoter operably linked to c acids encoding a desired protein, or a domain, nt, derivative or homolog, thereof, one or more origins of replication, and optionally, one or more 2012/061743 selectable s (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 bing the system). pQE vectors have a phage T5 promoter (recognized by E. coli RNA polymerase) and a double lac operator sion module to provide tightly regulated, high-level expression of recombinant proteins in E. 0012', a synthetic ribosomal binding site (RBS II) for efficient ation, a 6XHis tag coding sequence, to and T1 transcriptional terminators, ColEl origin of replication, and a beta- lactamase gene for conferring ampicillin resistance. The pQE vectors enable ent 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 rminally 6xHis-tagged proteins. Other exemplary plasmid vectors for ormation of E. 6012' cells, include, for example, the pET expression vectors (see, US. patent 4,952,496; available from Novagen, Madison, WI; see, also literature published by Novagen describing the system). Such plasmids include pET 1 la, which contains the T7lac promoter, T7 terminator, the inducible E. coli lac or, and the lac repressor gene; pET l2a-c, which contains the T7 promoter, T7 terminator, and the E. 601i ompT secretion signal; and pET 15b and pETl9b (Novagen, Madison, WI), which contain a His-TagTM leader sequence for use in purification with a His column and a thrombin cleavage site that s cleavage following purification over the column, the T7-lac promoter region and the T7 terminator.

Exemplary of a vector for mammalian cell expression is the HZ24 expression vector. The HZ24 sion vector was derived from the pCI vector backbone (Promega). It contains DNA encoding the Beta-lactamase resistance gene (AmpR), an Fl origin of replication, a Cytomegalovirus immediate-early er/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 ofolate reductase (DHFR) gene. 2. Expression Hyaluronan binding proteins and hyaluronan-degrading enzyme polypeptides, including soluble onidase polypeptides, can be produced by any method known to those of skill in the art including in vivo and in vitro methods. d proteins can be expressed in any organism suitable to produce the required s and forms of the proteins, such as for example, the amounts and forms needed for administration and ent. Expression hosts e 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.

Many sion 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 ally enhancers, translational signals, and transcriptional and translational ation s. 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.

Hyaluronan binding proteins and hyaluronan—dcgrading enzyme polypeptides, such as 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 e, but are not limited to, fusions ot'a signal sequence, atag such as for localization, ag. a hiss tag or a myc tag, or a tag for purification, for example, a GST fusion, and a sequence for directing protein ion and/or membrane association. 3. Prokaryotic Cells Prokaryotes, ally E. coli, provide a system for producing large amounts ofproteins. Transformation ofE. coli is a simple and rapid technique well known to those of skill in the art. Expression vectors for E. 0011' can contain inducible promoters, such promoters are useful for inducing high levels of protein sion and for expressing proteins that exhibit some toxicity to the host cells. Examples of inducible promoters include the lac er, the trp promoter, the hybn'd tac RECTIFIED SHEET (RULE 91) ISA/EP 2012/061743 promoter, the T7 and SP6 RNA promoters and the temperature regulated XPL promoter.

Proteins, such as any provided herein, can be expressed in the asmic environment ofE. 0012'. 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 B-mercaptoethanol and denaturants, such as ine-HCl 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 nment 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 ed can be obtained from the periplasm by osmotic lysis. Similar to cytoplasmic expression, in some cases proteins can become ble and denaturants and reducing agents can be used to facilitate solubilization and refolding. Temperature of induction and growth also can influence expression levels and lity, typically temperatures between 25 oC and 37 0C are used. lly, bacteria e aglycosylated proteins. Thus, if proteins require glycosylation for function, ylation can be added in vitro after purification from host cells. b. Yeast Cells Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe, Yarrowz'a lipolytz'ca, 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 gous recombination. lly, ble promoters are used to regulate gene expression. Examples of such promoters include GALl, GAL7 and GALS and metallothionein promoters, such as CUPl, AOXl or other Pichia or other yeast promoter. Expression s often include a selectable marker such as LEU2, TRPl, HIS3 and URA3 for selection and maintenance of the ormed DNA. Proteins expressed in yeast are often soluble. Co-expression with chaperonins such as Bip and protein disulfide isomerase can improve expression levels and solubility. onally, 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 romyces cerevisae and fusions with yeast cell e 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 ylation at Asn-X- Ser/Thr motifs. c. Insect Cells Insect cells, particularly using baculovirus expression, are useful for expressing polypeptides including the hyaluronan binding proteins and hyaluronan- degrading enzyme polypeptides, such as soluble hyaluronidase 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 er of baculovirus. Commonly used baculovirus s include the baculoviruses such as Autographa calz’form’ca nuclear polyhedrosis virus (AcNPV), and the Bombyx mori nuclear polyhedrosis virus (BmNPV) and an insect cell line such as Sf9 derived from Spodopteraflugz’perda, letz'a unipuncta (A78) and Danaus ppus (Dle). 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 tely processed in insect cells and can be used to secrete the expressed protein into the culture medium. In addition, the cell lines Pseudaletz'a unz’puncta (A78) and Danaus plexz'ppus (Dle) e proteins with glycosylation patterns similar to ian cell systems.

WO 63155 2012/061743 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 albopz'ctus) can be used for expression. The Drosophila othionein 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. ian Cells Mammalian expression s can be used to express proteins, including the hyaluronan binding proteins and hyaluronan-degrading enzyme polypeptides, such as soluble hyaluronidase polypeptides. Expression constructs can be transferred to mammalian cells by viral infection such as adenovirus or by direct DNA transfer such as mes, calcium phosphate, DEAE-dextran and by physical means such as electroporation and microinj ection. Expression vectors for mammalian cells lly include an mRNA cap site, a TATA box, a ational 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 riptional promoter-enhancers for evel 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 able marker genes include, but are not limited to, hygromycin B otransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, lycoside phosphotransferase, dihydrofolate reductase (DHFR) and thymidine kinase. For example, expression can be performed in the presence of methotrexate to select for only those cells expressing the DHFR gene. Fusion with cell e signaling molecules such as TCR-t; and chRI-y can direct expression of the proteins in an active state on the cell surface.

Many cell lines are ble for mammalian expression including mouse, rat human, monkey, chicken and hamster cells. Exemplary cell lines include but are not limited to CH0, T3, HeLa, MT2, mouse NSO (nonsecreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, SpZ/O, COS, NIH3T3, HEK293, 293 S, 2B8, and HKB cells. Cell lines also are available adapted to serum-free media which facilitates purification of ed proteins fi‘om the cell e media. Examples include CHO—S cells (Invitrogen, Carlsbad, CA, cat # 012) and the serum flee EBNA-l cell line (Pham et al, (2003) Biotechnol. Bioeng. 84:332-342). Cell lines also are available that are adapted to grow'in special media optimized for maximal expression. For example, D044 CHO cells are adapted to grow in suspension culture in a chemically defined, animal product—free medium. e. Plants 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 er 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 ne synthetase promoter, the ribose bisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters. able s such as hygromycin, phosphomannose isomerase and neomycin phosphotransferase are often used to facilitate selection and maintenance of transformed cells. ormed plant cells can be maintained in culture as cells, aggregates s tissue) or regenerated into whole plants. Transgenic plant cells also can include algae engineered to produce hyaluronidase polypeptides. Because plants have different glycosylation ns than mammalian cells, this can ce the choice of protein produced in these hosts.

RECTIFIED SHEET (RULE 91) ISA/EP 3. Purification Techniques Methods for purification of polypeptides, including hyaluronan binding proteins and onan-degrading enzyme polypeptides (e.g. 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 d 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 sion, s 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.

Proteins can be purified using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size on and size exclusion chromatography, ammonium sulfate precipitation and ionic exchange chromatography, such as anion exchange. Affinity purification techniques also can be utilized to e the ncy and purity of the preparations. For example, dies, receptors and other molecules that bind to onan binding proteins or 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 y purified with myc antibody, glutathione resin and Ni-resin, respectively. Purity can be assessed by any method known in the art including gel ophoresis and staining and spectrophotometric techniques. Purified rHuPH20 compositions, as provided herein, typically have a specific activity of at least 70,000 to 100,000 Units/mg, for e, about 120,000 Units/mg. The specific activity can vary upon modification, such as with a polymer. 4. PEGylation of Hyaluronan-degrading Enzyme Polypeptides Polyethylene glycol (PEG) has been widely used in biomaterials, biotechnology and medicine primarily because PEG is a patible, nontoxic, water-soluble r that is typically nonimmunogenic (Zhao and Harris, ACS Symposium Series 680: 45 8-72, 1997). In the area of drug delivery, PEG derivatives have been widely used in covalent attachment (i. e., "PEGylation") to proteins to reduce immunogenicity, proteolysis and kidney nce and to enhance solubility (Zalipsky, Adv. Drug Del. Rev. 16: 157-82, 1995). Similarly, PEG has been attached to low molecular weight, relatively hydrophobic drugs to enhance solubility, reduce ty and alter biodistribution. Typically, PEGylated drugs are injected as solutions.

A closely related application is synthesis of crosslinked degradable PEG networks or formulations for use in drug delivery since much of the same chemistry used in design of degradable, soluble drug carriers can also be used in design of degradable gels (Sawhney et al., Macromolecules 26: 581-87, 1993). It also is known that intermacromolecular complexes can be formed by mixing solutions of two complementary polymers. Such xes are generally stabilized by electrostatic interactions (polyanion-polycation) and/or hydrogen bonds (polyacid-polybase) between the polymers involved, and/or by hydrophobic ctions between the polymers in an aqueous surrounding (Krupers et al., Eur. Polym J. 32:785-790, 1996).

For example, mixing solutions of polyacrylic acid (PAAc) and polyethylene oxide (PEO) under the proper conditions results in the ion of complexes based mostly on hydrogen bonding. Dissociation of these complexes at physiologic conditions has been used for delivery of free drugs (i.e., non-PEGylated). In addition, complexes of complementary polymers have been formed from both lymers and copolymers.

Numerous reagents for PEGylation have been described in the art. Such reagents include, but are not limited to, N-hydroxysuccinimidyl (NHS) activated PEG, imidyl mPEG, mPEG2-N—hydroxysuccinimide, mPEG succinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEG succinimidyl ate, mPEG carboxymethyl 3-hydroxybutanoic acid succinimidyl ester, homobifilnctional PEG-succinimidyl propionate, homobifunctional PEG propionaldehyde, homobifianctional PEG butyraldehyde, PEG maleimide, PEG hydrazide, ophenyl-carbonate PEG, mPEG-benzotriazole carbonate, naldehyde PEG, mPEG butryaldehyde, branched mPEG2 butyraldehyde, mPEG acetyl, mPEG piperidone, mPEG methylketone, mPEG “linkerless” ide, mPEG Vinyl sulfone, mPEG thiol, mPEG yridylthioester, mPEG orthopyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, Vinylsulfone PEG-NHS, acrylate PEG- NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see e.g., dini et al., Bioconjugate Chem. 6:62-69, 1995; Veronese et al., J. Bioactz've Compatible Polymers 12:197-207, 1997; US. 5,672,662; US. 5,932,462; US. 6,495,659; US. 6,737,505; US. 4,002,531; US. 337; US. 5,122,614; US. 5,324, 844; US. ,446,090; US. 5,612,460; US. 5,643,575; US. 581; US. 5,795, 569; US. ,808,096; US. 5,900,461; US. 5,919,455; US. 5,985,263; US. 5,990, 237; US. 6,113,906; US. 6,214,966; US. 6,258,351; US. 6,340,742; US. 6,413,507; US. 6,420,339; US. 6,437,025; US. 6,448,369; US. 6,461,802; US. 6,828,401; US. 736; US. 2001/0021763; US. 2001/0044526; US. 2001/0046481; US. 2002/0052430; US. 2002/0072573; US. 2002/0156047; US. 2003/0114647; US. 2003/0143596; US. 2003/0158333; US. 2003/0220447; US. 2004/0013637; US 2004/0235734; W005000360; US. 2005/0114037; US. 171328; US. 2005/0209416; EP 1064951; EP 0822199; WO 01076640; WO 0002017; WO 0249673; WO 9428024; and WO 0187925).

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

While numerous reactions have been described for PEGylation, those that are most lly applicable confer directionality, e mild reaction conditions, and do not necessitate extensive downstream processing to remove toxic catalysts or bi- products. For instance, monomethoxy PEG (mPEG) has only one reactive al hydroxyl, and thus its use limits some of the heterogeneity of the resulting PEG- protein product mixture. Activation of the hydroxyl group at the end of the polymer opposite to the terminal y group is generally necessary to accomplish efficient protein PEGylation, with the aim being to make the derivatised PEG more susceptible to nucleophilic attack. The attacking nucleophile is y the epsilon-amino group of a lysyl residue, but other amines also can react (e.g. the N—terminal alpha-amine or the ring amines of histidine) if local conditions are favorable. A more ed attachment is possible in proteins containing a single lysine or cysteine. The latter ~190— residue can be targeted by PEG—maleimide for thiol-specific modification.

Alternatively, PEG hydrazide can be reacted with a periodate oxidized onan— degrading enzyme and reduced in the presence ofNaCNBH3. More specifically, PEGylated CMP sugars can be reacted with a hyaluronan-degrading enzyme in the presence of appropriate glycosyl-transferases. One technique is the “PEGylation” technique where a number of polymeric molecules are coupled to the polypeptide in question. When using this technique the immune system has difficulties in recognizing the epitopes on the polypeptide's surface responsible for the formation of antibodies, thereby reducing the immune response. For polypeptides introduced directly into the circulatory system of the human body to give a ular physiological effect (12¢. ceuticals) the l potential immune response is an IgG and/or IgM reSponse, while polypeptides which are inhaled through the respiratory system (tie. industrial polypeptide) potentially can cause an IgE response (i.e. ic response). One of the theories explaining the reduced immune response is that the polymeric molecule(s) (s) epitope(s) on the surface of the polypeptide responsible for the immune response leading to antibody formation. r theory or at least a partial factor is that the heavier the conjugate is, the more reduced immune response is obtained.

Typically, to make the tcd hyaluronan—degrading enzymes provided 2O herein, including the PEGylated hyaluronidases, PEG moieties are conjugated, via covalent attachment, to the polypeptides. Techniques for PEGylation include, but are not limited to, specialized linkers and coupling chemistries (see e.g, Roberts, Adv.

DrugDeliv. Rev. -476, 2002), attachment of multiple PEG moieties to a single conjugation site (such as via use of branched PEGs; see ag, Guiotto et al. , Bioorg.

Med. Chem. Lett. -180, 2002), site-specific PEGylation and/or mono- tion (see e.g., Chapman et al., Nature Biotech. 17:780-783, 1999), and site— directed enzymatic PEGylation (see e.g., Sato, Adv. Drug Deliv. Rev., 54:487—504, 2002). Methods and techniques described in the art can produce proteins having l, 2, 3, 4, 5, 6, '7, 8, 9, 10 or more than 10 PEGs or PEG derivatives ed to a single n molecule (see e.g., US. 2006/0104968).

As an exemplary illustrative method for making PEGylated hyaluronan- ing enzymes, such as PEGylated RECTIFIED SHEET (RULE 91) ISA/EP WO 63155 -l91- hyaluronidases, PEG aldehydes, succinimides and carbonates have each been applied to conjugate PEG moieties, typically succinimidyl PEGs, to rHuPH20. For example, rHuPHZO has been conjugated with exemplary succinimidyl monoPEG (mPEG) reagents ing mPEG—Succinimidyl Propionates (mPEG—SPA), mPEG— Succinimidyl ates (mPEG-SBA),‘and (for attaching "branched" PEGs) mPEG2-N-Hydroxylsuccinimide. These PEGylated succinimidyl esters contain different length carbon backbones between the PEG group and the activated cross- linker, and either a single or branched PEG group. These differences can be used, for example, to provide for different reaction cs and to ially restrict sites available for PEG attachment to rHuPHZO during the conjugation process.

Succinimidyl PEGs (as above) comprising either linear or branched PEGs can be conjugated to rHuPHZO. PEGs can used to te rHuPHZOs reproducibly containing molecules having, on the average, between about three to six or three to six PEG molecules per hyaluronidase. Such ted rHuPHZO compositions can be readily purified to yield compositions having c activities of approximately ,000 or 30,000 Unit/mg protein hyaluronidase activity, and being substantially free of non-PEGylated rHuPH20 (less than 5 % nonuPEGylated).

Using various PEG reagents, exemplary versions of hyaluronan-degrading enzymes, in particular soluble human recombinant hyaluronidases (e.g. rHuPH20), can be prepared, for e, using mPEG—SBA (30 kD), MB (30 kD), and branched versions based on NHS (40 kD) and mPEGZ-NHS (60 kD).

PEGylated versions H20 have been generated using NHS chemistries, as well as carbonates, and aldehydes, using each of the following reagents: mPEGZ-NHS- 40K branched, mPEG-NHS-IOK ed, mPEG-NHS—ZOK branched, mPEGZ- NHS-60K branched; mPEG-SBA-SK, mPEG—SBA-ZOK, BA-30K; mPEG- SMB-ZOK, mPEG-SMB-SOK; mPEG-butyraldehyde; mPEG-SPA-ZOK, mPEG—SPA- 30K; and PEG-NHS—SK-biotin. PEGylated hyaluronidases have also been prepared using PEG reagents available from Dowpharma, a division of Dow Chemical Corporation; including onidases PEGylated with Dowpharma‘s p-nitrophenyl- carbonate PEG (30 kDa) and with propionaldehydc PEG (30 kDa).

In one example, the PEGylation includes conjugation of mPEG-SBA, for example, mPEG—SBA—BOK (having a molecular weight of about 30 kDa) or another RECTIFIED SHEET (RULE 91) ISA/EP -l92- succinimidyl esters of PEG ic acid derivative, to a e hyaluronidase.

Succinimidyl esters of PEG butanoic acid derivatives, such as mPEG-SBA-30K y couple to amino groups of proteins. For example, covalent conjugation ofm- PEG-SBA-30K and rHuPH20 (which is approximately 60 KDa in size) provides stable amide bonds between rHuPH20 and mPEG, as shown in Scheme 1, below.

Scheme 1 H3CO+CH2CH20)—CH2CH2CH2CO—N|| + H2N—rHuPH20 mPEG-SBA l H3CO+CH2CHZO)’CH2CH2CH2C—N—H H rHuPH20 PEGylated rHuPH20 Typically, the mPEG-SBA-30K or other PEG is added to the hyaluronan- degrading enzyme, in some instances a hyaluronidase, at a PEG:polypeptide molar ratio of 10:1 in a suitable buffer, e.g. 130 mM NaCl /10 mM HEPES at pH 6.8 or 70 mM phosphate buffer, pH 7, followed by sterilization, e.g. sterile filtration, and continued conjugation, for e, with stirring, overnight at 4 CC in a cold room.

In one e, the ated PEG- hyaluronan-degrading enzyme is concentrated and buffer-exchanged.

Other methods of coupling succinimidyl esters of PEG butanoic acid derivatives, such as mPEG-SBA-30K are known in the art (see e.g., US. 5,672,662; US. 6,737,505; and US. 2004/0235734). For example, a polypeptide, such as a onan-degrading enzyme (e.g. a hyaluronidase), can be coupled to an NHS activated PEG derivative by reaction in a borate buffer (0.1 M, pH 8.0) for one hour at 4 CC. The resulting PEGylated protein can be purified by ultrafiltration.

Alternatively, tion of a bovine alkaline phosphatase can be accomplished by mixing the phosphatase with mPEG-SBA in a buffer containing 0.2 M sodium phosphate and 0.5 M NaCl (pH 7.5) at 4 0C for 30 minutes. Unreacted PEG can be —l93— removed by ultrafiltration. Another method reacts polypeptide with mPEG-SBA in deionized water to which triethylamine is added to raise the pH to 7.2-9. The resulting mixture is stirred at room temperature for several hours to complete the PEGylation.

Methods for PEGylation of onan-degrading polypeptides, ing, for example, animal—derived hyaluronidases and bacterial hyaluronan—degrading enzymes, are known to one of skill in the art. See, for e, an Patent No.

EP 0400472, which describes the PEGylation of bovine testes hyaluorindase and oitin ABC lyase. Also, U.S. Publication No. 2006014968 describes PEGylation of a human hyaluronidase derivedfrom human PH20. For example, the PEGylated hyaluronan-degrading enzyme generally contains at least 3 PEG moieties per molecule. For example, the hyaluronan—degrading enzyme can have a PEG to protein molar ratio n 5:1 and 9:1, for example, 7:1.

G. METHODS OF ASSESSING ACTIVITY AND MONITORING EFFECTS OF ANTI—HYALURONAN AGENTS Anti-hyaluronan agents, for example hyaluronan-degrading enzymes, such as a hyaluronidase or modified hyaluronidase (e.g. PH20 or PEGPHZO) act as therapeutic agents either alone, or in combination with secondary agents such as herapeutic drugs, for the treatment of hyaluronan-associated diseases and conditions, in particular cancers (see for example, US 2010/0003238 and W009/12891 7). In addition, as described elsewhere herein, therapy with an anti- hyaluronan agent, for example a onan-degrading enzyme, can be anied by treatment with a corticosteroid to minimize the systemic, for example musculoskeletal, side effects of the PEGyIated hyaluronidase. The HABP companion diagnostics, such as TSG—6-LM or TSGLMch or variants thereof, provided herein can be used in conjunction with an anti-hyaluronan agent therapy, for example a hyaluronan-degrading enzyme therapy, used for the treatment of onan- associated diseases or disorders, such as , in order to monitor responsiveness and efficacy of treatment with the agent (e.g. a hyaluronan-degrading enzyme). In addition, adjunct or supplementary methods also can be utilized to assess the s of anti-hyaluronan agents (e.g. hyaluronan-degrading enzymes) in treatment alone or in combination with corticosteroids. It is within the level of one of skill in the art to RECTIFIED SHEET (RULE 91) ISA/EP ~194- assess rationhof side effects by corticosteroids, as wells as efficacy, tolerability and pharmacokinetic studies of anti-hyaluronan agent y, including hyaluronan— ing enzyme therapy. This n es description of adjunct or supplementary methods that can be used to assess efficacy, responsiveness, tolerability and/or cokinetics of a hyaluronan—degrading enzyme therapy. 1. Methods to Assess Side Effects In wivo assays can. be used to assess the efficacy of corticosteroids on the amelioration or elimination of the musculoskeletal side effects that can be caused by an anti~hyaluronan agents, for example a hyaluronan-degrading enzyme, such as a ' hyaluronidase or hyaluronidase modified to exhibit increased systemic half—life (e.g.

PHZO 01' PEOPI-LZO). Side emus that can be assessed Include, [bi cxmuplc, illusclc and joint pain, stiffness of upper and lower extremities, ng, myositis, muscle soreness and ness over the entire body, weakness, fatigue and/or a decrease in range of motion at knee and clbowjoints. Assays to assess side effects can include animal models wherein the animal can be observed for reduced movement, behavior or posture s, radiographic findings, histopathological changes and other notable clinical ations. Other assays can include clinical trials in human subjects wherein patients can be questioned ing symptoms, assessed by physical examination, imaging (for example by MRI or PET) or by I'adiologic evaluation. Amelioration of a side effect caused by stration of an anti— hyaluronan agent (rag. a hyaluronan—dcgrading enzyme agent) is observed when the side effect is ameliorated, eliminated, lessened or reduced in the presence of the corticosteroid compared to in its abscnce.

In such examples, the dose of anti-hyaluronau agent (6. g. a hyaluronan— degrading enzyme) and/or corticosteroid can be varied to identify the optimal or minimal dose required to achieve activity While ameliorating side effects. Such studies are within the level of one of skill in the art. Further, the dosage regime can be varied. For example, studies can be performed using a dosage schedule of hyaluronan-degrading enzyme monthly, ly, once a week, twice a week, three times a week, four times a week or more. Further, the corticosteroid can be administered prior to, concurrently and/or uent to administration of the anti- hyaluronan agent (9.g. a hyaluronan-degrading enzyme).

RECTIFIED SHEET (RULE 91) ISA/EP -l95- For example, in vivo animal models can be utilized to assess the ability of osteroids, such as dexamethasone, to ameliorate or eliminate the side effects associated with yaluronan agent (6.g. hyaluronan-degrading enzyme) administration. Animal models can include non-human primates such as cynomolgus monkeys or rhesus macaques, dogs, for example beagle dogs, or any other animal that exhibits adverse side effects in response to treatment with an anti-hyalruonan agent (6.g. a hyaluronan-degrading enzyme, such as a PEGylated hyaluronidase, for example PEGPHZO) treatment. The animal models can be dosed with an anti- hyaluronan agent (e.g. a onan-degrading enzyme) in the presence or absence of corticosteroid and musculoskeletal effects observed or measured.

For example, animals such as cynomolgus monkeys, beagles or other similar animal model capable of able or measurable musculoskeletal events can be treated with hyaluronan-degrading enzyme in the presence or absence of corticosteroid. In one example, a group of animals, for example cynomolgus monkeys or beagles, is administered with an anti-hyaluronan agent, such as a hyaluronan-degrading enzyme alone, for example a ted hyaluronidase, such as by intravenous administration. For example, administration can be twice weekly.

Treatment can continue until changes in limb joint range-of-motion are observed at the knee and elbow joints or stiffness or reduced mobility is observed. Then, another group of animals can be treated with the anti-hyaluronan agent (6.g. hyaluronan- degrading enzyme) and corticosteroid stered, such as by oral doses of dexamethasone or other corticosteroid, given on the same day as the anti-hyaluronan agent (6.g. hyaluronan-degrading enzyme) administration. The groups of animals can then be compared for example, via physical ation t range-of—motion or other reduced mobility, histopathology of the joints, palpation for stiffness, or g known to those of skill in the art, to assess the ability of the osteroid, such as dexamethasone, to ameliorate the anti-hyaluronan mediated, such as hyaluronan-degrading enzyme-mediated, oskeletal side effects. Dose, dosing frequency, route of administration, and timing of dosing of corticosteroid, such as dexamethasone, can be varied to optimize the effectiveness of the corticosteroid.

In another example, the efficacy of corticosteroids such as dexamethasone on the amelioration or ation of the adverse side effects associated with an anti- WO 63155 -l96- onan agent (6.g. a hyaluronan-degrading enzyme) administration can be assessed in human patients with solid tumors. For example human ts can be dosed to e the ability of corticosteroid to ameliorate and/or ate anti- hyaluronan agent-mediated (e.g. hyaluronan-degrading enzyme-mediated) adverse events including, but not limited to any one or more of the following: muscle and joint pain/stiffness of upper and lower extremities, cramping, muscle, myositis muscle soreness and tenderness over the entire body, weakness and fatigue. Patients can be treated with an anti-hyaluronan agent (6.g. a hyaluronan-degrading enzyme) with or without co-treatment with a corticosteroid such as dexamethasone. During and after administration of an anti-hyaluronan agent (6.g. a hyaluronan-degrading enzyme), side effects of both treatment groups can be assessed. A physician can determine the severity of the symptoms by al examination of the subject including for example, patient complaints, vital signs, changes in body weight, lZ-lead ECG, echocardiogram, clinical chemistry, or imaging (MRI, PET or radiologic evaluation).

The severity of symptoms can then be quantified using the NCI Common Terminology Criteria for Adverse Events (CTCAE) g system. The CTCAE is a descriptive terminology utilized for Adverse Event (AE) reporting. A grading (severity) scale is provided for each AE term. The CTCAE displays Grades 1 through , with clinical descriptions for severity for each adverse event based on the following general guideline: Grade 1 (Mild AE); Grade 2 (Moderate AE); Grade 3 (Severe AE); Grade 4 (Life-threatening or disabling AE); and Grade 5 (Death related to AE). The ability of a corticosteroid to ameliorate adverse side s associated with administration of an anti-hyaluronan agent (6.g. a hyaluronan-degrading enzyme) can be ed by the observation of a reduction in grading or severity on the CTCAE scale in one or more adverse side effects in subjects treated with the anti-hyaluoman agent (e.g. hyaluronan-degrading enzyme) and corticosteroid as compared to ts treated with the same anti-hyaluronan agent (6.g. hyaluronan-degrading enzyme) alone, z'.e., the ty of the side effects, is d from Grade 3 to Grade 1 or Grade 2.

In another example, human patients can be dosed to assess tolerability by escalating the dose of an anti-hyaluronan agent (6.g. a hyaluronan-degrading ) and assessing the dose-limiting toxicity as measured by severity of side effects. In such an example, a maximum tolerated dose ofan anti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme) that can be tolerated in the presence of an ameliorating agent such as a corticosteroid can be determined. Treatment regimens can include a dose escalation wherein each patient receives a higher dose of hyaluronan—degrading enzyme at the same dose level of corticosteroid. Patients can be monitored for adverse events to ine the highest dose of hyaluronan- ing enzyme that can be administered with a corticosteroid before side effects are no longer tolerated. Tolerability can be measured based on the severity of symptoms ng during and after treatment. Doses of an anti-hyaluronan agent (e.g. a hyaluronan-degrading enzyme) can be ted until adverse effects reach a predetermined level, for example, Grade 3. Dosing regimens can also include a tapering of the amount of corticosteroid administered to examine the ued need for corticosteroid and the possibility of acclimation to the anti-hyaluronan agent with respect to resulting side effects. 2. ting Biomarkers Associated With Activity of an Anti- Hyaluronan Agent (e.g. a Hyaluronan-Degrading Enzyme Activity) As described herein, the extent and level ofHA phenotypes is a biomarker that is associated with and correlates to efficacy and activity of an anti-hyaluronan agent (e. g. hyaluronan—degrading enzyme). For example, for cancer patients with tumors such as advanced solid tumors, reduced tumor— and stroma-associated is a biomarker . of activity of an administered hyaluronan-degrading enzyme. An HABP binding assay to detect HA present in tissue (e. g. tumor biopsy) or bodily fluids (8. g. plasma) as described ere herein 'can be performed to evaluate and monitor the therapeutic effect of an yaluronan (e.g. hyaluronan-degrading enzyme).

In addition, assays can be performed separately or in conjugation with HABP assays described herein used to detect HA in tissue (e.g. tumor biopsy) or bodily fluids (e.g. plasma) to further assess the effects of an anti-hyaluronan agent (2. g. a hyaluronan—degrading enzyme) on hyaluronan inhibition or degradation activity. In particular, for ent of a onan-associated disease or ion, such as cancer, clinical measures or biomarkers associated with activity of an yaluronan agent, for example a hyaluronan-degrading enzyme activity include, but are not limited to, reduced tumor metabolic ty, increased apparent ion and RECTIFIED SHEET (RULE 91) ISA/EP -l98- enhanced tumor perfusion and/or increase in HA catabolites. Additional assays to measure such kers can include, but are not limited to, measurements of hyaluronan catabolites in blood or urine, ements of hyaluronidase activity in plasma, or measurements of interstitial fluid pressure, vascular volume or water content in tumors. It is within the level of one skilled in the art to perform such assays.

These assays can be med in animal models treated with a hyaluronan- degrading enzyme or in human ts. For example, animal models of hyaluronan- associated diseases, disorders or conditions can be utilized to assess the in viva affect of administration of an anti-hyaluronan agent, for example a hyaluronan-degrading enzyme, such as a hyaluronidase or hyaluronidase modified to exhibit increased half- life (6.g. PH20 or PEGPHZO). Another agent, such as a chemotherapeutic agent can also be ed in the assessment of activity. Exemplary hyaluronan-associated diseases for which an appropriate animal model can be utilized include solid tumors, for example, late-stage cancers, a metastatic cancers, erentiated cancers, ovarian cancer, in situ carcinoma (ISC), squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer and other cancers. Also exemplary of hyaluronan-associated es and disorders are inflammatory diseases, disc pressure, cancer and edema, for example, edema caused by organ transplant, stroke, brain trauma or other .

Animal models can e, but are not limited to, mice, rats, rabbits, dogs, guinea pigs and non-human primate models, such as cynomolgus monkeys or rhesus es. In some examples, immunodeficient mice, such as nude mice or SCID mice, are transplanted with a tumor cell line from a hyaluronan-associated cancer to establish an animal model of that cancer. ary cell lines from hyaluronan- associated cancers include, but are not limited to, PC3 prostate carcinoma cells, BxPC-3 pancreatic adenocarcinoma cells, MDA-MB-23l breast oma cells, MCF-7 breast tumor cells, BT474 breast tumor cells, Tramp C2 prostate tumor cells and Mat-LyLu prostate cancer cells, and other cell lines described herein that are hyaluronan associated, e.g. contain elevated levels of hyaluronan. An anti-hyaluronan agent, such as a hyaluronan-degrading enzyme, can then be administered to the animal with or without a osteroid such as dexamethasone, to assess the effects of ~199- the corticosteroid on anti-hyaluronan activity by measuring, for example, hyaluronan levels or content. onan content can be measured by staining tumor tissue samples for hyaluronan or by measuring soluble hyaluronan levels in plasma. Other measurements of anti-hyaluronan activity include the assessment oftumor volume, formation or size of halos, interstitial fluid pressure, water content and/or vascular volume.

In other examples, dogs such as beagle dogs, can be treated with an anti- hyaluronan agent in the presence or absence of a corticosteroid, such as thasone. Tissues such as skin or al muscle tissue are biopsied and stained for hyaluronan and evaluated visually. s from s treated with an anti-hyaluronan agent alone are then compared to tissues from aminals treated with the anti-hyaluronan agent and corticosteroid to measure the effect of the osteroid on anti-hyaluronan activity.

Assays for activity of an anti-hyaluronan agent, such as a onan- degrading enzyme activity, also can be med in human subjects. For example, assays to measure a biomarker associated with an anti-hyaluronan agent (eg. a hyaluronan—degrading enzyme) activity can be performed on human subjects known or suspected of having a hyaluronan-associated disease or condition (e.g. cancer) and that have been treated with a hyaluronan-degrading enzyme (e.g. PEGPHZO). a. Assays to assess the activity of a onan Degrading Enzyme The activity of a hyaluronan degrading enzyme can be assessed using methods well known in the art. For example, the USP XXII assay for hyaluronidase determines activity indirectly by measuring the amount of undegraded hyaluronic acid, or hyaluronan, (HA) substrate remaining after the enzyme is allowed to react with the HA for 30 min at 37 °C (USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia tion, Inc, Rockville, MD). A Hyaluronidase Reference Standard (USP) or National ary (NF) Standard Hyaluronidase solution can be used in an assay to ascertain the activity, in units, of any hyaluronidase. In one example, activity is measured using a urbidity assay. This is based on the formation of an insoluble precipitate when hyaluronic acid binds with serum albumin. .l The activity is measured by incubating hyaluronidase Or a sample containing RECTIFIED SHEET (RULE 91) ISA/EP hyaluronidase, for example blood or plasma, with sodium hyaluronate ronic acid) for a set period of time (6.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 onal 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, onidase activity is measured using a microtiter assay in which residual biotinylated hyaluronic acid is ed following incubation with hyaluronidase or a sample containing hyaluronidase, for example, blood or plasma (see e.g. Frost and Stern (1997) Anal. Biochem. 251:263-269, US. 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 coupled 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 n 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 et al., (1995) Anal. m. 229:35-41; Takahashi et al., (2003) Anal.

Biochem. 322:257-263).

The y of an active hyaluronan ing enzyme, such as a modified soluble hyaluronidase (eg PEGylated PH20) to act as a spreading or diffilsing agent, e. g. for chemotherapeutics, also can be assessed. For e, trypan blue dye can be injected, such as subcutaneously or ermally, with or without a hyaluronan degrading enzyme 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 the hyaluronan degrading enzyme to act as a spreading agent (see e.g. U.S. Published Patent No. 20060104968). b. ement of HA catabolites In another example, blood and urine can be collected at different time points throughout patient treatment and assayed for catabolites of hyaluronan. The presence of catabolites is indicative of the ation of hyaluronan and is thus a measure of the activity of hyaluronidase. Plasma enzyme also can be assessed and measured over time following administration. For example, HA lites, which are HA- disaccharide- breakdown products, can be assessed using high—perfonnance liquid chromatography (HPLC) to separate and measure saccharide peak areas. The Example 15 exemplifies this assay. e. metabolic activity A reduction in tumor metabolic activity is associated with anti-hyaluronan agent (e.g. hyaluronan—degrading enzyme) activity. Tumor metabolic activity can be assessed using standard procedures known in the art. For example, [18F]— fluorodeoxyglucose positron emission tomography (FDG-PET) can be used. PET is a non-invasive diagnostic that provides images and quantitative ters of perfusion, cell viability, proliferation and/or metabolic ty of tissues. The images result from the use of different biological substances (e. g. sugars, amino acids, metabolic precursors, hormones) labelled with positron emitting radioisotopes. For example, FDG is an ue of glucose and is taken up by living cells via the first stages of normal glucose pathway. In cancers, increased glycolytic activity exists resulting in ng of FDG in the cancer cell. A decrease in FDG trapping correlates with a decreased tumor metabolic activity and anti-tumorigenic activity.

Guidelines for PET imaging are known to one of skill in the art and should be followed by any treating physician or technician. d. Increased apparent diffusion and enhanced tumor perfusion Additional methods of assessing anti-hyaluronan agent (e.g. hyaluronan- degrading enzyme) ty include assays that assess the diffusion of water in tissues. ‘ As discussed elsewhere herein, tissues that accumulate hyaluronan generally have a higher titial fluid pressure than normal tissue due to the concomitant accumulation of water. Thus, tissues that accumulate HA, such as tumors, have high interstitial fluid pressure, which can be measured by various methods known in the art. For example, diffusion MRI, such as ADC MRI or DCE MRI, can be used.

Diffusion ofwater can be assessed by these procedures, and is directly ated to - presence uronan—rich tissues, such as solid tumors (see e.g. Chenevert et a1. (1997) Clinical Cancer Research, 3 :1457-1466). For example, tumors that iflitehyraluronmi havea distinguishable increase in ADC MRI or DCE MRI.

RECTIFIED SHEET (RULE 91) ISA/EP ~202~ e of increased perfusion. Such assays can be med in the presence and absence ofa hyaluronan—degrading enzyme, and reSults compared. Methods of measuring diffusion are a useful measure of assessing cellular changes following such therapies. 3. Tumor Size and Volume Activity of an yaluronan agent (eg. hyaluronan-degrading enzymes) is associated with reductions in tumor size and/or volume. Tumor size and volume can be monitored based on techniques known to one of skill in the art. For example, tumor size and volume can be monitored by radiography, ultrasound imaging, necropsy, by use of calipers, by microCT or by 18F-FDG-PET. Tumor size also can be assessed visually. In particular examples, tumor size ter) is ed ly using calipers.

In other examples, tumor volume canbe measured using an average of measurements r diameter (D) obtained by caliper or ultrasound assessments.

For example, tumor volume can be determined using VisualSonics Vevo 770 high- resolution ultrasound or other similar ultrasound. The volume can be determined from the formula V = D3 x n / 6 (for diameter measured using calipers) 0r V = D2 x d x 1r/ 6 (for diameter measured using ultrasound where d is the depth or thickness). For example, caliper measurements can be made ofthe tumor length (l) and Width (w) and tumor volume calculated as length x width2 x 0.52. In another example, microCT scans can be used to measure tumor volume (see e.g. Huang et al. (2009) PNAS, 106134266430). As an example, mice can be injected with Optiray Pharmacy ioversol injection 74% contrast medium (2.g. 741 mg of ioversol/mL), mice anesthetized, and CT scanning done using a MicroCat 1A scanner or other similar AD scanner rag. llVl 16K) (Aw KV, ow pm, we roranon Steps, torai angle or roranon = 196). The images can be reconstructed using software (e.g. RVA3 software program; ImTek). Tumor volumes can be determined by using available software (e.g. Amira 3.1 software; Mercury Computer Systems). Tumor volume or size also can be ined based on size or weight of a tumor.

The t of tumor growth inhibition can be ated based on the volume using the on: % TGI = [1 ~ ) + (Cu-CON x 100%, where “Tn” is the average tumor volume fg the treatment groupiatday “n” after the final dose of RECTIFIED SHEET (RULE 91) ISA/EP hyaluronan—degrading enzyme; “To” is the average tumor volume in that treatment group at day 0, before ent; “Cn” is the average tumor volume for the corresponding control group at day “n”; and “Co” is the average tumor volume in the control group at day 0, before treatment. Statistical analysis of tumor volumes can be determined. 4. Pharmacokinetic and Pharmacodynamic Assays Pharmacokinetic or codynamic studies can be performed using animal models or can be performed during studies with patients to assess the pharmacokinetic properties of an anti-hyaluronan agent, for example a hyaluronan degrading enzyme, such as a hyaiuronidase or modified hyaluronidase (e. g. PEGPH20). Animal models include, but are not limited to, mice, rats, rabbits, dogs, guinea pigs and man e models, such as cynomolgus monkeys or rhesus macaques. In some instances, pharmacokinetic or pharmacodynamic studies are med using healthy animals. In other examples, the s are performed using animal models ofa * disease for which therapy with hyaluronan is considered, such as animal models of any hyaluronan-associated disease or disorder, for example a tumor model.

The pharmacokinetic properties of an anti-hyaluronan agent (ag. a onan-degrading , such as a modified hyaluronidase) can be assessed by measuring such parameters as the maximum (peak) concentration (Cmax), the peak time (ie. when maximum concentration occurs; Tmax), the minimum concentration (i. e. the minimum concentration n doses; Cman), the elimination half—life (Tl/2) and area under the curve (i. e. the area under the curve generated by plotting time versus concentration; AUC), following administration. The absolute bioavailability of the agent or enzyme (e.g. a hyaluronidase) can be determined by comparing the area under the curve following aneous delivery (AUCSC) with the AUC following enous delivery (AUCiv). Absolute bioavailability (F), can be calculated using the a: F = ([AUC]sc >< dosesc) / ([AUCLv X doseiv). A range of doses and different dosing frequency of dosing can be administered in the phannacokinetic studies to assess the effect of increasing or decreasing concentrations of an anti- hyaluronan agent, for example a hyaluronan-degrading enzyme, such as a hyaluronidase or modified onidase (e.g. PEGylated PHZO) in the dose.

RECTIFIED SHEET (RULE 91) ISA/EP -2o4- H. KITS AND ARTICLES OF CTURE Provided herein are kits for use in selecting patients for treatment with an anti- hyaluronan agent (e.g. a hyaluronan degrading enzyme), for predicting the efficacy of treatment with an anti—hyaluronan agent (e. g. hyaluronan degrading enzyme), for determining the prognosis of a patient with an HA—associated diseases, or for monitoring the efficacy of treatment with an anti-hyaluronan agent (a g. a hyaluronan degrading enzyme) for the ent of HA-associated diseases, in particular cancer.

The kits provided herein contain an HABP reagent provided herein for the detection and quantitation of hyaluronan in a sample and ally, reagents for performing the s. For example, kits can additionally contain reagents for collection of tissues, preparation and sing of tissues, and reagents for quantitating the amount ofHA in a , such as, but not limited to, ion reagents, such as dies, buffers, substrates for enzymatic staining, chromogens or other materials, such as slides, containers, microtiter plates, and optionally, instructions for performing the methods.

Those of skill in the art will recognize many other le containers and plates and reagents that can be used for contacting the various materials. Kits also can n control samples representing tissues with different levels ofHA or reference samples stained for HA content for ison and classification of the test samples. The HABP diagnostic provided can be provided in a lyophilized or other stable formulation of the diagnostic agent. In some examples, the kit includes a device, such as an automated ar imaging system (ACIS) fluorometer, luminometer, or spectrophotometer for assay detection.

Also provided are combinations ofan HABP reagent provided herein, including the improved HABP reagents provided, and a hyaluronan degrading enzyme. As described herein, HABPs can be employed as companion diagnostic agents for treatment with a hyaluronan degrading enzyme. Such combinations optionally can be packaged as kits for the for use in selecting patients for treatment with an anti-hyaluronan agent (e.g. a hyaluronan degrading ) and treating such patients with the anti-hyaluronan agent (6.g. a hyaluronan degrading enzyme), for predicting the y of ent with an anti—hyaluronan agent (e. g. a hyaluronan degrading enzyme) in a t and treating such patients with the anti-hyaluronan agent (e. g. hyaluronan degrading enzyme), for determining the prognosis of a patient RECTIFIED SHEET (RULE 91) ISA/EP “205- with an HA-associated diseases and treating such patients with the anti-hyaluronan agent (9. g. hyaluronan degrading enzyme), or for monitoring the efficacy of treatment of a patient with an anti-hyaluronan-degrading enzyme (e.g. a onan degrading enzyme) for the treatment of HA—associateddiseases, in particular cancer, and treating such patients with the anti-hyaluronan agent (6. g. hyaluronan degrading enzyme) based'on efficacy, of treatment.’ Combinations, which can be ed as kits, can include, one or more additional agents for therapy, such as an anti-cancer agent or for the treatment or a side effect of therapy, including a corticosteroid for the treatment of musculoskeletal sides effects associated with treatment with an anti-hyaluronan agent (ag. hyaluronan degrading enzyme). The kits can include packing materials for the packaging of the yaluronan agent (e. g. hyauronan degrading enzyme) or the one or more additional therapeutic agents. For example,the kits can contain containers including single chamber and dual chamber containers. The containers e, but are not limited to, tubes, bottles and syringes. The ners can r include als for administration, such as a needle for Subcutaneous administration. The anti-hyaluronan agent (e.g. a hyauronan degrading enzyme) or the one or more additional therapeutic agents can be ed together or separately. The kit can, optionally, e instructions for administration including dosages, dosing regimens and instructions for modes of administration.

Kits ed herein also can include ts for detecting the expression of one or more additional proteins or encoding RNAs in the sample, such as, for example, one or more additional cancer markers, such as, for example, but not limited to, carcinoembryonic antigen (CEA), Alpha-Fetoprotein (AFP), CA125, CA19—9, prostate specific antigen (PSA), human chorionic gonadotropin (HCG), HERZ/neu antigen, CA27.29, CYFRA 21-2, LASA—P, CA15-3, TPA, 8-100 and (IA—125. 1. EXAMPLES The following examples are included for illustrative es only and are not intended to limit the scope of the ion.

RECTIFIED SHEET (RULE 91) ISA/EP Example 1 rHuPH20 Expressing Cell Lines A. Generation'of an Initial Soluble rHuPH20-Expressing Cell Line Chinese r Ovary (CHO) cells were transfected with the H224 d (set forth in SEQ ID NO:52). The HZZ4 plasmid vector for expression of soluble rHuPH20 contains a pCl vector backbone (Promega), DNA encoding amino acids 1- 482 of human PH20 hyaluronidase (SEQ ID N0149), 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 fl origin of replication, a Cytomegalovirus immediate-early er/promoter region (CMV), a chimeric intron, and an SV40 late polyadenylation signal . The DNA encoding the soluble rHuPI-l20 uct contains an NheI site and a Kozak consensus sequence prior to the DNA encoding the methionine at amino acid position 1 ofthe native 35 amino acid signal sequence of human PHZO, 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 BamHI restriction site. The construct pCI-PHZO- IRES-DHFR—SV40pa (HZ24), therefore, results in a single mRNA species driven by the CMV promoter that encodes amino acids 1-482 ofhuman 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 mal entry site .

Non-transfected CHO cells growing in GIBCO Modified CD-CHO media for DHFR(—) cells, supplemented with 4 mM ine and 18 ml/L Pluronic F68/L (Gihco), were seeded at 0.5 x 106 cells/ml in a shaker flask in preparation for transfection. Cells were grown at 37° C in 5% C02 in a humidified tor, shaking at 120 rpm. Exponentially growing non-transfected CHO cells were tested for viability prior to transfection.

Sixty million viable cells of the non-transfected CHO cell culture were pelleted and resuspended to a density of 2 ><107 cells in 0.7 mL of 2x transfection buffer (2x HeBS: 40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mM KCl, 1.4 mM Nazi-IP04, 12 mM dextrose). To each aliquot of resuspended cells, 0.09 mL (250 pg) of the linear HZ24 plasmid (linearized by ght ion with Cla I (New RECTIFIED SHEET (RULE 91) ISA/EP d 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 lasmid mixes were electroporated with a capacitor rge of 330 V and VI 960 uF or at 350 V and 960 uF.

The cells were removed from the cuvettes after electroporation and transferred into 5 JILL of IVIudil'ied CD—CHO media for ) cells, supplemented with 4 mM Glutamine and 18 ml/L Pluronic 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.

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. Cells expressing the highest levels of hyaluronidase activity were collected from the tissue culture well, counted and d to 1 ><104 to 2 ><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 iters of CD-CHO media (GIBCO) containing 4 mM GlutaMAXTM-l supplement (GIBCOTM, Invitrogen Corporation) and t hypoxanthine and thymidine ments were added to the wells containing cells (final volume 0.2 mL).

Ten clones were identified from the 5 plates grown without methotrexate. Six of these H224 clones were expanded in culture and transferred into shaker flasks as single cell suspensions. Clones 3D3, 3E5, 2G8, 2D9, 1E1 1, 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 ound 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 plates containing 50 nM methotrexate and 5 plates without methotrexate.

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 nes (>50 mL), RECTIFIED SHEET (RULE 91) ISA/EP 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 in shaker flasks and gave rise to clones producing in excess of 1,000 Units/ml hyaluronidase activity (clone 3D35M; or Genl 3D35M). A master cell bank (MCB) of the 3D35M cells was then prepared.

B. Generation of a Second Generation Cell Line Expressing Soluble rHuPH20 The Genl 3D35M cell line described in Example 1A was adapted to higher methotrexate levels to produce generation 2 (Gen2) . 3D35M cells were seeded from established methotrexate-containing es into CD CHO medium containing 4 mM GlutaMAX-lTM and 1.0 uM 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 e plates containing medium with 2.0 uM 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-lTM and 2.0 uM rexate for 20 passages.

A master cell bank (MCB) of the 3E10B cell line was created and frozen and used for subsequent s.

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

The resultant 2B2 cells are dihydrofolate ase nt (dhfr—) DG44 CHO cells that express soluble recombinant human PH20 (rHuPH20). The soluble -209— PH20 is present in 2B2 cells at a copy number of approximately 206 copies/cell.

Southern biot analysis of Spe I-, Xba I- and BamH I/Hind III-digested genomic 2B2 cell DNA using a rHuPH20-specific probe revealed the following restriction digest : 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 ed 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 I/I—Iind III. Sequence is of the mRNA transcript indicated that the d 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 ed 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 2 Production and Purification of rHuPH20 A. Production of Gen2 soluble rHuPH20 in 300 L Bioreactor Cell Culture A vial of HZZ4-2B2 cells (Example 13) was thawed and expanded from shaker flasks through 36L spinner flasks in CD-CHO media (Invitrogen, Carlsbad, CA) supplemented with 20 uM methotrexate and GlutaMAX—lTM (Invitrogen).

Briefly, 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 y reached greater than 1.5 x 106 cells/mL, the culture was ed 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 r than 1.5 x 106 cells/mL, the culture was ed 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 ceil density reached r than 1.5 x 106 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 RECTIFIED SHEET (RULE 91) ISA/EP WO 63155 culture was expanded into a 36 L spinner flask in 32 L culture volume and incubated at 37°C, 7% C02.

A 400 L reactor was sterilized and 230 mL of CD-CHO media was added.

Before use, the r was checked for contamination. Approximately 30 L cells were transferred from the 36L r 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 set point, 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 (4>< CD-CHO + 33 g/L Glucose + 160 mL/L Glutamax-lTM + 83 mL/L Yeastolate + 33 mg/L rHuInsulin) was added. At 168 hours (day 7), 10.8 L of Feed #2 (2>< CD-CHO + 33 g/L e + 80 mL/L Glutamax-lTM + 167 mL/L Yeastolate + 0.92 g/L Sodium Butyrate) was added, and culture temperature was changed to 365°C. At 216 hours (day 9), 10.8 L ofFeed #3 (1>< CD-CHO + 50 g/L e + 50 mL/L GlutamaX-lTM + 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>< CD-CHO + 33 g/L Glucose + 33 mL/L GlutamaX-lTM + 250 mL/L Yeastolate + 0.92 g/L Sodium Butyrate) was added, and culture temperature was changed to 355° C. The on 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 viva, ission Electron Microscopy (TEM) and enzyme activity.

The e was pumped by a altic pump through four Millistak filtration system modules (Millipore) in parallel, each ning a layer of diatomaceous earth graded to 4-8 um and a layer of diatomaceous earth graded to 1.4-1.1 um, followed by a cellulose membrane, then through a second single Millistak filtration system (Millipore) containing a layer of aceous earth graded to 0.4-0.11 um and a layer of diatomaceous earth graded to <0.1 um, followed by a cellulose membrane, and then through a 0.22 um final filter into a sterile single use e 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 10>< with a tangential flow filtration (TFF) apparatus using four Sartoslice TFF 30 kDa molecular weight cut-off (MWCO) polyether sulfone (PES) filters rius) followed by a 10X buffer exchange with 10 mM Tris, 20 mM Na2804, pH 7.5 into a 0.22 pm final filter into a 50 L sterile storage, bag.

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% Tn'ton X-100, 0.3% TNBP for 1 hour in a 36 L glass reaction vessel immediately prior to purification on the Q .

B. Purification of Gen2 soluble rHuPHZO 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, tivity and endotoxin (LAL) assay. The column was equilibrated with 5 column volumes of 10 mM Tris, 20 mM NaZSO4, pH 7.5. Following viral inactivation, the concentrated, diafiltered harvest (Example 2A) was loaded onto the Q column at a flow rate of 100 cm/lnz The column was washed with 5 column volumes of 10 mM Tris, 20 1nM NaZSO4, pH 7.5 and 10 mM Hepes, 50 mM NaCl, pH 7.0. The protein was eluted with 10 mM Hepes, 400 mM NaCl, pH 7.0 into a 0.22 pm final filter into sterile bag. The eluate sample was tested for bioburden, n concentration and hyaluronidase activity. A230 ance readings were taken at the beginning and end of the ge.

Phenyl-Sepharose (Pharmacia) hydrophobic interaction chromatography was next performed. A Phenyl-Sepharose (PS) column (19-21 L resin, H=29 cm, D= 30 cm) was prepared. The wash was collected and d for pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 column volumes of 5 mM potassium ate, 0.5 M ammonium sulfate, 0.1 mM CaClZ, pH 7.0. The protein eluate from the Q sepharose column was supplemented with 2M ammonium sulfate, 1 M potassium phosphate and l M CaClz stock solutions to yield final trations of 5 mM, 0.5 M and 0.1 mM, respectively. The protein was loaded RECTIFIED SHEET (RULE 91) ISA/EP ~212- 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 CaClZ 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 pm final filter into a sterile bag. The flow through was sampled for den, protein concentration and enzyme activity.

An aminophenyl boronate column (ProMeclics) 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 ’10 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 um sulfate, pH 7.0. The column was washed with 20 mM bicine, 0.5 M um 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 NaCl, pH 6.9 and passed through a sterile filter into a e bag. The eluted sample was tested for bioburden, protein concentration and enzyme activity.

The hydroxyapatite (HAP) column (Biorad) was prepared. The wash was collected and tested for pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 mM potassium phosphate, 100 mM NaCl, 0.1 mM CaClz, pH 7.0. The aminophenyl te purified protein was supplemented to final concentrations of 5 mM potassium phosphate and 0.1 mM CaClz 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 NaCl, 0.1‘rnM CaCiz. The column was next washed with 10 mM potassium phosphate, pH 7, 100 mM NaCl, 0.1 mM CaClz. The protein was eluted with 70 mM potassium phosphate, pH 7.0 and passed through a 0.22pm sterile filter into a sterile bag. The eluted sample was tested for den, protein concentration and enzyme activity.

The HAP d protein was then passed through a Viral removal filter. The sterilized t filter rius) was first prepared by washing with 2 L of 70 mM potassium phosphate, pH 7.0. Before use, the d buffer was sampled for pH and conductivity. The HAP purified protein was pumped via a peristaltic pump through RECTIFIED SHEET (RULE 91) ISA/EP -2l3- the 20 nM Viral removal filter. The filtered n in 70 mM potassium ate, pH 7.0 was passed through a 0.22 um 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.

Example 3 Determination of Hyaluronidase Activity of Soluble rHuPH20 Hyaluronidase ty of soluble rHuPH20 in samples such as cell cultures, plasma, purification fractions and purified ons was determined using either a imetric assay, which is based on the formation of an insoluble precipitate when hyaluronic acid binds with serum albumin, or a biotinylated-hyaluronic acid substrate assay, which es the amount of enzymatically active rHuPH20 or PEGPH20 by the digestion of biotinylated hyaluronic acid (b-HA) substrate non-covalently bound to plastic well iter plates.

A. Microturbidity Assay Hyaluronidase activity of soluble rHuPH20 is measured by incubating soluble rHuPH20 with sodium hyaluronate ronic 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 ity resulting from enzyme activity on the sodium hyaluronate substrate is a measure of the soluble rHuPH20 hyaluronidase activity. The method is med using a calibration curve generated with dilutions of a soluble rHuPH20 assay g reference standard, and sample actiVity measurements are made relative to this calibration curve.

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 NaCl, 50 mM PIPES, pH 5.5) and 25.0 mL of sterile water for injection (SWFI), and diluting 0.2 mL of 25 % Buminate solution into the mixture and ing for 30 seconds. This was performed within 2 hours of use and stored on ice until needed. The samples were diluted to an estimated l-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 less than 20 uL. —214— The minimum sample volumes needed to perform the assay were as follows: In- process Samples, FPLC Fractions: 80 uL; Tissue Culture Supematants: 1 mL; Concentrated Material: 80 uL; Purified or Final Step Material: 80 uL. The dilutions were made in triplicate in a Low n Binding 96-well plate, and 30 uL of each on was transferred to Optilux black/clear bottom plates (BD BioSciences).

Dilutions ofknown soluble rHuPH20 with a concentration of 2.5 U/mL were prepared in Enzyme t 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, l.0 U/mL, l.5 U/mL, 2.0 U/mL, and 2.5 U/mL. nt blank” wells that contained 60 uL of Enzyme Diluent on were included in the plate as a negative control. The plate was then covered and warmed on a heat block for 5 s at 37 oC. The cover was removed and the plate was shaken for 10 seconds. After shaking, the plate was returned to the heat block and the MULTIDROP 384 Liquid Handling Device was primed with the warm 0.25 mg/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 CC for 2-4 hours, or until completely dissolved). The reaction plate was erred to the MULTIDROP 384 and the on was initiated by pressing the start key to dispense 30 uL 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 0C for 10 minutes.

The MULTIDROP 384 was prepared to stop the reaction by priming the machine with Serum Working Solution and changing the volume setting to 240 uL. (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 uL of serum Working ons was dispensed into the wells. The plate was removed and shaken on a plate reader for 10 seconds. After a r 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.

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

B. Biotinylated Hyaluronan Assay The biotinylated-hyaluronic acid assay es the amount of enzymatically active rHuPH20 or PEGPH20 in biological s by the digestion of a large lar weight (~ 1.2 megadaltons) biotinylated hyaluronic acid (b-HA) substrate non-covalently bound to plastic multi-well microtiter . The rHuPH20 or PEGPH20 in standards and samples are allowed to incubate in a plate coated with b- HA at 37 0C. After a series of washes, remaining uncleaved/bound b-HA is treated with Streptavidin Horseradish Peroxidase ate (SA-HRP). Reaction between lized SA-HRP and the chromogenic substrate, 3,3’,5,5’-tetramethylbenzidine (TMB), produces a blue colored solution. After stopping the reaction with acid, formation of the soluble yellow reaction product is determined by reading the absorbance at 450 nm using a microtiter plate spectrophotometer. The decrease in absorbance at 450 nm resulting from enzyme activity on the biotinylated hyaluronic acid (b-HA) substrate is a measure of the e rHuPH20 hyaluronidase activity.

The method is med using a calibration curve generated with dilutions of a soluble rHuPH20 or PEGPH20 reference standard, and sample activity measurements are made relative to this calibration curve.

Dilutions of the sample and calibrator were prepared in Assay Diluent. The Assay Diluent was prepared by adding 1 % v/v pooled plasma (from the appropriate s) to 0.1 % (w/v) BSA in HEPES, pH 7.4. This was prepared daily and stored at 2-8 C’C. Depending upon the species type as well as the anticipated hyaluronidase level, single or multiple dilutions were ed to ensure at least one sample dilution would fall within the range of the calibration curve. To guide the selection of test sample dilution(s), information known about the dose of onidase administered, the route of stration, approximate plasma volume of the species and the time point were used to estimate the hyaluronidase activity levels. Each sample dilution was mixed as it was prepared by brief pulse-vortexing and pipet tips were changed in between each on. In general, the dilutions began with an initial 50 or lOO-fold dilution followed by additional serial dilutions. A seven-point calibration curve of rHuPH20 or PEGPH20 (depending upon the treatment administered) was prepared -2l6- ranging in concentration from 0.004 to 3.0 U/mL for rHuPH20 and from 0.037 to 27 U/mL for PEGPH20. One-hundred microliters (100 uL) of each test sample dilution and calibration curve point was applied to triplicate wells of a 96-well microtiter plate (Immulon 4HBX, Thermo) that had been previously coated with 100 uL per well of b-HA at 0.1 mg/mL and blocked with 250 uL of l .0 % (w/v) Bovine Serum n in PBS. s) were covered with an adhesive plate seal and incubated at 37 0C for approximately 90 minutes. At the end of the incubation , the adhesive seal was d from the plate, samples were aspirated and the plate washed five (5) times with 300 uL per well Wash Buffer (10 mM Phosphate , 2.7 mM Potassium Chloride, 137 mM Sodium Chloride, pH 7.4, with 0.05 % (v/v) Tween 20, PBST) using an automated plate washer (BioTek ELx405 Select CW, Program ‘4HBXl ’).

One hundred microliters of StreptaVidin-HRP ate Working Solution [Streptavidin-HRP conjugate (1:5,000 V/V) in 20 mM Tris-HCl, 137 mM Sodium Chloride, 0.025 % (v/v) Tween 20, 0.1 % (w/v) Bovine Serum Albumin] was added per well. The plate was sealed and allowed to incubate at ambient temperature for approximately 60 minutes without shaking and protected from light. At the end of the incubation period, the adhesive seal was d from the plate, samples were aspirated and the plate washed five (5) times with 300 uL per well Wash Buffer as bed above. TMB solution (at t temperature) was added to each well and allowed to incubate protected from light for approximately five (5) minutes at room temperature. TMB Stop Solution (KPL, Catalog # 5006) was then added as 100 uL per well. The absorbance of each well at 450 nm was determined using a microtiter plate spectrophotometer. The response of the Calibration Curve on each plate was modeled using a 4-parameter ic curve fit. The hyaluronidase actiVity of each unknown was calculated by interpolation from the calibration curve, corrected for sample dilution factor, and reported in U/mL.

Example 4 Preparation of PEGylated rHuPH20 In this example, rHuPH20 was PEGylated by reaction of the enzyme with linear N-hydroxysuccinimidyl ester of methoxy poly(ethylene glycol) butanoic acid (mPEG-SBA-30K). -2l7- A. Preparation of BA-30K In order to generate PEGPH20, rHuPH20 (which is approximately 60 KDa in size) was covalently conjugated to a linear N-hydroxysuccinimidyl ester of methoxy poly(ethylene glycol) butanoic acid (mPEG-SBA-3OK), having an approximate molecular weight of 30 kDa. The structure ofmPEG-SBA is shown below, where n z 681.

H300+CH20H20}CH20H20Hch—N mPEG-SBA Methods used to prepare the mPEG-SBA-3OK that was used to PEGylate rHuPH20 are described, for example, in US. Patent No. 5,672,662. Briefly, the mPEG-SBA-3OK is made according to the ing procedure: A solution of ethyl te (2 equivalents) dissolved in dioxane is added drop by drop to sodium hydride (2 equivalents) and toluene under a nitrogen atmosphere. mPEG methane sulfonate (1 lent, MW 30 kDa, Shearwater) is dissolved in toluene and added to the above mixture. The resulting mixture is refluxed for approximately 18 hours. The reaction mixture is concentrated to half its al volume, extracted with 10% aqueous NaCl solution, extracted with l % aqueous hydrochloric acid, and the aqueous extracts are combined. The collected s layers are extracted with dichloromethane (3x) and the organic layer is dried with magnesium sulfate, filtered and evaporated to dryness. The resulting residue is dissolved in IN sodium hydroxide containing sodium chloride and the mixture is stirred for 1 hour. The pH of the e is adjusted to approximately 3 by addition of 6N hydrochloric acid. The mixture is extracted with dichloromethane (2x).

The organic layer is dried over magnesium sulfate, filtered, concentrated, and poured into cold diethyl ether. The precipitate is collected by ion and dried under vacuum. The resulting compound is ved in dioxane and refluxed for 8 hours and then concentrated to dryness. The resulting residue is ved in water and extracted with romethane (2x), dried over magnesium sulfate, and the 2012/061743 solution is concentrated by rotary evaporation and then poured into cold diethyl ether.

The itate is collected by filtration and dried under vacuum. The resulting compound (1 equivalent) is dissolved in romethane and N-hydroxysuccinimide (2.1 equivalents) is added. The solution is cooled to 0 oC and a solution of dicyclohexylcarbodiimide (2.1 equivalents) in dichloromethane is added dropwise.

The solution is stirred at room temperature for approximately 18 hours. The reaction mixture is filtered, concentrated and precipitated in diethyl ether. The precipitate is collected by filtration and dried under vacuum to afford the powder mPEG-SBA-30K which is then frozen at S -15 CC.

B. Conjugation of mPEG-SBA-30K to rHuPH20 To make the PEGPH20, mPEG-SBA-3OK was coupled to the amino s) ofrHuPH20 by covalent conjugation, providing stable amide bonds between rHuPH20 and mPEG, as shown below, where n z 681.

H20H20>70H20H20H200—N|| ‘1’ HZN—rHuPHZO mPEG-SBA j H3OO+CH20H20>VCHZCHZCHzc—N—H H PEGylated rHuPH20 Prior to conjugation, the rHuPH20 purified bulk n made in Example 2B was concentrated to 10 mg/mL, using a 10 kDa polyethersulfone (PES) tangential flow ion (TFF) cassettes (Sartorius) with a 0.2 m2 filtration area, and buffer exchanged against 70 mM Potassium Phosphate at pH 7.2. The concentrated protein was then stored at 2-8 0C until use.

To conjugate the rHuPH20, the mPEG-SBA-3OK (Nektar) was thawed at room ature in the dark for not longer than 2 hours. Depending on the batch size, a sterile 3” stir bar was placed into a 1 or 3 liter Erlenmeyer flask and buffer exchanged rHuPH20 protein was added. Five grams of dry mPEG-SBA-30K powder per gram ofrHuPH20 (10:1 molar ratio ofmPEG-SBA-3OK: rHuPH20) was added to ~219- the flask under a vaccuum hood and the mixture was mixed for 10 minutes or until the mPEG-SBA-BOK was complete dissolved. The stir rate was set such that vortexing occurred without foaming.

The solution was then d under a class 100 hood by pumping the on, via peristaltic pump, h a 0.22 pm polystyrene, cellulose e filter capsule (Corning 50 ml. Tuhetop filter) into a new 1 or 3 liter Erlenmeyer flask containing a sterile 3” stir bar. The volume of the PEGPHZO reaction mixture was determined, by mass (1 g/mL density) and the 0.22 pm filter used for filtration was examined in a post—use integrity test.

The mixture was then placed on a stir plate at 2-8 °C and mixed for 20 d: 1 hours in the dark. The stir rate was again set such that vortexing occurred without g. The entire eyer container was wrapped in foil to protect the solution from light. After mixing, the reaction was quenched by adding 1M glycine to a final concentration of 25 mM. s were removed from the container to test pH and conductivity. The pH and conductivity were then adjusted by adding to a solution of mM Tris Base (5.65 UL) and 5 mM Tris, 10 mMNaCl, pH 8.0 (13.35 L/L) to proceed with Q Sepharose purification.

A QFF Sepharose (GE Healthcare) ion exchange column (Height = 21 .5-24.0 cm, Diameter = 20 cm) was prepared by equilibration with 5 column volumes (36 L) of 5 mM Tris, 10 mM NaCl, pH 8.0. The conjugated product was loaded onto the QFF column at a flow rate of 95 cm/hr. The column was then washed with 11 L of equilibration buffer (5 mM Tris, 10 mM N201, pl—l an) at a flnw rate of 9.5 cm/hr followed by a wash with 25 L of equilibration buffer at a flow rate of268 cm/hr. The protein product was then eluted with SmM Tris, 130 mM NaCl, pH 8.0 at a flow rate of 268 cm/hr. The resulting purified PEGPl-IZO was trated to 3.5 mg/mL, using a 30 kDa polyethersulfone (PES) tangential flow filtration (TFF) cassettes (Sartorius) with a 0.2 m2 filtration area, and buffer ged against 10 mM Histidine, 130 mM NaCI at pH 6.5. The resulting material was tested for enzyme activity as described in Example 3. The ted rHuPHZO material at a concentration of 3.5 mg/mL (final enzyme activity 140,000 U/rnL) was filled, in 3 mL volumes, into 5 1nL glass vials with a siliconized bromobutyl rubber stopper and aluminum flip-off seal, and frozen (frozen overnight in a ~20 °C r, then put in a —80 °C freezer for longer storage). ' RECTIFIED SHEET (RULE 91) ISA/EP The PEGylated rHuHP20 contained approximately 4.5 moles of PEG per mole of rHuPH20.

B. Analysis of PEGylated rHuPH20 The PEGylated rHuPH20 (PEGPH20) material was assayed by gel ophoresis. Three batches of PEGPH20, made as described in Example 4A above, revealed an identical pattern of multiple bands, representing unreacted PEG and multiple species of PEGPH20 conjugates, which migrated at different distances.

Based on comparison with migration of a molecular weight marker, the bands representing the species ranged from approximately 90 KDa to 300 KDa, with three dark bands migrating above the 240 KDa marker. These data indicated that the PEGPH20, generated by covalent conjugation of mPEG-SBA-30K, contained a heterogeneous mixture of PEGPH20species, likely including mono-, di- and tri- PEGylated proteins. The lack of a visible band at 60 KDa suggested that all the n had reacted with the PEG, and that no detectable native rHuPH20 was present in the mixture.

Example 5 Competency of Tumor Cells to Form llular Matrix and Relationship to Tumor Cell Hyaluronan (HA) Content, Levels of Hyaluronan Synthase (HAS), and Hyaluronidase (Hyal) Expression A. Comparison of Tumor Cell HA Content, Expression of HAS 1, 2, 3 and Hyal 1 and 2, and Pericellular Matrix ion In this Example, the amount of endogenous HA synthesis enzymes, hyaluronan synthase (HAS) 1, 2, and 3, onidase (Hyal) l and (Hyal) 2 and the amount of hyaluronan (HA) accumulation in tumor cells was compared to show that each correlated to pericellular matrix formation by the tumor cells. 1. Cell Lines Used in the Study Ten cell lines from tumors of various tissue origin (e.g., prostate, breast, ovarian, pancreatic, and lung) and species origin (e.g., human, mouse and rat) were examined in the study. The following cell lines were obtained from the American Type e tion (ATCC): 4T1 mouse breast tumor (ATCC CRL—2539), PC-3 human prostate adenocarcinoma (ATCC 35), BxPC-3 human pancreatic arcinoma (ATCC CRL-l687), MDA MB 231 human breast adenocarcinoma (ATCC HTB-26), Mat-Lylu rat malignant te carcinoma (ATCC JHU-92), AsPc- 1 human pancreatic adenocarcinoma (ATCC CRL-1682), DU-145 human prostate carcinoma (ATCC HTB-81), and MIA PaCa 2 human pancreatic carcinoma (ATCC CRL-1420). The ATCC cell lines were grown in recommended culture medium containing 10% PBS at 37 0C in a humidified incubator supplied with 5% C02/95% air. MDA-MBLuc (Cat. No. D3H2LN) cells, which s the North American Firefly Luciferase gene, were sed from Caliper Life Sciences Inc. and grown in RPMI containing 10% PBS.

The DU-145/HAS2 and MDA-MBLuc/HAS2 cell lines were generated by transduction of the DU-145 and MDA-MBLuc cell lines with a irus encoding hyaluronan synthase 2 (HAS2) (SEQ ID NO: 195). To generate the HAS2 retrovirus, N—terminal His6-tagged hHAS2 cDNA (SEQ ID NO: 196) was inserted into the Aer and NotI sites of the vector pLXRN (SEQ ID NO: 197; Clontech, Cat.

No. ), which includes the neomycin resistance gene, to create pLXRN-hHAS2 (SEQ ID NO: 201). The pLXRN-hHAS2 His plasmid was then co-transfected with pVSV-G envelope vector (SEQ ID NO: 198 Clontech, part of Cat. No. 631530) into GP-293 cells using Lipofectamine 2000 reagent (Life Technologies). A DU-145 Mock cell line also was generated by co-transfection of the empty pLXRN d and pVSV-G envelope vector.

The virus titer was determined by quantitative PCR method (Retro-XTM qRT- PCR Titration Kit; ch, Catalog No. 631453) using the following primers (Clontech Catalog No. #K1060-E): pLXSN 5’ primer (1398—1420): 5’-CCCTTGAACCTCCTCGTTCGACC—3’ (SEQ ID NO: 199); pLXSN 3’ primer (1537—1515): 5 ’-GAGCCTGGGGACTTTCCACACCC— 3’(SEQ ID NO: 200).

To ish HAS2 sion cell lines, 70% confluent cancer cells, DU-145 or MDA MB 231 Luc, were incubated with a 60:1 to 6:1 ratio of retrovirus in DMEM (Mediatech) containing 10% FBS for 72 hours. The cultures were maintained in selective medium containing 200 ug/mL of G418. Stable xpressing cancer cells were generated after 2 weeks of G418 conditional medium selection. 2. Quantification of Hyaluronic Acid A hyaluronan binding protein (HABP)-based assay was employed to determine the amount uronan produced by the tumor cells. HABP-based assays are preferable to chemical methods for measuring HA as a tumor microenvironment (TME) biomarker because the HABP preferentially detects HA ed of at least 15 (n-acetyl glucose—glucuronic acid) disaccharides, which is competent to bind hyaladherins (HA binding proteins) (see, e.g., dt S, et a1. (2011) Glycobz‘ology 21: 175-183).

Tumor cells were seeded at l x 106 cells in 75 cm2 flasks and incubated for 24 hours. Tissue culture supematants were ted for quantitation of HA using an enzyme—linked HABP sandwich assay (R&D Systems, Catalog No. DY3614), which uses recombinant human aggrecan as a HA capture and detection reagent (recombinant human aggrecan Gl-IGD-GZ domains, ValZO-Gly676 ofAccession No.

NP_037359 (SEQ ID NO: 202) with a C-terminal 10-HIS tag, R&D Systems, Catalog No. 1220-PG). The assay for HA detection was performed according to the manufacturer's instructions. Briefly, assay plates were coated with inant human an, and s (lie, tissue culture supematants) containing HA were added to the plate (three independent replicates of each cell line were tested) The plates were washed and the bound HA was detected using biotinylated recombinant human aggrecan. After removing the unbound probe, streptavidin conjugated to horseradish peroxidase (HRP) was added as a ary detection reagent. After g the plate, the bound HRP was detected by incubation with the 1:1 HZOQ/Tetramethylbenzidine ate solution (R&D Systems) and quantitated by optical density detection at 450 nm using a SpectraMax M3 Multi-Mode Microplate Reader (Molecular Devices , CA). Concentration of HA in the culture media for each tumor cell type was expressed as mean HA concentration (ng/mL) in culture media (Table 5). 3. Quantification of HASl, HASZ, HAS3, HYALI and HYAL2 mRNA expression RNA was extracted from cell pellets using an RNeasy® Mini Kit (Qiagen GmbH) according to the manufacturer’s instructions. The extracted RNA was then quantified using a NanoDrop ophotometer (NanoDrop Technologies, RECTIFIED SHEET (RULE 91) ISA/EP Wilmington, DE). Quantitative real-time PCR (qRT-PCR) using gene-specif1c primers was used to quantitate the relative mRNA levels of each hyaluronan synthase and hyaluronidase. qRT-PCR primers were purchased from Bio Applied logies Joint, Inc, (San Diego, CA). The DNA sequences for the primers used in the individual PCR reactions were as follows: Table 4: Primer sequences used for qRT-PCR analysis of HAS and HYAL gene ex n ression '—TACAACCAGAAGTTCCTGGG-3' 5'-CTGGAGGTGTACTTGGTAGC-3' HAS1 SEQ ID NO: 395 SEQ ID NO: 396 '-GTATCAGTTTGGTTTACAATC-3' 5'-GCACCATGTCATATTGTTGTC-3' HA82 SEQ ID NO: 397 SEQ ID NO: 398 SEQ ID NO: 399 SEQ ID NO: 400 SEQ ID NO: 401 SEQ ID NO: 402 SEQ ID NO: 403 SEQ ID NO: 404 H(SEQIDNO:405) (SEQ ID NO: 406) For the PCR reactions, samples were mixed with iQ SYBR Green master mix (Bio-Rad) and the designated primer pairs for each gene. The PCR ons were performed on a Bio-Rad Chromo 4 qPCR device. First strand synthesis was performed under the following conditions: 42 CC for 2 minutes for the DNA elimination reaction, 42 CC for 15 minutes for reverse-transcription, and 3 minutes at 95 °C for inactivation of reverse-transcriptase. For cation, 3 minutes initial denaturation at 95 CC, 45 cycles of 15 s denaturation and 1 minute annealing extension at 58 0C were used. The gene expression CT value from each sample was calculated by izing with the internal housekeeping gene GAPDH and relative values were plotted. Table 5 lists the CT values for each tumor cell type for each gene assayed. 4. Assay For Pericellular Matrix Formation Monolayer cultures of the ten cell lines were grown and tested for aggrecan- facilitated pericellular matrix formation. To visualize an-mediated HA pericellular matrices in vitro, particle exclusion assays were used as previously described in Thompson CB, et al. (2010) M01 Cancer Ther 9: 3052-3064, with some —224— modifications. Briefly, cells were seeded at l x 105 cells per well in a six-well plate for 24 hours, and then treated with culture cell media alone or media containing 1000 U/mL rHuPH20 at 37 CC for 1 hour. Pre-treatment with rHuPH20 inhibits formation of the pericellular matrix; thus, it was employed as a negative control for pericellular matrix formation for each cell type. The cells were then incubated with 0.5 mg/mL of bovine aggrecan (Sigma-Aldrich) at 37 0C for 1 hour. Subsequently, media were removed and replaced with 108/mL suspension of 2% glutaraldehyde-fixed mouse red blood cells (RBCs), isolated from Balb/c mouse (Taconic, Hudson, NY), in PBS, pH 7.4. The particles were allowed to settle for 15 minutes. The cultures were then imaged with a phase-contrast cope coupled with a camera scanner and imaging program (Diagnostic Instruments). Particle exclusion area and cell area were measured using the SPOT Advance program (Diagnostic Instruments, Inc., Sterling Heights, MI). Pericellular matrix area was calculated as matrix area minus cell area, and expressed as um2 (Table 5). 5. Results: Comparison of Tumor Cell HA Content, and HAS and HYAL Expression t0 Pericellular Matrix Formation The concentration ofHA in conditioned media as determined by the HABP- based ion assay was found to correlate with the area of aggrecan-mediated pericellular matrix formed by the tumor cells in monolayer culture (Table 5, P < 0.0029). Further, cell lines that were engineered to s hyaluronan se 2 (HAS2), DU-145/HAS2 and MDA-MB-23 1/HAS2, displayed increased HA production and enhanced pericellular matrix formation in vitro compared to the respective parental cell lines. In contrast, no correlation was found between pericellular matrix ion and relative levels of HAS 1, 2, or 3 or Hyal 1 or 2 mRNA expression. These s indicate that the direct measurement of tumor cell- associated HA specifically provides a predictor for pericellular matrix formation.

Table 5. Quantitation of HA tion, pericellular matrix formation, HAS and Hyal ex n ression in tumor cell lines . 3 Hyaluronidase HAS lsoform mRNA Tumor Cell Line PM1 HA in CM2 isoform mRNA“ HASl HASZ HAS3 H all H all 4T1 0 473.83 NE NE NE NE NE -23l/HAS2 1088.55 372.20 2.48 19.90 0.09 0.14 0.53 PC3 0 294.45 1.41 0.34 6.32 0.14 1.19 DU-l45/HAS2 981.00 7417.00 1.08 7.81 0.65 0.34 1.04 BxPC3 967.20 467.12 1.00 1.00 1.00 1.00 1.00 2012/061743 MDA-MB-231 WT 770.45 256.90 3.39 0.54 0.05 0.13 0.64 MatLylu 760.55 265.91 NE NE NE NE NE AsPC-l 524.20 66.47 1.87 1.65 1.28 0.81 1.91 DU~145 WT 252.10 41.79 1.01 0.03 1.51 0.17 0.70 a-2 129.40 0.00 0.46 0.00 0.04 0.28 - 0.72 Correlation Coefficient - 0.0029 0.23 0.34 0.71 0.66 0.36 S earman P value) NE: not evaluated ’Pericellular matrix area (urns) assessed via particle exclusion assay.

Mean HA concentration (ng/mL) in culture media (11 = 3, independent es). 3' 4 Hyaluronan synthase (HAS) and hyaluronidase (Hyal) expression as determined by real—time RT- PCR. Ct values were normalized by GAPDH mRNA and the fold differences are relative to BXPC3 expression.

Example 6 Measurement of Tumor Cell Hyaluronan Concentration and Relationship to Anti—tumor Activity of PEGPHZO In this example, the concentration ofhyaluronan in different tumor cell lines was assessed by histochemical is and compared to the ability of PEGPHZO to inhibit tumor growth graft tumors generated from the tumor cell lines and tumor colony growth of a HA rich tumor cell line.

A. Comparison of HA Content with PEGPH20 Efficacy in Various Xenograft Tumor Models 1. Xenograft Tumor Models Fourteen tumor cell line-derived xenograft tumors were generated from the following tumor cell lines: DU—l45 human prostate carcinoma (ATCC HTB-81 mock transfected with empty pLXRN plasmid, see Example 5.A.l ), DU-145 HAS2 (see Example 5.A.1), MDA MB 231 human breast adenocarcinoma (ATCC HTB-26), MDA-MB-231\Luc (D3H2LN, Caliper Life Sciences), MDA MB 231 Luc/HASZ (see Example 5.A.1), SKOV3 human ovarian carcinoma (ATCC HTB-77), AsPc-l human pancreatic adenocarcinoma (ATCC CRL-1682), MIA PaCa 2 human atic carcinoma (ATCC CRL-l420), 4T1 mouse breast tumor (ATCC CRL~253 9), BxPC-3 human pancreatic adenocarcinoma (ATCC CRL—1687), Mat—Lylu rat malignant prostate oma (ATCC JI—IU-92), and PC-3 human prostate adenocarcinoma (ATCC 35). Tumor cell lines were maintained as described in Example 5.

LUM 697 and LUM 330 human tumor explants were ed from Crown Bioscience, Beijing China.

RECTIFIED SHEET (RULE 91) ISA/EP Six to eight week old nu/nu (Ncr) athymic nude mice (Taconic) or Balb/c (Harlan) or Balb/c nude mice (Shanghai Laboratory Animal Center, CAS (SLACCAS); see Example 7) were housed in micro-isolator cages, in an environment-controlled room on a r light/12-hour dark cycle, and received sterile food and water ad libitum. All animal studies were conducted in ance with ed IACUC protocols.

For generation of tumors, mice were inoculated with tumor cells peritibially (intramuscular injection adjacent to the right tibia periosteum), subcutaneously (s.c., right hind leg), or in mammary fat pad according to Table 6 below.

Table 6. Animal Tumor Models Tumor Type Animal Models Mice Inoculation Cell Number (Source) Source Site Dul45 Mock Prostate Ca. (H) \Icr nu/nu; female Taconic peritibially 5 x 106/0.05mL MDA MB 231 Breast Ca.(H) \Icr nu/nu; female Taconic peritibially lxlxlmm3 tissue cube SKOV3 n Ca. (H) \Icr nu/nu; female Taconic s.c. 1x106/0.lmL MDA MB 23 lLuc Breast Ca.(H) \Icr nu/nu; female Taconic bially 1 x 106/0.lmL MDA MB 231 6 peritibially. . .

Breast Ca. (H) \Icr nu/nu, female. Taconic l x 10 /0.lmL Luc/HASZ AsPc-l Pancrflalgic Ca. \Icr nu/nu; female Taconic s.c. 1x107/0.lmL MIA PaCa 2 Pancr(eIaIt)10 Ca. \Icr nu/nu; female Taconic s.c. 1x107/0.lmL mammary fat 5 4Tl Breast Ca. (M) BALB/c; female Harlan 5.0 X 10 cells/0.05 mL BXPC3 Pancrflalgic Ca. Ncr nu/nu; female Taconic s.c. 1x107/0.lmL MatLylu c permblally. . . 5 Prostate Ca. (R) Ncr nu/nu; male 2.0 x 10 cells/0.04 mL PC3 Prostate Ca. (H) Ncr nu/nu; male Taconic peritibially 1 x 106/0.05mL DUl45 HASZ Prostate Ca. (H) Ncr nu/nu; male Taconic peritibially 5 x 106/0.05mL LUM858 Lung Ca. (H) BALB/c nude SLACCAS s.c. 3x3x3mm3 cube LUM 697 Lung Ca. (H) BALB/c nude SLACCAS s.c. 3x3x3mm3 cube LUM 330 Lung Ca. (H) BALB/c nude SLACCAS s.c. m3 cube H: Human; M: Mouse; R: Rat For peritibial tumors, tumor volumes were determined using VisualSonics Vevo 770 high-resolution ultrasound. For subcutaneous and mammary fat pad tumors, tumor s were calculated by caliper measurement of the length (L) and width (W) of the solid tumor masses. Tumor volume (TV) was ated as: (L X W2)/2. s were selected for PEGPH20 treatment when tumor volumes reached ~400-500 mm3. Animals were then randomized into treatment and control groups (n 2 6 mice/group).

Treatment with 0 and analysis of tumor growth inhibition (TGI) were performed as described (Thompson et al.). Mice were treated with e (10 mM Histidine, pH 6.5, 130 mM NaCl) or PEGPH20 for 2—3 weeks according to the schedule shown in Table 7. Tumor volumes were measured twice . When tumor size exceeded 2,000 mm3 animals were removed from the study and humanely euthanized.

Table 7: PEGPH20 Treatment and Tumor Growth tion PEGPH20 PEGPH20 Dosing TGI Ani dose amount per Frequency (day X) mal (mg/kg) 0.1 mL dose “um Du145 Mock ~10,000 U twice weekly 6 0.7% MDA MB 231 . ~3,000 U twice weekl 6 0% d17 U1 SKov3 ~3,500 U 0% cm U1 MDA MB . ~3,ooou 23% (d14) 10 231Luc MDA MB 231 . ~3,ooou 43% (d14) 10 Luc/HASZ AsPc-1 . ~3,ooou 18% d11 MIA PaCa2 . ~3,ooou 24% d15 4T1 . ~3,ooou 61% d14 BXPC3 . ~3,000 U 45% d25 MatL Iu . ~3,000 U 34% d9 PC3 ~10,ooou 65% d18 DU145 HASZ ~10,ooou 50% d18 LUM858 (see . ~3,000 U twice weekly 5 16% (d14) 10 Ex.7 LUM 697 (see . ~3,000 U twice weekly 5 97% (d14) _\ O Ex.7 LUM 330 (see . ~3,000 U twice weekly 5 44% (d16) 10 Ex. 7 The TGI for each tumor model was calculated based on the volume from the study termination day as ted in Table 7. Percent Tumor Growth Inhibition (TGI) for each respective tumor model was calculated using the ing equation: % TGI = [1 — (Tn-T0) + (Cu-Co)] x 100% where “Tn” is the average tumor volume for the treatment group (animals receiving PEGylated rHuPH20) at day “11” after the final dose of PEGylated rHuPH20; “To” is the average tumor volume in that treatment group at day 0, before treatment; “C11” is the average tumor volume for the ponding control group at day 4; a). 11 and “C0” is the average tumor volume in the control group at day 0, before treatment. Statistical analysis of tumor volumes between the control and treatment groups was performed using a one-way ANOVA test with P value of P S 0.05 defined as statistically significant. 2. Histochemistry Staining of HA in Tumor Tissue and Semi- quantification of HA Content At the termination of the tumor grth inhibition study, each of the fourteen xenograft tumors ted were analyzed for HA content by histochemistry using biotinylated hyaluronan binding protein (B-HABP) as a probe for HA detection and digital quantification.

Tumor tissues were harvested, fixed in 10% neutral buffered formalin solution (NBF), embedded in paraffin, and cut into 5 um sections. For hemical analysis, the ns were deparaffinized and rehydrated. nous peroxidases were blocked with peroxo-block solution (Invitrogen, CA, USA) for 2 minutes. Non- specific staining was blocked using 2% BSA in 2% normal goat serum PBS for 1 hour at room temperature (RT) prior to incubation with 2.5 ug/ml biotinylated HA-binding protein (B-HABP, Catalog No. 400763, Seikagaku, Tokyo, Japan) for 1 hour at 37 CC.

To confirm specificity of ng, a subset of sections were pre-treated with rHuPH20 (1000 U/mL) in PIPES buffer (25 mM PIPES, 70 mM NaCl, 0.1% BSA, pH 5.5) at 37 CC for 1h before addition of B-HABP. After washing to remove the primary reagent, samples were incubated with streptavidin-horseradish peroxidase solution (BD Pharmingen, g No. ) for 30 minutes at RT and detected with 3, 3’- diaminobenzidine (DAB; Dako, Catalog No. K3467). Sections were then counterstained in Gill’s hematoxylin (Vector Labs, Catalog No. H-3401), dehydrated and mounted in Cytoseal 60 medium (American MasterTech).

An Aperio T2 ope (Aperio) was used to generate high-resolution images of tissue sections. Images were quantitatively analyzed with Aperio Spectrum software using a pixel count algorithm for brown color (HA) count. The tissue core in the sections with less than 10% of tumor cells or more than 50% of necrotic tissue was excluded for the tion. PC3 (HAI3) xenograft tumor s were used as a positive l. A ratio of strong positive (brown) stain area to the sum of total stained area was calculated and scored as +3, +2, +1, or 0 when the ratio was more than 25%, 10-25%, less than 10%, or 0, respectively.

WO 63155 Spearman’s rank correlation coefficient was used to evaluate the relationship between HA expression and response to PEGPH20 treatment. 3. Results: ison of Tumor HA content and Tumor Growth Inhibition Following PEGPHZO Treatment The results presented in Table 8 e the level of HA measured in tissue ns taken from the xenograft tumor and the percentage tumor growth inhibition (TGI) by PEGPHZO. The results are from tumors treated with at least 1 mg/kg PEGPH20. Doses higher than 1 mg/kg did not increase tumor growth inhibition. In the PC-3 (I-IA”) and BXPC3 (HAH) animal models, no significant increase in efficacy was observed at doses greater than 10 ug/kg and 100 pig/kg, respectively.

Despite the diversity oftumor cell types (human, murine and rat origin), there was a cant correlation (P < 0.001, Spearman’s r = 8, n=l4) between increasing B-HABP—mediated HA staining intensity and in viva antitumor activity of O (Table 8).

Table 8: Tumor HA staining intensity and corresponding PEGPHZO- mediated lrowth inhibition.

' Models Tumor Type HApositive TGI (%) Source Pixels % DU145 Mock Prostate Ca. (H) MDA MB 231 Breast Ca. (H) SKOV3 Ovarian Ca.(H) MDA MB c Breast Ca. (H) MDA MB 231/Luc/HAS2 Breast Ca. (H) AsPc—l Pancreatic Ca. (H) MIA PaCa 2 Pancreatic Ca. (H) LUM330 (see Ex.7) Lung Ca. (I-I) 4T1 Breast Ca. (M) BXPC3 Pancreatic Ca. (H) - MatLylu Prostate Ca. (R) PC3 Prostate Ca. (H) DU145 HASZ Prostate Ca. (H) LUM697 (see Ex.7) Lun Ca. H) H: Human; M: Mouse; R: Rat B. Comparison ofHA Content with PEGPH20 Efficacy in a Xenograft Tumor Model of HASZ Overexpression The effect of sing HA production in a tumor cell on sing the sensitivity of tumors to treatment with PEGPHZO was filrther examined in tumor xenografis that express exogenous hyaluronan synthase 2 (HAS2). As shown in RECTIFIED SHEET (RULE 91) ISA/EP Example 5, HA tion by the DU-145 tumor cell line could be increased by transduction of the cells with a gene encoding HASZ, which led to enhanced pericellular matrix formation in vitro. Additionally, the DU142-HASZ displayed increased HA staining and sed tumor inhibition by PEGPH20 in the xenograft models described above. In this Example, the efficacy of PEGPGZO treatment over time was compared in the DU-145 versus DU—I45-I—IASZ xenografis.

The mouse aft models were prepared as described above in Example 6A. Briefly, mice were inoculated with either 5/vector controls or DU- 145/HAS2 cells as indicated in Table 6. When. the tumors reached approximately 500 mm3 in size, the mice were divided into treatment groups (n = 8) and treated with vehicle alone or PEGPH20. For the PEGPH20 treatment, the mice were injected via tail vein at a dose of 4.5 mg/kg twice weekly for 3 weeks. Tumor volume was monitored by caliper measurement as described above. The xenograft tumors'were analyzed for HA content by histochemistry using biotinylated hyaluronan binding protein (B-HABP) as described above 24 hours after the last treatment with The verexpressing DU-145 te tumor aft grew more aggressively in nude mice than the parental cell line transfected with empty vector (DU-145 Mock), similar to previous reports (Table 7) (Thompson et al. (2010)).

PEGPH20 inhibited tumor growth in DU-145~HAS2 tumors (TGI=50%, P < 0.001, n=8), but not in DU—145 vector control tumors .7%, P > 0.05, n=8). In addition, histochemistry staining with B-HABP of PEGPH20-treated tumors showed HA removal in tumor samples ed to control tumors. These data suggest that accumulation ofHA in the ECM facilitates tumor development, and that ed tumor-associated HA accumulation is associated with the anti-tumor activity of PEGPH20.

C. Dose Related s of PEGPH20 Treatment in Hyaluronan-Rich Tumors In this experiment, the dose dependent effect of PEGPH20 on tumor growth inhibition ofHA-rich tumors was examined. Mouse xenograft models were prepared as described above in Example 6A. Briefly, mice were inoculated with either BxPC-3 human pancreatic adenocarcinoma (ATCC CRL-1687) or PC~3 human prostate adenocarcinoma (ATCC CRL—1435) cells according to Table 6. When the tumors RECTIFIED SHEET (RULE 91) ISA/EP reached approximately 500 mm3 in size, the mice were divided into ent groups (n = 10) and treated with vehicle alone or PEGPH20. For the PEGPH20 treatment, the mice were systemically injected tail vein at a dose of 0.01, 0.1, 1, 4.5 and 15 mg/kg (350, 3,500, 35,000, 157,500 and 500,000 U/kg, respectively) twice weekly for 2 weeks. Tumor volume was monitored by caliper measurement as described above.

It was observed that the maximum effective dose of PEGPH20 is below 1mg/kg. Significant tumor inhibition was observed for all doses of PEGPH20 in the PC-3 xenograft model (P < 0.001 for 0.1, l, 4.5, 15 mg/kg doses; P < 0.01 for the 0.01 mg/kg dose ed to vehicle) and for all doses greater than 0.01 in the BxPC-3 xenograft model (P < 0.001 for 0.1, l, 4.5, 15 mg/kg doses compared to vehicle). No significant increase in efficacy was ed at doses greater than 0.01 ug/kg (PC3, HA”) or 0.1 mg/kg (BxPC3, HA”).

D. Effect of PEGylated 0 on Colony Growth of Hyaluronan-Rich Tumor Cells in Vitro To ine whether PEGPH20 can inhibit anchorage-independent grth and proliferation of hyaluronan-rich prostate tumor cells (PC3) in vitro, a three- dimensional clonogenic assay was performed on cells. PC3 cells, at approximately 80% confluency, were trypsinized, harvested, and washed once in completed medium.

Cell density was adjusted to 8x104/mL cells and ded in Matrigel® (BD Biosciences, San Jose, CA) on ice. 0.025 mL of this cell / Matrigel® mixture were seeded onto a 48 well cell culture plate that had been pre-coated with Matrigel® at 0.1 mL per well, and solidified at 37°C for 1 hour. For continuous exposure, over 17 days, to control API buffer and various concentrations of PEGPH20, 0.6 mL/well of completed medium containing API , 1, 3, 10 and 100 U/mL of PEGPH20 were added to the top of the appropriate well. The wells were incubated at 37 CC, in a humidified atmosphere with 5% C02 in air for 17 days, fresh treatment medium, including the appropriate concentration of enzyme, where appropriate, was replaced every 3-4 days during the 17 day period.

On day 17, growth of colonies was assessed by ing images with a Nikon Eclipse TE2000U inverted microscope d to an Insight re digital camera (Diagnostic Instruments, Michigan). The colony number and diameter of each colony in um were measured using Image] software (open source software, a publicly ble program for display and is of images, for calculating area and pixel value) and coupled calibration on (colony volumes were calculated using colony diameter and using the formula: 4/3 TE r3.

Average colony volume of wells for each condition were determined and the s of PEGPH20 on colony volume ed by comparing the average colony volume in the control sample (API (active pharmaceutical ingredient) buffer (10 mM Hepes and 130 mM NaCl, pH 7.0) without enzyme) to the samples that were incubated in the presence of PEGylated rHuPH20. Inhibitory ratios were calculated using the formula: (mean volume of control - mean volume of treated) / (mean volume of control)*100.

PEGylated rHuPH20 induced a dose-dependent tion of growth, evidenced by lower colony volume compared with control. Based on inhibitory ratios calculated using the above formula, the cultures incubated in the presence of PEGPH20 at 1, 3, 10, and 100 U/mL exhibited an average reduction in colony volume of 39%, 67%, 73%, and 75% tively (p<0.01 for the 3 U and 10 U samples; p<0.001 for the 100 U samples; n=6), compared to cultures incubated with control buffer. Statistical ences were analyzed using the Mann-Whitney Test.

The IC50 of PEGPH20 in reducing colony volume, determined using the Graphpad Prism®4 m (GraphPad Software, Inc., La Jolla, CA), was approximately 1.67 U/mL. The average number of es was 10.17::1.56 per well in vehicle-treated (control) cultures and 11.50 :: 0.89 per well in the cultures treated with PEGPH20 100U/mL. The difference in colony number was not significant between the control and the 100U/mL cultures (n=6, p>0.05). These results indicate that PEGPH20 can inhibit proliferation and/or survival of onan rich cancer cells.

In an independent experiment, PC3 cells were seeded in reconstituted basement membrane (Matrigel) as described above and continuously exposed to vehicle or 0.1, 1, 10, 100, and 1000 U/mL of PEGPH20 for 19 days. Images were then digitally captured and colony volume was assessed using the Image] program. tion of colony volume compared to control was 22, 45, 63, 73 and 74%, respectively (P < 0.01for 1 U/ml, P < 0.001 for 10 U/mL and above compared to vehicle; n=3).

WO 63155 Example 7 ment of HA as a Biomarker for ting Response of Human NSCLC Tumors to PEGPH20.

A. Expression of HA in NSCLC Patient biopsies Previous work has shown that elevated accumulation ofHA occurs in non- small cell lung cancer (NSCLC) (Hernandez JR, et al. (1995) IntJ Biol Markers 10: 149-155 and Pirinen R, et al. (2001) IntJ Cancer 95: 12-17). By contrast, NSCLC- derived cell lines exhibit low levels suggesting the NSCLC cells lines lose HA sion during passaging in vitro. Thus, HA expression in primary tumor biopsies was examined.

A tissue microarray (TMA) panel of 190 NSCLC biopsies (US Biomax, Inc.) were examined for histotype and HA accumulation. HA content was determined by B-HABP hemistry staining as described in Example 6. Samples were scored as +3, +2, +1 or 0 when the ratio of strong positive (brown) stain area to the sum of total stained area was more than 25%, 10-25%, less than 10% or 0, respectively. In this panel, adenocarcinoma (ADC), squamous cell oma (SCC), and large cell carcinoma (LCC) cell types were observed at frequencies of 32% and 3%, , 51%, respectively, classified based on pathology diagnosis ed by US Biomax (Table 9). Other unidentified subtypes comprised about 11% of the 190 samples examined.

Analysis of tumor-associated HA accumulation showed that all histotypes have subsets of cells which express the HA+3 high HA phenotype, with an overall rate of approximately 27% (Table 9). In ular, 40% of SCC cases yed the HA+3 phenotype, while 11% ofADC and 33% of LCC cases were scored as HA+3 . 34% of SCC cases displayed the HA+2 phenotype, while 48% ofADC and 50% of LCC cases were scored as HA”. 25% of SCC cases displayed the HA+1 phenotype, while 36% ofADC and 17 % of LCC cases were scored as HAIZ. In this dataset, none of the normal lung tissue samples expressed the HA+3 phenotype, although detectable HA was observed in most samples of normal lung tissue.

Table 9. Distribution of HA ex n ression in human lun cancer samles HA os1t1ve incidence N (A) -______ Table 9. Distribution of HA ex u n in human lun_ cancer sam les HA ositive incidenceN 9/0 mum— HA scores were defined based on % of positive HA staining intensity 2 ADC: Adenocarcinoma 3 ,SCC: Squamous Cell Carcinoma 4 LCC: Large Cell Carcinoma B. Expression of HA in NSCLC Patient Explants and Prediction of PEGPHZO Efficacy In order to prospectively test the relationship n HA overexpression and antitumor se ofNSCLC to PEGPHZO-mediated HA depletion, human NSCLC patient tumor explants representing different degrees ofHA lation were selected and assessed for responsiveness to PEGPHZO ent in a xenografi tumor model. Primary ts characterized for HA accumulation were used for this study because explant models contain a more representative sampling of the genetic diversity of intact tumors, and should retain aspects of native tumor~like stroma.

Tumor biopsies were obtained fi‘om sixteen NSCLC patients, and were maintained at a low passage number subcutaneously in nude mice (Crown Bioscience, Beijing, China). The NSCLC tumor explants were screened for HA accumulation in explant tissues from passages 1-4 and were assigned. an HA phenotype (i. e. , +1, +2 or +3) by B-HABP histochemistry staining as described above. Three squamous cell— type (SCC) explants were prospectively selected for afi transplantation, representing the HA+3 (LUM697), HA+2 (LUM330), and HA“ (LUM858) ypes.

When the seed tumors for the selected tumor explants reached 500-700 mm3 in size, the mice were sacrificed and the tumors were extracted and minced into 3X3 ><3 mm3 fragments. One fragment for each tumor was subcutaneously implanted into the right rear flank of a female Balb/c nude mouse (n=10 for each group) as indicated in Table 6. Tumor volumes were determined by caliper measurements of the st longitudinal diameter h(L)) and the greatest transverse diameter (width(W)) and estimated using the calculation of (L X W2)/2. When the average tumor size reached 500 mm3 (range 300—600 mm3), the animals were randomized into two groups. For RECTIFIED SHEET (RULE 91) ISA/EP therapy, animals were treated with vehicle or PEGPH20 at 4.5 mg/kg twice weekly for 5 doses as shown in Table 7 above. The percentage tumor growth inhibition (% TGI) and statistical analysis were performed as described Example 6.

The rank-order ofHA ype (z'.e., +1, +2 or +3) as determined by hemistry was found to predict the degree of tumor growth inhibition by PEGPH20 (Table 9). For e, the percentage growth inhibition was 97% for LUM697 (HAH), 44% for LUM330 (HA+2), and 16% for LUM858 (HAH). In addition, tumor regression was observed in the LUM697 (HAH) tumor explant group, but not the LUM330 (HA+2) and LUM858 (HAH) groups: 4 of 10 animals with LUM697 (HAH) tumors had decreased tumor burden compared to pretherapy.

Example 8 Effect of PEGPH20 Treatment on Xenograft Tumor Cell DNA Synthesis and the Tumor Microenvironment (TME) A. Effect of HA Depletion on Tumor Cell DNA Synthesis To test whether HA depletion has antiproliferative effects on tumor cells in viva, PC-3 (HAH) tumor xenografts d with 0 were examined for levels ofDNA synthesis.

Six to eight week old nu/nu (Nor) athymic nude mice intraperitibially implanted with PC-3 tumor cells as described in Example 6 (1 X 106 cell in 0.05mL per mouse). Tumor volume was monitored by caliper measurement. When the tumors reached ~400 mm3, the mice were d with vehicle or PEGPH20 (1 mg/kg (35,000 U/kg) or 4.5 mg/kg (157,500 U/kg); about 700 U/dose or 3150 U/dose based on 20 g mouse body weight), 100 uL via tail vein injection twice weekly for two weeks. The 24 hours before study termination, the mice were administered 10 mg/kg BrdU (0.2mL) (Invitrogen, Cat#00-0103) intraperitoneally. Tumors were excised from the mice, fixed in 10% buffered formalin and embedded in paraffin. Tissues were cut into 5 um ns, and cell proliferation was assessed after staining with an anti-BrdU antibody (BrdU Staining Kit; Invitrogen, Cat#93-3943) according to the manufacturer’s ctions. s treated with PEGPH20 were compared to vehicle-treated animals. A 58.3% reduction in synthetically active nuclei was observed in the PEGPH20 treated tumors compared to vehicle-treated tumors (percent BrdU positive nuclei was reduced WO 63155 from 4.8% to 2%). This result parallels the observed growth inhibition of prostate PC3 (HA8) or pancreatic BxPC3 (HA+2) xenografts as a result of PEGPH20 treatment (~50% TGI at doses of 1 mg per kg or more) (see Example 6).

B. Effect of HA Depletion on Expression of Tumor Microenvironment Associated Proteins Previous studies have shown that treatment of HA+3 tumors with PEGPH20 has a dramatic effect on tumor interstitial fluid pressure (IFP), and therefore on the fluid pressure differential between the tumor and its al enVironment (see Thompson et al. (2010)). al changes in the TME can have an impact gene expression (Shieh AC (201 1) Ann Biomed Eng 39: 1379-1389). In order to test whether removal ofHA has an impact on turnover or expression ofTME proteins, expression ofTME proteins, such as murine collagen I (Colldl), murine collagen V (ColSul), and tenascin C (TNC), which are found in the actively remodeling matrix, were ed. 1. Localization and Semi-quantification of Collagen in Tumor Tissue Tumor tissues with adjacent skin from PC-3 tumors generated in Example 8A were fixed in 10% neutral buffered formalin for 48 hours, processed using a tissue processor (TISSUE-TEKVIP, Sakura k, CA) and ed in paraffin block.

The paraffin-embedded tissue samples were cut into 5um sections, dewaxed, and rehydrated in deionized water. Antigen retrieval was processed by heating slides in EDTA buffer at pH 8.0, 100 0C for 25 min. Slides were rinsed in PBS-T, blocked with 2% normal goat serum in 2% PBS/BSA for 30 min, followed by incubation with rabbit polyclonal anti-collagen type 1 antibody (1:200, Abcam, Cat#ab34710) for 2 hours at room ature. The sections were then incubated in Texas red tagged goat abbit IgG (1:200, Vector Laboratories, Cat# F1-1000) for 1 hour at room temperature, and counter d and mounted with ProLong® Gold antifade t with DAPI (InVitrogen, CA). Micrographs were captured under a Zeiss Axioskop cope coupled with RT3 camera (Diagnostic Instruments, MI). Random 5 fields from each section were analyzed for collagen-positive intensity using Image-Pro plus program. 2. cDNA Arrays Analysis of Gene Expression in PC3 aft Tumor Tissue NCR nu/nu mice bearing PC3 tumors were generated and treated with vehicle or 0 as described in Example 6A. Animals were euthanized 8 and 48 hours post-treatment with vehicle or PEGPH20. Tumor tissues were excised in sterile conditions and snap frozen in liquid nitrogen. Total RNA was isolated from frozen tissue according to Asuragen’s standard operating procedures. The purity and quantity of total RNA samples were determined by absorbance readings at 260 and 280 nm using a op 0 UV spectrophotometer. The integrity of total RNA was qualified by t Bioanalyzer 2100 uidic electrophoresis.

Samples for mRNA profiling studies were processed by Asuragen, Inc. using Affymetrix Mouse 430 2.0 and Human Ul33 plus 2.0 arrays. 3. Results Tumor-specific reduction of Collal in the PC-3 tumors was observed following depletion of HA by treatment with PEGPH20. 80% reduction in Colldl ng compared to vehicle d tumors was ed (P < 0.05 t test). Collal staining in skin from PEGPH20 treated mice, however, remained stable. In addition, sed levels of murine (stromal) mRNAs for Colldl, Col50tl, and TNC were observed as measured by mRNA expression array analysis. TNC mRNA was most significantly impacted (66% se), followed by Collal (53% decrease) and Col50tl (45% decrease). These results suggest that depletion ofHA results in significant changes in the expression of proteins within the TME.

Example 9 Generation of TSG-6 Link Module IgG Fc Fusion Protein A fusion protein, TSGLM-Fc, containing the link module of TSG-6 and the Fc domain of IgG was generated. A mutant fusion protein TSGLM-Fc/AHep in which the heparin binding region of the TSG-6 link module was mutated, also was generated.

A. Vector construction of inant human TSG-6 link module fusion proteins DNA de novo synthesis (GenScript, NJ) was employed to generate nucleic acid encoding the TSGLM-Fc filsion protein. The nucleic acid contains a DNA encoding a human immunoglobulin light chain kappa (K) leader signal peptide ~238' sequence (SEQ ID NO:210), a 669bp-long cDNA fragment ofhuman IgG1 heavy chain (GI No.5031409; SEQ ID NO: 203, encoding the peptide sequence set forth in SEQ ID NO:204) and a 285 bp—Iong cDNA fragment of human TSG—6 link module region (SEQ ID NO:216, encoding the peptide sequence set forth in SEQ ID NO:207, which nnrregpnnrls to amino acid positions ’H tn 17.0 nf‘ the TSG—G prepmrein, GI No 315139000, set forth in SEQ ID N02205 (mRNA) and SEQ ID N02206 (protein)).

The human IgG1 heavy chain and human TSG-6 link module regions were connected with a 6 bp AgeI restriction enzyme cleavage site and a 12 bp sequence, GACAAAACTCAC (SEQ ID NO: 208), encoding four additional amino acids (DKTH; SEQ ID NO: 209) originally published as part of the IgGl Fc sequence (Nucleic Acids Research, 1982, Vol. 10, . Two unique restriction enzyme cleavage sites, NheI at 5’ end and BamHI at 3’end, were synthesized flanking the fusion protein sequence. The synthesized fragment has a sequence set forth in SEQ ID N02217. The fragment was codon optimized for improved protein expression and synthesized by de novo DNA synthesis. The codon Optimized fragment has a sequence set forth in SEQ ID N02] 1. The protein ce for the encoded TSG—6- LM—Fc fusion protein is set forth in SEQ ID NO: 212.

The synthesized codon zed fragment was inserted via NheI and BamHI cleavage sites into the pHZZ4 IRES bicistronic mammalian sion vector (SEQ ID NO: 52) using well-known recombinant DNA ures (restriction enzyme and ligation reagents obtained from New England Biolabs, Ipswich, MA) to generate 4-TSG—6-LM-Fc construct (SEQ ID N02213). Recombinant protein expression in this vector is driven by a CMV promoter.

In order to enhance the hyaluronan (HA) binding specificity and reduce binding to other GAG chains, a construct encoding a mutant fusion protein, TSG-6— LM-Fc/AHep, that contains 3 lysine to alanine mutations at amino acid positions 55, 69, 76 ofthe TSG—6 link module was constructed. The mutations reduce the heparin binding ty of the TSG-6 link module, while not affecting the HA g activity (see Mahoney DJ el al. (2005) J Biol Chem. 280227044—2‘7055, which reports 10—fold lower n binding activity for the triple ; K20A/K34A/K41A in the heparin binding site). TSG—6-LM—Fc/AHep was generated by mutagenesis of the nucleic acid fragment encoding the LM-Fc fusion n and insertion into RECTIFIED SHEET (RULE 91) ISA/EP the pHZ24 IRES vector to generate pHZ24-TSGLM-Fc/AHep (SEQ ID NO:2l8).

The ce of the TSGLM-Fc/AHep nt is set forth in SEQ ID NO: 214, which encodes the TSGLM-Fc/AHep fusion protein set forth in SEQ ID NO: 215.

B. Recombinant Protein Expression and ation FreeStyle CHO-S suspension cells rogen) were employed for expression of TSGLM-Fc and TSGLM-Fc/AHep fusion proteins. The yle CHO-S suspension cell line was ined in CHO-S CD e medium (Invitrogen) prior to transfection. For preparation of the cells for transfection and recombinant protein sion, FreeStyle CHO-S cells were cultured in FreeStyle CHO Expression Medium (Invitrogen) supplemented with 8 mM L-glutamine in shake flasks at 37°C in a humidified atmosphere of 8 % CO2 in air on an orbital shaker platform rotating at 125 rpm with loosened caps of flasks to allow for aeration. ent transfection of suspension cells was performed according to the manufacturer’s instructions. Briefly, cells were split at a density 6><105/ml 24 hours before transfection, and transfected using FreeStyle Max lipid with a DNA/lipid ratio at 1:1. After 96 hours ransfection, cells were harvested at 4,000g for 20 min, and supematants were collected. A time course analysis of protein expression level during the post-transient transfection revealed that the protein expression level reached a plateau after 96 hours post transfection. Thus, the recombinant protein was collected at 96 hour ransfection.

The sed TSGLM-Fc and TSGLM-Fc/AHep fusion proteins in the collected supernatants were affinity purified by Protein A resins (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Briefly, the collected supematants were adjusted to pH 7.4, 0.15 M NaCl with 1 M Tris-HCl, pH 7.4 (Teknova Catalog No. T1074) and 5M NaCl (Sigma) and diluted with binding buffer 3 fold before loaded onto a Protein A column. The eluted product was immediately neutralized with 1M Tris-HCl, pH 8.5, and dialyzed against Phosphate-Balanced Solution (PBS, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.46mM KH2P04, and pH 7.4) at 4 CC, and stored at -20 CC. The yield of the purified proteins from the supematants through a single step Protein A affinity column was between 3 to 5 mg/liter.

C. SDS~PAGE and Western Blot is of Expressed Recombinant Proteins The purity, size and identity of the purified fusion protein were determined by SDS-PAGE 4—20% gradient gel under reducing and Hen-reducing conditions and Western Blot analysis. 60 ng ofpurified protein was used in the is. The size of the purified fusion proteins were about 40 kDa under reducing conditions and about 80 kDa under non-reducing conditions, indicating the expressed proteins form homodimers via disulfide bonds in hinge region of IgG Fe. The purity of the protein samples were greater than 95%. The purified proteins were stable in PBS for at least one month at 4 °C t any visible degradation or loss of g activity.

The identity of the TSG—6 link module in TSG—6-LM-Fc and TSG—6-LM- Fc/AHep was assessed by Western blot with goat uman TSG-6 IgG (R&D Systems, lnc., Minneapolis, MN) followed by rabbit anti-goat IgG—HRP (EMD, San Diego, CA). Recombinant full length human TSG—6 protein (R&D Systems, Inc., Minneapolis, MN) was employed as a positive control. The pattern of detected proteins by n blot analysis under reducing and non-reducing conditions was the same as that of SDS-PAGE analysis except for a small amount of upper bands ed under the non-reducing condition, most likely representing ers of the recombinant proteins based on their molecular weight size.

The identity of the Fc portion in the purified recombinant proteins was confirmed by Westem blot analysis with bbit anti-human IgGFc (Jackson ImmunoResearch, West Grove, PA). The pattern of ed proteins was the same as for the SDS-Page and anti-TSG-6 analyses, indicating that the purified proteins contain both TSG-6 link module (LM) as well as .

To analyze whether the proteins were glycosylated, the purified proteins were treated with glycosidase PNGase F (0.5 units per ng protein), which s the N— linked oligosaccharides from proteins, and analyzed by SDS-PAGE and Western blot. A 5 kDa difference of molecular weights of proteins was observed between before and after treatment with PNGase F, indicating that the expressed proteins were ' glycosylated.

RECTIFIED SHEET (RULE 91) ISA/EP ~241- Example 10 Binding of TSG-6 to Hyaluronan and n Two formats were used to test the binding of both TSGLM-Fe and its mutant to HA and heparin. In one format, binding of TSGLM-Fc and TSG-6—LM- Ur Fe/AlIep to immobilized MA or Heparin on a miemplate was employed. In the second format, binding of biotinylated HA and heparin to immobilized inant TSG~6—LM-Fc and TSG—6—LM-Fc/AHep proteins on a mieroplate was employed.

A. Binding of recombinant TSGLM-Fc and TSG-6—LM-Fc/Al-lep to immobilized HA and Heparin Wild type and mutant TSG-é-LM Fe fusion homodimers were tested for their HA binding and heparin binding activities using either HA or heparin-coated microplates. Briefly, hyaluronan with an average MW of about 1000 kDa (Lifecore, , MN) or Heparin with an average MW of 15 kDa (Calbioehcm, San Diego, CA) at concentration of 100 ug/ml in 0.5 M sodium carbonate buffer, pH 9.6, was dispensed into 96-well plates in duplicate, 100 ll, and incubated at 4 °C overnight. Plates were blocked with 1% BSA in PBS to reduce non—specific binding.

'TSG—6-LM-Fc and TSG-6~LM-Fc/AHep purified protein samples were diluted to give rise to a concentration range From 0.31 to 40 nag/ml for g to HA coated plate, 0.78 to 100 ng/ml for binding to heparin coated plate. For each sample, 100 pl per well in ate was added to the microplate and incubated at room temperature for l hour. Plates were washed PBS with 0.05% Tween 20, 5 times to remove unbound protein. Hyaluronan or heparin bound 'l‘SG-o—LM-Fc and TSG-o-LM— Fe/AHep were ed with rabbit anti-human IgG Fe «HRP (Jackson ImmunoResearch, West Grove, PA) followed by TMB (3,3’,S,5'— tetramethylbenzidine) substrate (KPL, rsburg, MD). The samples were incubated 60 minutes with the rabbit anti-human IgG Fe —HRP antibody. After washing, bound HRP was detected with TMB solution over 10-15 minutes development time ed by addition phoric acid reagent to stop color development. Absorbance was measured at OD450 using a Molecular Devices, Spectra M3 spectrophotometer.

Both TSG—OLM-Fc and lA'l-Fc/AI-Iep displayed the same HA binding activity on the HA coated plate; and their ion curves of HA binding activity were RECTIFIED SHEET (RULE 91) ISA/EP 2012/061743 almost pped, ting that the two sed proteins bind HA with high affinity based on the EC50 values from titration curves of HA binding. The triple mutation in the heparin binding site has no effect on its HA binding. In contrast, the binding of the two proteins to the heparin coated plate showed a significant difference. The wild type TSGLM-Fc bound heparin although with relatively low binding activity compared to its binding to HA, which could be due to the size ence ofthe two GAG chains coated on the plates. The mutant TSGLM-Fc protein exhibited about 10% of heparin binding activity compared to that of wild type, which was consistent with the reported result for the triple-mutated TSG—6-LM r (Mahoney DJ et a]. (2005)).

B. Binding of biotinylated HA and Heparin to immobilized recombinant TSG LM—Fc and TSG—6-LM-Fc/AHep The GAG binding properties of wild type TGSé—LM-Fc and TSG—6-LM- Fc/AHep were further examined by coating microplates with the inant proteins and assessing their binding to biotinylated HA and ylated heparin.

For preparation of the microplates, TSG—6-LM-Fc and TSG-6—LM-Fc/AHep at a concentration of 2 ug/ml in 1 x PBS buffer was diSpensed into 96-well plates in duplicates, 100 til/well, and incubated at 4 °C overnight. Plates were blocked with 1% BSA in PBS to reduce ecific g.

TSG~6-LM—Fc and TSG—6-LM—Fc/AHep purified protein s were diluted to give rise to concentration range from 0.31 to 40 ng/ml for binding to HA coated plate, 0.78 to 100 ng/ml for binding to heparin coated plate. 100 pd per well for each sample in duplicate was added to the microplate and incubated at room temperature for 1 hour. Hyaluronan or heparin bound TSG—é—LM~Fc and TSGLM-Fc/AHep were detected with anti-human IgG Fc ~HRP (Jackson ImmunoResearch, West Grove, PA) followed by TMB (3,3‘,5,5'-tetramethylbenzidine) substrate (KPL, Gaithersburg, MD).

For biotinylation of HA, the carboxyl groups on HA were used for the conjugation Via hydrazide chemistry. , biotin-hydrazide was dissolved in DMSO at a concentration of 25 mM, and added at a volume ratio of 6:100 into an HA solution, containing 1000 kDa or 150 kDa molecular weight HA (Lifecore Biomedical, LLC Chaska, MN) at 1 rug/ml in 0.1 M MES, pH 5.0. 1-Ethyl[3— RECTIFIED SHEET (RULE 91) ISA/EP dimethylaminopropyl] iimide hydrochloride (ECD) and sulfo- NHydroxysuccinirnide (sulfo-NHS) were added in the ation reaction to a concentration of 40 uM and 850 Mid, respectively, to mediate the conjugation of biotin—hydrazide and HA. The reaction was kept at 4 °C overnight while stirring. The U! excess amount of chemicals was removed from biotinylated HA by dialysis.

Biotinylated heparin was purchased from EMD, San Diego (Catalog No. 375054).

Biotinylated hyaluronan or n were diluted in PBS with a concentration . range from 0.78 ng/ml to 100 ng/ml, dispensed 100 ul/well, and incubated at room temperature for 1 hour. Plates were washed with PBS with 0.05% Tween 20, 5 times to remove unbound protein. The bound biotinylated onan and heparin were detected with anti-Streptavidin—HRP (Jackson ImmunoResearch, West Grove, PA) followed by TMB substrate (3,3’,5,5‘-tetramethylbenzidine) substrate (KPL, Gaithersburg, MD) as described above. Absorbance was measured at OD450.

The binding results observed were r to the binding assay performed in Example 10A, which used immobilized HA and n and free TSGLM-Fc and TSGLM-Fc/AHep. There was no ence of binding activity of immobilized TSGLM-Fc and TSG-G-LM-Fc/AHep to biotinylated HA or in the g titration curves between TSG—6—LM—Fc and LM-Fc/AHep, and a significant reduction in the binding of mutant TSG-o-LM~Fc.’AHep to biotinylated heparin compared to that of wild type n also was observed. Therefore, the HA and heparin g properties of wild type TSG-6—LM—Fc and its mutant can be evaluated in either GAG coated or recombinant protein coated format; and both formats revealed similar binding ns.

C. Calculation of Binding Affinity of TSG—6-LM-Fc The HA binding affinity of TSG-6—LM-Fc was measured using Bio-Layer Interferometry (BLI) teclmology via Octet QKe instrument (ForteBio, Menlo Park, CA). The full length TSG-6 recombinant protein (R&D Systems, Inc., Minneapolis, MN) was used as control. Briefly, biotinylated HA with an average molecular weight of 150 cha was immobilized on streptavidin coated biosensors for 240 seconds.

TSGLM-Fc and TSG—6-LM-Fc/AHep was then ated with immobilized HA for 180 seconds at different concentrations in PBS at pH 6.0 or pH 7.4, followed by dissociation of bound proteins in PBS at pH 6.0 or pH 7.4 for 240 seconds. The RECTIFIED SHEET (RULE 91) ISA/EP results of binding kinetics were analyzed by the software provided by the manufacturer. Results for the calculated binding affinity are provided in Table 10.

Table 10. Bindin Affini of TSGLM-Fc Sam le ID InnnM kon llMs kdis Us Full R"2 LM-Fcam 54513—09 TSGLM-Fc 6.25 m 54513—09 TSGLM-Fc Example 11 Competitive Inhibition Assessment ofTSG-6 Binding to Hyaluronan and Heparin by Other Glycosaminoglycans The HA and heparin GAG binding sites ofthe TSG-6 link module are located at different regions of the link module. In order to determine r the two binding sites would interfere with each other during the ction with TSG-6 link module or in the presence of other GAG chains, a competitive inhibition assay was performed to assess binding of HA or heparin in the presence of other GAG chains.

HA and heparin coated 96-well microplates were prepared as described in Example 10A. TSGLM-Fc and TSGLM-Fc/AHep, at a concentration of40 ng/ml for the HA coated plates and 100 ng/ml for the n coated plates, were pre- incubated with four different GAG chains: HA (Lifecore Biomedical, LLC Chaska, MN), chondroitin sulfate A (EMD, San Diego, CA, Catalog No. 230687) chondroitin sulfate C (EMD, San Diego, CA, Catalog No. 2307) and heparin e (EMD, San Diego, CA, g No. 375095), at three different concentrations (0.11, 0.33, 1.0 rig/ml) or without GAG chain as control at room temperature for 10 minutes. The s were then sed (100 pl) in duplicate into the HA and heparin coated 96- well microplates and incubated at room temperature for 1 hour. Plates were washed with PBS with 0.05% Tween 20, 5 times, to remove unbound protein. Bound TSG LM-Fc and TSG—6-LM-Fc/AHep were ed with anti-human IgG Fc —HRP (Jackson ImmunoResearch, West Grove, PA) followed by TMB (3,3’,5,5’- tetramethylbenzidine) substrate (KPL, Gaithersburg, MD) as described above.

Absorbance was ed at OD450.

RECTIFIED SHEET (RULE 91) ISA/EP —245— For the HA coated plate, both TSGLM-Fc and TSGLM-Fc/AHep revealed similar itive inhibition patterns. Binding of TSGLM-Fc to the immobilized HA was efficiently inhibited by cubation of same amount of protein with the different doses of free HA (approximately 68%, 85%, and 93 % inhibition for the 0.11, 0.33, 1.0 ug/ml doses, respectively), but was not affected by pre-incubation with different doses of free heparin or chondroitin sulfate C. Some inhibition of LM-Fc and TSGLM-Fc/AHep was observed for pre- incubation with chondroitin sulfate A, though it was less than for HA (approximately 23%, 43%, and 63 % inhibition for the 0.11, 0.33, 1.0 ug/ml doses). Thus, an approximately 10 fold higher amount of chondroitin sulfate A was needed for inhibition. (In ndent experiments up to 30-fold higher amount of chondroitin sulfate A was needed for inhibition compared to HA). Because TSGLM-Fc and TSGLM-Fc/AHep showed similar inhibition with pre-incubation with chondroitin sulfate A, it is likely that the HA binding site in TSG-6 link module is responsible for the chondroitin sulfate A binding.

For the heparin coated plates, the binding of TSGLM-Fc to heparin was efficiently inhibited not only by pre-incubation with heparin, but also by pre- incubation with either HA or chondroitin sulfate A. This data shows that the binding of TSGFc-LM to HA could block its heparin binding activity. As expected, mutant TSGLM-Fc /AHep did not bind heparin and thus exhibited readings close to background for both control and cubation samples.

This study demonstrates that binding of link module of TSG-6 to HA is not affected by the presence of free heparin or preformed TSG-6 heparin x, while its g to heparin is significantly inhibited by the presence of free HA or preformed TSG-6 HA. Based on these observations, one can de that TSG LM binds to HA and heparin aneously or binding of TSGLM to HA is er than its binding to heparin. HA and TSGLM complex formation can cause protein conformation change or other arrangements of the n that are not favorable for its binding to heparin. 2012/061743 Example 12 Comparison of Glycosaminoglycan Binding Properties of TSG—6-LM-Fc, TSG—6- LM-Fc/AHep and HABP In this example, the city and binding activity of TSGLM-Fc, TSG- c/AHep and HA binding protein (HABP) to HA, heparin, and other GAGs were compared. For this experiment, biotinylated-TSGLM-Fc and biotinylated- TSGLM-Fc/AHep HA binding proteins were generated and ed to commercially available biotinylated-HA binding protein (HABP) (Seikagaku, Tokyo, Japan) for their binding activity on GAG chain coated plates.

A. Biotinylation of TSG—6-LM-Fc and TSG—6-LM-Fc /AHep A random labeling approach was used to conjugate the biotin to primary amine containing residues (Lys) in the protein directly t pre-incubation with free HA in order to protect HA binding sites. For biotinylation of TSGLM-Fc and TSGLM-Fc /AHep, direct conjugation of the primary amine active reagent NHS- PEG4-Biotin (Thermo Fisher Scientific, o, IL) was performed according to the manufacturer’s instructions. 0.5 mg protein in PBS at tration 1 mg/ml and 10 ul of 20 mM biotinylation reagent was used for the biotinylation reaction. The N- ysuccinimide ester (NHS) group of NHS-PEG4-Biotin reacts specifically and ntly with lysine and N—terminal amino groups at pH 7-9 to form stable amide bonds. The hydrophilic polyethylene glycol (PEG) spacer arm imparts water solubility that is transferred to the biotinylated le, thus reducing aggregation of labeled proteins stored in solution. The PEG spacer arm also gives the reagent a long and flexible connection to minimize steric hindrance involved with binding to aVidin molecules. Unreacted NHS-PEG4-Biotin was removed with dialysis against l><PBS and stored at —20 0C.

For comparison, the TSGLM-Fc and TSGLM-Fc /AHep proteins also were biotinylated using the oriented labeling ch, which conjugates the biotin units to sugar chains on the proteins by oxidation of polysaccharide chain on the protein using NaIO4 followed by biotin-hydrazide. Briefly, 1 ml protein at a concentration of 1 mg/ml in 0.1 M phosphate buffer was first oxidized by , pH 7.2, sodium periodate (NaIO4) at a final concentration of 5 mg/ml, at 4 CC for 30 minutes.

The reaction converts the two adjacent primary hydroxyl groups on sugars to —247— corresponding aldehyde reactive groups. The ed n was dialyzed against 0.1 M phosphate buffer, pH 7.2. The dialyzed protein was then mixed with 50 mM hydrazide-biotin prepared in DMSO at volume ratio 9 to 1 resulting in 5 mM hydrazide-biotin in the reaction and incubated at room temperature for 2 hours to form hydrazone bonds between aldehyde groups and hydrazide . The d protein was dialyzed against l><PBS and stored at —20 CC.

After conjugation and removal of free biotin, the HA binding activity of both biotin-TSGLM-Fc and biotin-TSGLM-Fc/AHep were tested together with non labeled corresponding proteins to examine if the labeling would cause reduced HA binding activity using the binding assay as described in Example 10A using HA coated plates. No difference in HA binding activity was found between labeled vs non labeled proteins.

B. Binding of biotinylated-TSG—6-LM-Fc, biotinylated-TSG—6-LM-Fc/AHep and biotinylated-HABP t0 GAGs For preparation of the GAG coated microplates, HA, Heparin, oitin e A, or chondroitin sulfate C, at a concentration of 100 ug/ml in 0.5 M sodium carbonate buffer, were sed, 100 ul/well, into 96-well plates in duplicate, and incubated at 4 OC overnight. Plates were blocked with 1% BSA in PBS to reduce non-specific binding. The three biotinylated proteins, biotinylated-TSGLM-Fc, biotinylated-TSGLM-Fc/AHep and biotinylated-HABP were diluted to concentrations ranging from 0.05 to 100 ng/ml for binding to HA, chondroitin sulfate A, and oitin sulfate C coated , and 0.23 to 500 ng/ml for binding to heparin coated plates. The diluted protein samples were dispensed onto the plates, 100 ul/well in duplicate, and incubated at room temperature for 1 hour. The ns bound to the GAG coated plates were detected with Streptavidin—HRP (Jackson ImmunoResearch, West Grove, PA) followed by TMB (3,3’,5,5’- tetramethylbenzidine) substrate (KPL, Gaithersburg, MD) as described above. ance was measured at OD450.

All three ylated GAG binding proteins exhibited strong HA binding activity on the HA coated plate. At 11.1 ng/ml n concentration, which represented one dilution lower than maximal binding concentrations (Le. 33.3 ng/ml and 100 ng/ml) for HA, binding of biotinylated-TSGLM-Fc and biotinylated-TSG- 6-LM~Fc/AHep to HA was approximately 14 fold over background, and B—HABP binding to HA was approximately 9 fold over background.

Both biotinyiated-HABP and biotinylated-TSG—6—LM—Fc/AHep displayed little binding activity against the heparin coated plate. Biotingilate¢xvild type TSG-6_ LM—Fc also showed negative in n g activity, suggesting that the random labeling approach with NHS-PEG4-Biotin caused a loss of heparin binding activity.

When LM-Fc was biotinylated by the oriented labeling approach as described above, binding to heparin was restored and the protein exhibited similar heparin g ty as that of non—labeled LM—Fc. Thus, biotin modification of lysines in heparin site of TSGLM-Fc should abolish its heparin binding activity.

All three proteins exhibited no binding activity to chondroitin sulfate C coated plate, but demonstrated strong binding towards chondroitin e A coated plate.

The biotinylated-TSG—é-LM-Fc and biotin-TSGLM-Fc/AHep were observed to exhibit a few fold higher binding activity than that of biotin-HABP. At 11.1 ng/ml protein concentration, binding of biotinylated-TSGLM-Fc and biotinylated-TSG LM—Fc/AHep to HA was approximately 20 fold and 12 fold over background, respectively and B-HABP g to HA was approximately 6 fold over background.

Nonetheless, both TSG—6-LM-Fc and TSGLM—Fc/AHep have much stronger preference for binding to HA as demonstrated in the GAG competitive assay. As shown in Example 11, at least 10 fold more of chondroitin sulfate A was needed to reach the similar competitive inhibition as HA. In addition, in a separate experiment, ylated—HABP was ed to biotinylated-TSG-6HLM-Fc in a GAG competitive assay, and similar inhibition patterns of four GAG chains (HA, Heparin Chondroitin es A & C) to the binding of biotin-HABP to HA versus the binding of TSG‘6-LM-Fc to HA were observed.

Example 13 Quantitation of Hyaluronan in K3-EDTA Human Plasma by Aggrecan Binding Assay The concentration ofhyaluronan was ined in clinical human plasma s using a sandwich binding assay. Plasma samples were obtained from 19 " subjects with solid tumor and various tumor types at advanced stage that were enrolled in a clinical study (Phase 1-101 and Phase 1-102; see Table 11) assessing RECTIFIED SHEET (RULE 91) ISA/EP —249— escalating dosage of PEGPH20 in patients in the ce or absence of dexamethasone. In addition, plasma samples also were obtained from twenty (20) normal patients (obtained from BioReclamation, Hicksville, NY). Prior to treatment with 0, baseline levels ofHA were determined as follows.

Immulon 4HBX 96-well flat bottom microtiter plates (Immulon/Thermo; Catalog No. 3855) were coated with a inant human aggrecan (rHu-aggrecan) R & D Systems, g No. 842162) as capture reagent. Prior to use, the rHu-aggrecan was reconstituted by adding 250 ul of reagent diluent to l vial and stored at 2-8°C for up to 1 month. Then, to generate a 0.5 ug/mL solution of grecan, a 240-fold dilution of the stock was ed (e.g. 41.7 uL stock to about 10 mL PBS).

Immediately after dilution, 100 uL was dispensed into each well of a 4HBX plate and the plate was covered with a plate sealer and incubated overnight or up to 3 days at room temperature. After incubation, each well in the plate was washed five (5) times with lXPBST wash buffer (1 X PBS, 0.05% Tween 20) using the ELX405 Select CW plate washer. The assay plate was then blocked with block buffer (5% Tween 20 in PBS) by adding 300 uL of block buffer to each well. The plate was covered with an adhesive plate cover and ted at ambient ature for at least 1 hour without shaking.

Prior to ting the plate with sample, plasma samples and a standard curve were prepared. Briefly, plasma test samples were obtained and stored at 560°C until es. Immediately prior to analyses, the test samples were thawed on wet ice and mixed briefly by vortexing just prior to dilution. Then, several serial dilutions of plasma test sample dilutions were prepared in order to ensure at least one sample dilution fell within the range of the calibration curve by dilution in Reagent Diluent (5% Tween-20 PBS solution, prepared by adding 6.5 mL Tween-20 (Sigma; Catalog No. P7949) to 123.5 mL Phosphate Buffered Saline (PBS; CellGro; Catalog No. 21- 03 l-CV)). To assess assay validity, two quality control s also were diluted for assay. The controls were pooled human plasma collected in Kg-EDTA (pooled human Kg-EDTA plasma; “low quality control”) and pooled human Kg-EDTA plasma spiked with exogenous hyaluronan (HA) (“high quality control”). The minimum required dilution (MRD) for human K3-EDTA plasma (used as a control) was 1:4.

Dilutions were in polypropylene tubes (e.g., BioRad Titer tubes; BioRad, Catalog No. 223-9391) and were made to a total volume (sample and diluent) of 500 ul. Each dilution was mixed as it was prepared by brief pulse-vortexing. Pipets were changed in between each dilution.

For the standard curve, a hyaluronan stock (132 kD, 1800 ng/mL; R& D s, Catalog No. 842164) was diluted by serial dilution in reagent diluent (5% Tween 20 in PBS) to final concentrations of 500 ng/mL, 167 ng/mL, 55.6 ng/mL, 18.5 ng/mL, 6.2 ng/mL, 2.1 ng/mL, and 0.68 ng/mL. A blank well containing t diluent also was ed in the standard.

Then, at the end of the block step, each well was washed five (5) times with 1XPBST wash buffer (1 X PBS, 0.05% Tween 20) using the ELx405Select CW plate washer. The test samples, controls and standard curve were added to the coated and blocked plate by adding 100 uL of each in triplicate to wells of the plate. The plate was covered with an adhesive plate sealer and incubated at ambient temperature for approximately 2 hours. After incubation, each well was washed five (5) times with 1XPBST wash buffer (1 X PBS, 0.05% Tween 20) using the ELx405Select CW plate To detect binding ofHA to the coated rHu-aggrecan, a biotinylated rHuAggrecan detection reagent (72 ug/mL; R& D Systems, Catalog No. 842163) was added to the plate. First, 10 mL of a 0.3 ug/mL biotinylated-aggrecan solution was made by diluting the stock solution 240-fold in reagent diluent (5% Tween/PBS).

Then, 100 uL of the detection reagent was added to each well. The plate was d with an adhesive seal and incubated at ambient temperature for imately 2 hours. Each well was washed five (5) times with 1XPBST wash buffer (1 X PBS, 0.05% Tween 20) using the ELx405 Select CW plate washer. Then, an Streptavidin- HRP (SA-HRP; R&D s, Catalog No. 890803) working solution was prepared in reagent diluent by diluting the stock ld. Then, 100 uL of the dilute SA-HRP working solution was added to each well. The plate was covered with an adhesive seal and incubated at ambient ature for approximately 20 minutes with shaking at 500 rpm. At the end of the SA-HRP incubation period, each well was washed five (5) times with 1XPBST wash buffer (1 X PBS, 0.05% Tween 20) using the ELx405 Select CW plate . Then, 100 uL of a TMB substrate (KPL; Catalog No. 5203), which was equilibrated to ambient temperature protected from light, was added to each well and incubated at ambient temperature for 20 minutes with shaking at 500 rpm. Then, 100 uL ofTMB stop solution (KPL; Catalog No. 50 06) was added to each well for at least 5 minutes but less than 30 minutes prior to determining the optical density at 450 nm (OD 450nm) using a microtiter plate spectrophotometer and SoftMaX Pro software.

Based on the OD450nm value, the concentration of intact hyaluronan for each sample was determined by olating from the standard curve. The results were multiplied by the sample dilution factor. The data was ed as the average of all values within the limits of quantitation of the calibration curve in ng/mL. The results are set forth in Tables 11 and 12. The results show that the median plasma HA in healthy humans was 0.015 ug/mL while in phase 1 ts it was 0.06 ug/mL. This represented a statistically cant difference with a p<0.0001.

Table 11 - Plasma HA from Subjects with Tumors Trial 101 t Tumor Type Age (n_/mL) 1 Histiocytoma 86 2 Colorectal 62 3 Rectal 60 4 Pancreatic 57 Bladder 63 6 Colon 66 7 Pancreatic 63 8 Carcinoid 56 9 Ovarian 70 Colon 60 1 1 Prostate 78 12 Non small cell lung cancer 61 13 Prostate 71 14 Prostate 55 Trial 102 Ovarian 55 16 Esophageal 71 17 NSCLC 65 18 colon w/ liver mets 72 19 colo-rectal 62 Table 12: Plasma HA from Health Sub'ects Subject Age Sex Result (n_/mL) -1 -4 11.2 18-3 63-2 13-4 13-4 12.8 24 F 12.6 18-4 13 28 F 18.5 14-5 16 54 M 11.7 21-9 18 38 M 8.3 37-5 Example 14 Histochemical Detection of HA Histochemical detection ofHA were obtained from a opsy tumor specimen and a post-treatment metastatic liver biopsy sample from a patient dosed for 4 weeks with 1.6 ug/kg PEGPH20 + dexamethasone. The pre-dose biopsy (pre biopsy) was an archived sample obtained in 2007 (3.5 years prior to the treatment with PEGPH20). The post-treatment biopsy sample was obtained 3 days after the last dose (8th dose) in a PEGPH20 plus dexamethasone treatment regimine from a female colon cancer patient with liver metastases. Specifically, the patient post- treatment biopsy was ed after one cycle of PEGPH20 treatment at 1.6 ug/kg on a twice weekly le for the cycle of administration with dexamethasone co- treatment. The treatment cycle was defined as a 28-day period, with PEGPH20 administered intravenously (IV) and dexamethasone administered orally. On each dosing day, a premedication n of 4 mg of dexamethasone was administered orally one hour prior to the PEGPH20, followed by a second dose of 4 mg dexamethasone 8-12 hours after PEGPH20 dosing.

Briefly, the tumor biopsies were fixed in normal buffered formalin (NBF) and um sections cut and stained using a biotin labeled hyaluronan g protein bio) (Seikagaku, Japan). After washing to remove the primary reagent, a labeled secondary reagent was used. Nuclei were r-stained using a DAPI (4',6- diamidinophenylindole) reagent. Micrographs were captured via a Nikon Eclipse TEZOOOU inverted fluorescent microscope coupled to a Insight FireWire digital camera (Diagnostic Instruments, Michigan) or ZEISS overhead scope (Carl Inc.) that has the same imaging system.

The histochemical staining of the samples with biotinylated-HA binding n trated a decrease in pericellular and stromal HA levels after one cycle of PEGPH20 treatment. The results are summarized in Table 13. The H score represents the relative intensity of pericellular and stromal HA. The data demonstrates the y of PEGPH20 to e tumor-associated HA as demonstrated by a reduction ofHA staining in the tumor biopsy after treatment.

TABLE 13. Histochemical Detection of HA Pericellular tumor Stroma % total area cells (% cells stained) (% area stained) mn-Inn-n mm---_ mummm r associated stroma Example 15 HPLC Method For the Estimation of Hyaluronan (HA) level in Plasma This Example describes a method for the determination of the HA- disaccharide content in plasma as a measure ofHA catabolites, which are the breakdown products after enzymatic activity of PEGPH20. The method employs the hydrolysis ofHA with Chondroitinase ABC to release the HA-disaccharides, derivatize them with 2-amino acridone (AMAC) and analyze them on a e-phase HPLC coupled with fluorescence detection. Quantitation of the HA-disaccharides is accomplished by comparison with HA-disaccharide standards. This assay was used to measure the enzymatic activity of PEGPH20 by monitoring concentrations of ~254- hyaluronan lites in plasma ofpatients that were selected at schedules times from patients after treatment with PEGPHZO. l. Working Standards In the method, a working standard solution was generated. First, adilute stock solution (DSS) was generated from an accharide Stock Solution (SS). The HA disaccharide SS was generated by adding 1 mL of water to a Vial ofHA-Disac (V- labs, Cat. No. C3209) containing 2 mg of lyophilized powder to make a uniform suspension. To generate dilute stock ons, 5 ul of the SS solution was diluted with 125 pl ofwater to generate a D881 solution (containing 200 pmoles/ul HA- Disac; 200 nmoles/ml ac). old, serial dilutions in water were made to generate DSSZ (containing 40 pmoles/ul HA—Disac; 40 nmoles/ml HA—Disac) and then DSS3 (containing 8 pmoles/ul HA-Disac; 8 nmoles/ml HA-Disac). Next, working standard ons were generated as set forth in 25% human serum albumin (HSA) (ABO Pharmaceuticals, Cat. No. 1500233) or Normal Mouse plasma (Bioreclamation, Cat. No. MSEPLEDTAZ—BALB—M) as set forth in Tables 14 and 15.

TABLE 14: Workin_ rd Solution in HSA III-flewWSS# D833 D532 DSSl Water 25% HAODisac Ll in 150 :1 W880 m-———n- WSSI ____—_ _———__- —_—-__— ——_—-—- ____-—— =_—_—_—WSS8 —_——_— —_—_—_— _———_—— TABLE 15: Workingfitandard Solution in Normal Mouse Plasma HAODisac (pmoles Plasma in 150 pl) ( l 100-00 W852 RECTIFIED SHEET (RULE 91) ISA/EP TABLE 15: Workin Standard on in Normal Mouse Plasma WSS# DSS3 DSSZ DSSl Water HAODisac (pl) (pmoles in 150 pl) 6.25 ___—— 1250 ___—- ___—___ ___—___ ___-__— ___—__- ___—__— ___—__— 2. Hydrolysis and Derivation of Samples Next, the sample was hydrolyzed. The sample (e, g, plasma) was prepared by taking approximately 100 pg of protein in a polypropylene tube and adjusting the volume to 340 pl with water. A matrix blank also was prepared by taking dilution buffer (1.59 g HEPES, 5.07 g NaCl, 1800 mL water, pH 7.0) equal to the volume of the sample and adjusting the volume to 340 pl. Hydrolysis of the samples and matrix blank were effected by adding 60 p1 of TFA to the sample tube and matrix blank tube and the contents were mixed and incubated at 100 °C for 4 hours. The vials were allowed to cool to room temperature. The vials were evaporated to s using a speed vac. Then, 300 pl of water was added to each tube and vortexed to resuspend the samples.

For tion ofhydrolyzed samples, blanks and working samples, 45 pl of each sample (sample, blank or working sample) was evaporated to dryness in a speed vac. Then, 10 p1 of SAS was added to the dried sample, blank and g rds.

Then, 50 pl ABA/NaCNBH3 labeling solution was added. The tubes were vortexed and centrifuged briefly. Then, 440 pl of Mobile Phase A was added and the tubes were mixed well. Mobile Phase A was prepared as follows: 132 mL of l M ammonium e buffer (Sigma, Cat. No. A7330) was added to a I L volumetric flask and water added to fill the flask. ing derivation, nominal on—column loads per 20 pl of injection for the working standards is as set forth in Table 16.

RECTIFIED SHEET (RULE 91) ISA/EP , TABLE 16 WSS# Fuc(pmol) Gal (pmol) Man mol mol umol —_-.i- ' ‘ 3. HPLC The HPLC column was equilibrated at a flow rate of 1.0 mL/min with the initial mobile phase settings as outlined in Table 17. The system was allowed to equilibrate until the ne was steady. HPLC analysis was performed with the instrument parameters as outlined in Table 17.

TABLE 17: HPLC Instrument ters Parameter Values Bakerbond C18 reversed phase column, Column Tem - erature 5m°><3w3‘8536Hgm33 Mobile Phase A 0.2% n—butylamine, 0.5% oric acid, 1% tetrah drofuran in water Mobile Phase B on C33\ ' O(3"i"—to hase A, 50% acetonitrile 1-0 mL/min 1 Fluorescence; Excitation 360 nm, . Emission 425 nm 4-6 °C Gradient MI.— The sequence for sample analysis was as follows: WSSS (1 injection) for column conditioning/equilibration/detector gain; water injection (1 injection); WSS3 (3 ions); W88} (1 injection); WSSZ (1 injection); WSS4(1 injection); WSSS (1 injection); Water (1 injection); Matrix Blank (1 injection); Sample 1 (1 injection); Sample 2 (1 injection); WSS3 (3 injections); Water (1 injection). The system was considered suitable when there was acceptable separation quality; the signal to noise RECTIFIED SHEET (RULE 91) ISA/EP ~257- ratio for the shorter monosaccharide peak in the W881 sample was equal to or more than 10; the relative standard deviation (RSD) of the peak areas for each monosaccharide rd for the 6 injections of WSSB was equal or less than 4%; the correlation coefficient (r) was 0.99 (r was measured using software to plot the peak area of each working standard t the on-colurnn load (expressed as pmol) using the first three injections of the WSS3 standard and calculating the slope, intercept and correlation coefficient for the working standards using a linear least square regression model); the peak areas for peaks corresponding to monosaccharides were no more than 2% of the peak area measured for WSSS; and the peak areas for peaks corresponding to ccharides in water injection were no more than 0.5% of the peak areas measured for WSSS. 4. Sample Analysis The average corrected peak area for each ccharide in each sample preparation was determined. Valley~to-valley integration was used for the GalN peak.

To determine this, the linear curves generated from the working standards were used to calculate the amount of each monosaccharide loaded for each sample preparation.

For each type of monosaccharide, the average molar ratio of monosaccharides per protein molecule for each sample was calculated. Then, for each sample, the overall sum of the average molar ratios for all five monosaccharides was determined. The calculations were performed based on the following: lar weight (MW) ofnon- glycosylated onidase protein is 51106 g/mol; the total volume of each sample was 500 pl; the sample dilution factor is 0.15; the volume of each injection is 20 pl; and the sion factor from mg to pg is 109. The calculations were performed as follows for each monosaccharide: The amount ofmonosaccharide for each preparation was calculated using the following equation: ccharide (pmol) = Peak Area— Intercept Slope The number of monosaccharides per protein molecule was calculated by using the following formula: Monosaccharides per protein ratio = Monosaccharide (pmol) x MW x 500 pl . 0.1 mgx109x20 plXOJS RECTIFIED SHEET (RULE 91) ISA/EP For each sample, the results for each sample were ed as the monosaccharides per protein ratio for each monosaccharide along with the sum of the five monosaccharide ratios.

. Results The disaccharide assay described above was used to measure HA and its catabolites fiom patients enrolled in phase I clinical studies that ed IV doses of 0 at doses that ranged from 0.5 ug/kg to 50 ug/kg over a dosage regime cycle with or without dexamethasone. Plasma HA concentrations prior to 0 dosing were typically less than 1 ug/mL or below the level of quantification (0.5 ug/mL) for all patients in the study.

Plasma collected from a a patient that received a single 50 ug/kg dose of PEGPH20 was assessed over time after treatment. The results show that plasma concentrations of onan increased significantly. In this patient, while 0 concentrations declined with a terminal half-life of 2 days, elevated concentrations of HA catabolites accumulated more slowly and persist for up to 2 weeks post- PEGPH20 treatment with a maximum HA plasma concentration observed about 200 hours post-PEGPH20 dose.

Plasma also was collected from 12 additional patients beginning 24-hours post initial PEGPH20 dose that were either treated with 0.5 ug/kg PEGPH20 twice weekly (1 patient), 0.5 ug/kg every 21 days (3 patients), 0.75 ug/kg every 21 days (4 patients), 1.0 ug/kg every 21 days (3 ts), or 1.5 ug/kg every 21 days (1 patients). The s show that ing single or multiple doses of PEGPH20 that ranged from 0.5 ug/kg to 1.5 ug/kg, HA catabolite levels increased in a dose- dependent manner over the course of a week. Maximal HA concentrations (Cmax) and one-week area-under-the-curve-estimates (AUC0_16gh) were also determined for each patient to quantify the pharmacodynamic response. The results showed that systemic re to HA catabolites, as measured by maximum plasma concentration or area- under-the-curve increased with increasing dose of PEGPH20. In addition, blood samples from patients administered with PEGPH20 where dexamethasone was added to a dosing regime as a premedication to eliminate or ameliorate the musculoskeletal effects caused by PEGPH20 administration. The treatment cycle was defined as a 28- WO 63155 day period, with PEGPHZO administered enously (IV) and dexamethasone stered orally. Dosing of PEGPH20 and dexamethasone took place on days 1, 4, 8, 11, 15, 18, 22 and 25 of the 28-day cycle. On each dosing day, a premedication regimen of 4 mg of dexamethasone was stered orally one hour prior to the PEGPHZO, followed by a second dose of 4 mg dexamethasone 8-12 hours after PEGPHZO dosing. Plasma was taken at various time points after administration of PEGPHZO during the first week of treatment. Consistent with the observations in the samples from patients receiving only PH20 described above, plasma HA concentration vs. time data sed after administration ofPEGPHZO.

Concentrations of plasma HA measured during the first week of dosing increased with increasing dose of PEGPHZO. In three patients that completed a full cycle of treatment and received 8 doses of PEGPHZZO, the results showed sustained increased plasma HA concentrations in samples from all three patients measured throughout the dosing period.

These s are consistent with the expected mechanism ofPEGPH20 ty and support the role of HA as a ker for PEGPH20 phannacodyanmics.

Example 16 Magnetic Resonance Imaging Diffusion weighted MRI was performed using a single shot spin-echo sequence to estimate pixel-by-pixel values for apparent diffusion coefficient.

Dynamic contrast enhanced magnetic resonance imaging (DOE-MRI) included imaging during infusion with a contrast agent. Calibration was accomplished using a two part phantom containing an inner tube and ter mixture. Scans were performed eatment and post—treatment. 1. Apparent Diffusion Coefficient ic Resonance Imaging (ADC-MRI) Apparent diffusion coefficient magnetic Resonance imaging RI) measures the volume of water that has moved across the cell membrane based upon a calculation derived from the pre-and post-treatment scans. ADC-MRI scans were completed for a total of 10 ofthe 14 patients in a phase I clinical study assessing PEGPH20 treatment without dexamethasone premedication and in 4 of the 5 ts in a phase 1 clinical study assessing PEGPH20 treatment with dexamethasone RECTIFIED SHEET (RULE 91) ISA/EP premedication. Analysis of the images acquired from each patient was performed by a radiologist at Imaging Endpoints (Scottsdale, AZ), and quantitative estimates of ADC were computed for tissues in each patient. A summary of the ADC-MRI findings associated with tumor regions is shown in Table 18. As shown in the Table, increases in ADC-MRI were observed in 7 of 14 (50%) of patients ing PEGPHZO dosing. Increased ADC values are consistent with the mechanism of action of IZO. ADC values, however, did not change in 5 of 14 patients, and valnes decreased in 2 of 14 patients. ‘ Table 18: ADC—MRI Summa Change In Tumor ADC‘ Dose & ncy Post-Dose Scan Days MRI from baseline Exam le 15 50 /k_ HEDJ-P no chane no chan_e DJ increase 4}. se in] mh nodes 0.5 J f, k0;21 da 0 cle la increase DJ increase . b3 no chane 0.75 n_/k_; 21 day c cle D3, D30 increase 1.0 ; 21 day c cle LII increase 1.5 1 _;21da c cle La.) no chan_e Exam le 14 1.6 ng/kg + D3’ D29 . dexamethasone; 2x/wk .0 ug/kg + D1’ D4 . increase D1 dexamethasone; 2x/wk 16 its/kg + D25 D25 deCICaSC D25‘ dexamethagone; 2X/Wk 2. Dynamic Contrast ed Magnetic Resonance Imaging (DCE- MRI) Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) measures blood flow that indicates a change in tumor’s vascularity. Scans were completed in 4 patients in a phase I clinical study assessing PEGPHZO treatment with dexarnethasone premedication. Analysis es acquired from each patient was performed by a radiologist at Imaging Endpoints (Scottsdale, AZ), and quantitative estimates of volume transfer ient (Ktrans), blood volume (Vp) and extracellular RECTIFIED SHEET (RULE 91) ISA/EP volume fraction (Ve) were computed for tissues in each patient. A y of the DCE-MRI findings associated with tumor regions is set forth in Table 19. cant increases in the Ktrans parameter were observed in the two ts that were scanned on the day of PEGPH2O dosing. The increase in Ktrans within hours of dosing is consistent with preclinical data that show PEGPHZO causes vascular decompression and increased blood flow son er a]. (2010) Mol. Cancer Then, 9:3052-64.

Table 19: DCE-MRI Summa Dose & Frequency ' Post-Dose Scan Days Change in Tumor DCE- MRI from baseline 1.6 rig/kg + D3, D29 decrease in ktrans at D29 1 dexamethasone; 2x/wk .0 ug/kg + increase in ktrans, Ve, Vp dexamethasone; 2x/wk (8 hr). Return to baseline (D4) dexamethasone; 2x/wk Increase in Vp on D25 vs.

' D2. No chan_e in Ktrans 1.6 pig/kg + D1, D2 Increase in Ktrans (8hr, dexamethasone; Zx/wk 24hr. Increase in Ve for lung tumor but not liver tumor (D1, D2). No Chan e inV DCE-MRI imaging also was performed on a further patient with a pancreatic tumor enrolled in a phase I clinical study receiving 3.0 rig/kg + dexamethasone/wk in IO a cycle of administration for 28 days. Imaging was performed pre-dose and ose as s: 8 hours (Day 1), 24 hours (Day 2) and 3 days post the 4th weekly PEGPH20 dose in cycle 1 (end of cycle 1). The results are set forth in Table 20. The results shows that PEGPH20 increases tumor Ktrans ed by serial DCE-MRI.

Table 20: DCE-MRI Results from t Dosed 3.0 ug/kg + dexamethasonelwk Baseline Day 1 Day 2 End of Cycle 0.057 0.147 0.242 0.212 3. FDG-PET Imaging Positron emission tomography (PET) using FDG, an analogue of glucose, was used to give tissue metabolic activity in terms of regional glucose uptake. The FDG PET imaging was performed on a patient with metastatic rectal carcinoma with lung RECTIFIED SHEET (RULE 91) ISA/EP metastasis ed in a phase I clinical study receiving 3.0 ug/kg + dexamethasone; 2x/wk in a cycle of administration for 28 days. Imaging was performed se, 8 hours post-dose, 24 hours post-dose and at the end of the cycle (1 day after the 8th dose). The FDG standardized uptake value (SUV) was determined using standard methods. The results are set forth in Table 21. The results showed that the patient exhibited sed tumor metabolic activity post-PEGPH20 treatment of landmark pulmonary metastases.

TABLE 21: FDG-PET Results From Patient Dosed 3.0 __/k + dexamethasone; 2x/wk Anatomical Baseline A A Site (SUV) baseline baseline to 8 h to Day Superior . . -3 8% t of left lower lobe left lung . . -40% base right upper . . -4l% lobe at right perihilar lobe 4. Summary The results show that various tumor imaging modalities can be used to demonstrate and r activity of PEGPH20 in tumor tissue.

Example 17 TSG—6-Fc Tumor-Targeted g for HA-Rich Cancer Diagnosis and Treatment Hyaluronan-rich tumor-bearing mice or control mice were administered with TSG LM-Fc/AHep labeled with DyLight 755 Fluor Labeling reagent (TSGLM-Fc/ AHep DUSS), and mice were imaged to assess tumor-binding and distribution of TSG- 6-LM-Fc/ AHep DL755. Specificity also was assessed by comparing staining and distribution to an 55 control. For generation of BXPC3 peritibial tumor- g mice, mice were inoculated with BXPC-3 human atic adenocarcinoma (ATCC CRL—l687) tumor cells subcutaneously (s.c., right hind leg) at 1 x107 cells/0.1 mL. For generation of HA+3Dul45-Has2 and HA-DUl45 tumor-bearing mice, mice were inoculated with both Dul45-Has2 cells (generated as described above) and Dul45 cells bially (intramuscular injection nt to the right tibia periosteum on either side) at 5 X 106 L TSGLM-Fc/ AHep DUSS was generated by fluorescently ng TSG LM-Fc/AHep (generated as described in Example 9) with DyLight 755 using the Thermo Scientific DyLight 755 Amine-Reactive Dye kit (Catalog No. 84538; Thermo ific, Rockford, IL) ing to the manufacturers protocol. ””55 A. bution of TSG—6-LM-Fc/ AHep with and without pretreatment with PEGPH20 Mice g an HA+2BXPC3 peritibial tumor at about 18-20 mm in diameter DL755' were injected intravenously with 1 ug, 5 ug or 10 ug TSGLM-Fc/ AHep In one group of mice, mice were pretreated with intravenous administration of PEGPH20 at 4.5 mg/kg three (3) hours prior to administration of TSGLM-Fc/ AHepDL755.

A cent whole body image system (IVIS Lumina XR, Caliper Life Sciences, Mountain View, CA) was used to track fluorescence in the animal.

Selective excitation of DyLight755 was done using a D745 nm band-pass filter, and the d fluorescence was collected through a long-pass D800 nm filter. The 3 groups of mice (non-injected, TSGLM-Fc/ AHep DL755, and PEGPH20 + TSG LM-Fc/ AHep DL755) were imaged at various timepoints post TSGLM-Fc/ AHep DL755 (1 hours, 4 hours, day 1, day 2, day 3, day 4, day 5 and day 6). For imaging, non-injected control mice also were assessed. Fluorescent images were captured with a super cooled, high sensitivity, digital camera. Fluorescent images were later analyzed with Living Image (Caliper Life Sciences, Mountain View, CA).

The results show that by 1 hour and 4 hours after injection, TSGLM-Fc/ DUSS AHep was detected as circulating in the blood stream, and also was detected as starting to bind to the tumor. The binding to the tumor was dose-dependent, with increased staining intensity observed with the 10 ug dose. Less tumor binding was detected by imaging in mice treated with PEGPH20 at all doses and time points. At later time points after injection (e.g. day l or day 2), liver g also was detected, although this was less in the mice injected with the lug low dose of TSGLM-Fc/ DL755 reached peak levels between day AHep DUSS. TSGLM-Fc/ AHep l and 2 as assessed by image analysis. DL755 In low-dose treated mice, TSGLM-Fc/ AHep was eliminated day 3 after ion. TSGLM-Fc/ AHep DUSS was sill circulating in high-dose treated mice 5 days post injection, and all binding to the tumor was shed 6 days after injection.

DUSS In sum, the in viva imaging results show that TSGLM-Fc/ AHep binding was dose-dependent and reached peaked levels 1-2 days post-inj ection.

Further, HA removal by PEGPH20 resulted in less TSGLM-Fc/ AHep DUSS binding. TSGLM-Fc/ AHep DUSS binding was eliminated from the tumor 6 days post injection.

DL755 B. Comparison of TSG—6-LM-Fc/ AHep binding between Du145 tumor +/- H2152 HA+3Dul45-Has2 and HA-DU145 tumor-bearing mice were injected intravenously with 5 ug TSGLM-Fc/ AHep DUSS’ The mice were imaged daily post TSGLM-Fc/ AHep DUSS injection. Although a low-level background staining of DL755 45 tumor was detected, there was much more TSGLM-Fc/ AHep binding to HA-rich Dul45-Has2 as assessed by image results. The binding peaked at day 1-2 as determined by staining intensity. Thus, the results show that the more HA that is present in the tumor, the more TSGLM-Fc/ AHep DL755 binds to the tumor.

DL755 C. Targeting Specificity of TSGLM-Fc/ AHep The specificity of TSGLM-Fc/ AHep DUSS for HA-rich tumors was r DL755 assessed by comparing binding of TSGLM-Fc/ AHep or 55 t0 HA+2BXPC3 bial tumor-bearing mice. HA++ BXPC3 peritibial tumor-bearing mice were injected intravenously with 5 ug TSGLM-Fc/ AHep DL755 or with 5 ug IgGDL755. The mice were imaged daily after ion. The imaging results showed little to no detectable staining of IgGDL755 to the tumor, and thus r binding of TSGLM-Fc/ AHep DUSS to PC3 tumor than IgGDL755.

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.

Claims (55)

CLAIMS :

1. A TSGlink module (LM) multimer, comprising: a first polypeptide containing a TSGLM linked directly or indirectly via a linker to a first multimerization domain; and a second polypeptide containing a TSGLM linked directly or indirectly via a linker to a second multimerization domain, wherein: the first and second multimerization domains each comprise a sequence of amino acids, whereby the first and second polypeptides form a multimer; and the TSGLM multimer has a g affinity to hyaluronan (HA) with an association constant of at least 107 M-1.

2. The TSGLM multimer of claim 1, wherein the link module is the only TSG-6 portion of the first polypeptide and the second polypeptide.

3. The TSGLM er of claim 1 or claim 2, n the first and second link module are the same or different.

4. The LM er of any of claims 1-3, wherein each of first and second polypeptides do not comprise the full-length of TSG-6.

5. The TSGLM multimer of any of claims 1-4, wherein the TSGLM comprises the sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 418 or a sequence of amino acids comprising at least 85% amino acid sequence identity to the sequence of amino acids set forth in SEQ ID NO: 207, 360, 417 or 418 that specifically binds to HA.

6. The TSGLM multimer of any of claims 1-5, wherein the TSGLM contains amino acid cations to reduce or eliminate binding to n.

7. The TSGLM multimer of claim 6, n binding to heparin is reduced at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, , 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold or more.

8. The TSGLM multimer of claim 6 or claim 7, wherein the LM comprises an amino acid replacement at an amino acid position corresponding to amino acid residue 20, 34, 41, 54, 56, 72 or 84 set forth in SEQ ID NO:360, y a corresponding amino acid residue is identified by alignment to a TSGLM set forth in SEQ ID NO:360.

9. The TSGLM multimer of claim 8, wherein the amino acid replacement is to a non-basic amino acid residue selected from among Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Ala (A), Val (V), Ile (I), Leu (L), Met (M), Phe (F), Tyr (Y) and Trp (W).

10. The TSGLM er of any of claims 6-9, wherein the TSGLM ses an amino acid replacement corresponding to amino acid replacement K20A, K34A or K41A in a TSGLM set forth in SEQ ID NO:360 or the replacement at the corresponding residue in another TSGLM.

11. The TSGLM multimer of any of claims 6-10, wherein the TSGLM comprises amino acid replacements corresponding to amino acid replacements K20A, K34A and K41A in a TSGLM set forth in SEQ ID NO:360 or the replacement at the corresponding residue in another TSGLM.

12. The TSGLM multimer of any of claims 6-11, wherein the TSGLM comprises the sequence of amino acids set forth in SEQ ID NO:361 or 416 or a sequence of amino acids comprising at least 85% amino acid sequence identity to the ce of amino acids set forth in SEQ ID NO: 361 or 416 that ically binds to HA.

13. The TSGLM er of any of claims 1-12, wherein the multimerization domain is selected from among an immunoglobulin constant region (Fc), a leucine zipper, complementary hydrophobic regions, complementary hydrophilic s, compatible protein-protein interaction domains, free thiols that forms an intermolecular disulfide bond between two molecules, and a protuberance-into-cavity and a satory cavity of cal or similar size that form stable multimers.

14. The TSGLM multimer of claim 13, wherein the multimerization domain is an Fc domain or a variant thereof that effects multimerization.

15. The TSGLM multimer of any of claims 1-14, wherein the first and second polypeptide comprise a LM and an immunoglobulin Fc domain.

16. The TSGLM multimer of any of claims 1-15, sing the sequence of amino acids set forth as amino acids 24-349 of SEQ ID NO: 212 or 215 or a sequence of amino acids that exhibits at least 85% amino acid sequence identity to amino acids 24-349 of SEQ ID NO:212 or 215.

17. The LM multimer of any of claims 1-16, wherein the TSGLM multimer comprises a ce of amino acids encoded by the sequence of nucleotides set forth in any of SEQ ID NOS: 211, 214 or 217, or a sequence of nucleotides that exhibits at least 85% sequence identity to the sequence of nucleotides set forth in any of SEQ ID NOS: 211, 214 or 217.

18. The TSGLM multimer of any of claims 1-17 that has a g ty to HA with an association constant (Ka) of at least 1 x 108 M-1.

19. A nucleic acid molecule encoding the TSGLM multimer of any of claims 1- 18, wherein: the nucleic acid le encodes a fusion polypeptide containing a TSGLM linked directly or ctly via a linker to a erization domain; and the multimerization domain is a polypeptide that interacts with itself to form a stable n-protein interaction, whereby the encoded protein forms a multimer containing at least two TSG-6 link modules.

20. The nucleic acid molecule of claim 19, comprising the sequence of nucleotides set forth in any of SEQ ID NOS:211, 214 or 217, or a sequence of nucleotides that exhibits at least 85% sequence identity to the sequence of nucleotides set forth in any of SEQ ID NOS: 211, 214 or 217.

21. A vector, comprising the nucleic acid molecule of claim 19 or claim 20.

22. An isolated cell or cell culture, comprising the nucleic acid molecule of claim 19 or claim 20.

23. A method of producing a TSGLM multimer, comprising: introducing the nucleic acid molecule of claim 19 or claim 20 into a cell; culturing the cell under conditions whereby the fusion polypeptide is expressed by the cell; and optionally recovering the TSGLM multimer.

24. A method for selecting a subject for treatment of a tumor with an antihyaluronan agent, comprising: contacting a tissue or body fluid sample previously obtained from a subject who has a tumor or cancer with a TSGLM er of any of claims 1-18; and detecting binding of the TSGLM multimer to the sample, thereby determining the amount of hyaluronan in the sample, wherein if the amount of hyaluronan in the sample is at or above a predetermined threshold level, selecting the t for treatment with an antihyaluronan agent.

25. The method of claim 24, wherein the predetermined threshold level is high

26. The method of claim 24 or claim 25, wherein: the predetermined old level is at least or above 0.025 µg HA/ml of sample, 0.030 µg/ml, 0.035 µg/ml, 0.040 µg/ml, 0.045 µg/ml, 0.050 µg/ml, 0.055 µg/ml, 0.060 µg/ml, 0.065 µg/ml, 0.070 µg/ml, 0.08 µg/ml, 0.09 µg/ml. 0.1 µg/ml, 0.2 µg/ml, 0.3 µg/ml or higher; or the sample is a tumor tissue sample, and the predetermined threshold is an HA score of at least +2 (HA+2) or at least +3 (HA+3); or the sample is a tumor tissue sample, and the predetermined threshold level is at least a percent HA positive pixels in tumor (cells and ) to total stain in tumor tissue of at least 10%, 10% to 25% or greater than 25%.

27. The method of claim 26, wherein the predetermined threshold level is an HA score of at least +3 (HA+3) (high levels).

28. A method for predicting efficacy of treatment of a subject with an antihyaluronan agent, sing: contacting a tissue or body fluid sample from a subject who is or has been d with an anti-hyaluronan agent with a LM multimer of any of claims 1-18; and detecting g of the TSGLM multimer to the sample, thereby determining the amount of hyaluronan in the sample, wherein detection of a decrease in hyaluronan compared to before treatment with the anti-hyaluronan agent or before the us dose of antihyaluronan agent tes that the treatment is effective.

29. The method of any of claims 24-28, n the anti-hyaluronan agent is a hyaluronan ing enzyme or is an agent that inhibits hyaluronan synthesis.

30. The method of claim 29, wherein the anti-hyaluronan agent is a hyaluronan degrading enzyme that is a onidase.

31. The method of any of claims 24-30, wherein the yaluronan agent is a soluble PH20 hyaluronidase or a C-terminally truncated form of a human PH20 hyaluronidase that lacks all or a portion of the GPI attachment site.

32. The method of claim 31, wherein the soluble hyaluronidase is a human PH20.

33. The method of any of claims 30-32, wherein the PH20 or truncated form thereof comprises the sequence of amino acids set forth in any of SEQ ID NOS: 4-9, 47, 48, 150-170 and 183-189 or a ce of amino acids that exhibits at least 85% sequence identity to any of SEQ ID NOS: 4-9, 47, 48, 150-170 and 183-189.

34. The method of any of claims 24-33, wherein the anti-hyaluronan agent is a hyaluronan-degrading enzyme that is modified by conjugation to a polymer.

35. The method of claim 34, wherein the polymer is PEG and the hyaluronan degrading enzyme is PEGylated.

36. A method of diagnosis of a hyaluronan-associated disease or condition, comprising: contacting a tissue or body fluid sample previously obtained from a subject with a TSGLM multimer; and detecting binding of the TSGLM multimer to the sample, thereby determining the amount of hyaluronan in the sample, wherein the subject is diagnosed with a hyaluronanassociated disease or condition if the amount of onan is above a predetermined level or above the hyaluronan level of a reference sample.

37. The method of claim 36 , wherein the hyaluronan-associated disease or condition is a tumor or cancer.

38. The method of claim 36 or claim 37, wherein the predetermined or reference level is the median level of hyaluronan present in healthy or normal tissue or fluid samples from a population of control ts.

39. The method of any of claims 36-38, wherein: the predetermined threshold level is greater than 0.015 µg HA/ml of sample; or the sample is a tumor or tissue sample, and the predetermined threshold level is an HA score of HA+1, HA+2 or HA+3.

40. The method of any of claims 24-39, wherein the TSGLM is modified to reduce or eliminate binding to heparin.

41. The method of claim 40, wherein binding to heparin is reduced at least 1.2- fold, ld, 2-fold, 3-fold, 4-fold, , 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30- fold, 40-fold, 50-fold, 100-fold or more.

42. The method of claim 40 or claim 41, wherein TSGLM comprises an amino acid replacement selected from an amino acid replacement corresponding to amino acid replacement K20A, K34A or K41A in a LM set forth in SEQ ID NO:360 or the replacement at the corresponding residue in another TSGLM.

43. The method of claim 40 or claim 41, wherein TSGLM comprises amino acid replacements corresponding to amino acid replacements K20A, K34A and K41A in a TSGLM set forth in SEQ ID NO:360 or the replacement at the corresponding e in another TSGLM.

44. The method of any of claims 24-43, wherein the multimerization domain is selected from among an immunoglobulin constant region (Fc), a leucine zipper, complementary hydrophobic regions, complementary hydrophilic regions, ible protein-protein interaction domains, free thiols that forms an intermolecular disulfide bond between two molecules, and a erance-into-cavity and a compensatory cavity of identical or similar size that form stable ers.

45. The method of claim 44, wherein the multimerization domain is an Fc domain or a variant thereof that effects multimerization.

46. The method of any of claims 42-45, n the TSGLM multimer is a fusion protein that ns a TSGLM and an immunoglobulin Fc .

47. The method of claim 46, n the TSGLM-Fc has the sequence of amino acids set forth as amino acids 24-349 of SEQ ID NO: 212 or 215 or a sequence of amino acids that exhibits at least 85% amino acid sequence identity to amino acids 24-349 of SEQ ID NO:212 or 215 and specifically binds to HA.

48. The method of claim 46 or claim 47, wherein the TSGLM-Fc comprises the sequence of amino acids encoded by the sequence of nucleotides set forth in any of SEQ ID NOS: 211, 214 or 217, or a sequence of nucleotides that exhibits at least 85% sequence ty to the sequence of nucleotides set forth in any of SEQ ID NOS: 211, 214 or 217.

49. The method of any of claims 24-48, wherein the TSGLM multimer binds to HA with a binding affinity that has a dissociation constant (Kd) of at least less than or less than 1 x 10-7 M.

50. The method of any of claims 24-49, wherein the TSGLM multimer is conjugated to a detectable moiety that is detectably labeled or that can be detected.

51. The method of any of claims 24-50, wherein: the sample previously obtained from a subject is a stromal tissue sample; or the sample is a fluid sample that is a blood, serum, urine, sweat, semen, saliva, cerebral spinal fluid, or lymph .

52. The method of any of claims 24-51, wherein the sample is a stromal tissue sample from a tumor.

53. The method of claim 52, wherein the tumor is of a cancer selected from among breast cancer, pancreatic , n cancer, colon cancer, lung cancer, non-small cell lung cancer, in situ oma (ISC), squamous cell carcinoma (SCC), thyroid cancer, cervical cancer, uterine cancer, prostate cancer, testicular cancer, brain cancer, bladder cancer, stomach cancer, hepatoma, melanoma, glioma, retinoblastoma, mesothelioma, myeloma, lymphoma, and leukemia.

54. The method of claim 52 or claim 53, wherein the tumor is of a cancer that is a late-stage cancer, a metastatic cancer and an undifferentiated cancer.

55. The method of any of claims 24-54, wherein onan is detected by a solid phase binding assay, by histochemistry, or by in vivo imaging.