WO2004082610A2 - Modulation of hyaluronan and cd44 interaction and uses thereof in treating disorders - Google Patents

Abstract

Methods of modulating the interaction of hyaluronan and CD44, screening assays to identify agents for modulating the interaction, and methods of treatment of disorders mediated by CD44-HA interaction are provided herein.

Description

10286-012W01 / BWH 899

MODULATION OF HYALURONAN AND CD44 INTERACTION AND USES THEREOF IN TREATNG DISORDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S.S.N. 60/454,719, filed, March 14, 2003, the contents of which are hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This invention relates to methods of modulating the interaction of hyaluronan and CD44, screening assays to identify agents for modulating the interaction, and methods of treatment of disorders mediated by CD44-HA interactions.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

The work described herein was funded, in part, through a grant from the National Institutes of Health (Grant No. CA84156 awarded to Robert Sackstein). The United States government may, therefore, have certain rights in the invention.

BACKGROUND

Hyaluronan (HA, also termed hyaluranic acid or hyaluronate), is a high molecular- weight, negatively charged, uniformly repetitive, linear glucosaminoglycan composed of disaccharides of glucuronic acid and N-acetylglucosamine. HA is found in extracellular matrices, at the cell surface and inside cells (Toole, B. P., Semin. CellDev. Biol.12:79-87, 2001). The hyaluronan synthases (identified as Hasl, Has2, and Has3) are integral plasma membrane proteins responsible for HA production (DeAngelis, P.L., Cell Mol Life Set 56:670-682, 1999; Weigel, P.H. et al, JBiol Chem. 272:13997-14000, 1997). It has been suggested that HA plays an important role in several biological processes including embryonic development and morphogenesis (Toole, B. P., Semin. CellDev. Biol.12:79-87, 2001; Camenisch, T.D., et al., J Clin Invest. 106:349-360, 2000), wound healing (Oksala O., et al., J Histochem Cytochem. 43:125-135, 1995; Noble, P.W., Matrix Biol. 21:25-29, 2002), tumor invasion and metastasis (Auvinen, P.K., et al, Int J Cancer. 74:477-481, 1997; Ropponen K., et al, Cancer Res. 58:342- 347, 1998; Hiltunen, E ., et al, Cancer Res. 62:6410-6413, 2002; Toole B.P., Glycobiol. 10286-012W01 / BWH 899

12:37R-42R, 2002). It has also been shown that activation of various components of intracellular signaling pathways result from interaction of HA with CD44 (a major HA-receptor) (Toole, B. P., Semin. CellDev. BiolΛ2:79-S7, 2001; Kamikura D.M., et a\., Mol Cell Biol. 20:3482-3496, 2000; Oliferenko, S., et al., J Cell Biol. S:l l59-l l64, 2000). Other studies, in contrast, imply a role for HA on the (lumenal) aspect of blood vessels. CD44 on lymphocytes interacts with HA on endothelium and participates in preferential homing of activated cells to tertiary sites of inflammation (Mohamadzadeh, M., et al, J Clin invest.101 :97-108, 1998; DeGrendele, H.C., et al, Science. 278:672-675, 1997; Nandi, A., et al., JBiol Chem. 275:14939-14948, 2000).

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the treatment of choice for many hematologic malignancies, aplastic anemia and certain genetic diseases. Acute graft- versus-host disease (aGVHD) is a major complication of this curative therapy, resulting in significant morbidity and mortality. This can occur in up to 80% of recipients depending on the degree of HLA disparity between donor and host. The skin, gastrointestinal tract and the liver are the principal target organs affected by aGVHD. Although liver and gut involvement alone may occur in some cases, the skin is involved in over 80% of patients with aGVHD and acute cutaneous GVHD (acGVHD) frequently occurs without other organ involvement. Steroids are the primary treatment for aGVHD reactions.

SUMMARY

The invention is based, in part, on the observation that binding of CD44 to hyaluronan mediates pathologic cell adhesion interactions. In particular, it was found that by reducing synthesis or expression of hyaluronan or the ability of HA to interact with CD44, various disorders mediated by CD44-HA interaction can be treated. It was also found that grafts versus host disease (GVHD) is a disorder mediated by CD44-HA interaction and that the methods described herein can be used to treat GVHD in a subject.

Accordingly, in one aspect, the invention features a method for treating a disorder characterized by CD44-hyaluronan interactions. The method includes administering to a subject an agent that decreases the expression or synthesis of hyaluronan, to thereby treat the disorder. The method can further include identifying a subject having or at risk for having a CD44- hyaluronan mediated disorder prior to the administering. 10286-012W01 / BWH 899

In one embodiment, the method includes administering the agent at a dose sufficient to reduce the expression or synthesis of hyaluronan, and/or inhibit interaction of CD44 with hyaluronan. In one embodiment, the agent is administered at a dose that does not alter the interaction of CD44 with another ligand, e.g., E-selectin, L-selectin, or both, hi a related embodiment, the agent is administered at a dose that does not alter expression of a specific glycoform of CD44, e.g., a glycoform having sialylated epitopes, e.g., an HCELL glycoform.

In one embodiment, the agent degrades hyaluronan. For example, the agent can be a glycosidase, e.g., hyaluronidase.

In another embodiment, the agent is a sugar analog, e.g., any synthetic sugar analog that interferes with HA synthesis, e.g., a fluorinated sugar analog, e.g., 3-F-GlcNAc or 4-F-GlcNAc.

In yet another embodiment, the agent inhibits a hyaluronan synthase (e.g., HAS1, HAS2, or HAS3). For example, the agent can be a small inhibitory RNA (siRNA) that inhibits a hyaluronan synthase.

In other embodiments, the agent is any available agent that interferes with the synthesis and expression of HA. For example, butyrate is an agent that interferes with synthesis of HA.

In one embodiment, the agent is administered topically, subcutaneously, intradermally, intravenously, orally, transmucosally, or rectally. hi one embodiment, the agent is administered locally (e.g., to a specific lesion). In another embodiment, the agent is administered systemically.

In one embodiment, the method further includes the step of determining a change in CD44-hyaluronan interaction in the subject. In another embodiment, the method further includes the step of evaluating the presence of hyaluronan after administration, e.g., to evaluate further dosing.

In one embodiment, the CD44-HA mediated disorder is a cancer. The cancer can be, for example, a cutaneous cancer such as melanoma, basal cell and squamous cell carcinoma, or Kaposi's sarcoma. The cancer can be a leukemia or a lymphoma (e.g., chronic myeloid leukemia, chronic lymphatic leukemia, chronic granulocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myelocytic leukemia, Hodgkin's disease, non- Hodgkin's lymphomas, Burkitt's lymphomas, and mycosis fungoides). The cancer can be a neoplastic disorder or a disorder of solid tumors such as an adenocarcinoma of the lung, kidney, uterus, prostate, bladder, ovary, colon; a sarcoma such as liposarcoma, synovial sarcoma, 10286-012W01 / BWH 899

rhabdomyosarcoma, Ewing's tumor, neuroepithelioma; or another tumor such as retinoblastoma, Wilm's tumor, and mesothelioma. hi a preferred embodiment, the cancer is an ovarian cancer.

In one embodiment, the CD44-HA mediated disorder is an inflammatory disorder, e.g., an autoimmune, allergic, transplantation-associated, or pathogen-associated disorder. The inflammatory disorder can be a cutaneous inflammatory disorder, e.g., contact dennatitis, atopic dermatitis, a cutaneous infection (e.g., a bacterial, viral, fungal, or parasitic infection of the skin), psoriasis, acute cutaneous graft versus host disease, acne, a drug-related hypersensitivity reaction, pemphigus vulgaris). The inflammatory disorder can be a disorder of the pancreas, e.g., insulin-dependent diabetes mellitus. h another embodiment, the inflammatory disorder is an inflammatory disorder of bones or joints (e.g., rheumatoid arthritis, ankylosing spondylitis), of the nervous system (e.g., multiple sclerosis), of the gut (e.g., inflammatory bowel disease, Crohn's disease, ulcerative colitis), of the endocrine system (e.g., Grave's disease, Hashimoto's thyroiditis), or of the renal and hepatic system, or a multi-organ inflammatory disorder (e.g., lupus erythematosus). In one embodiment, the inflammatory disorder is sarcoidosis. h one embodiment, the disorder is a disorder caused by a pathogen, e.g., a bacterial, viral, fungal, or parasitic pathogen. h one embodiment, the disorder is a vascular disorder.

In another aspect, the invention features a method for treating a disorder characterized by CD44-hyaluronan interactions. The method includes administering to a subject an agent that decreases binding of hyaluronan to CD44, thereby treating the disorder. The method can further include identifying a subject having or at risk for having a CD44-hyaluronan mediated disorder prior to the administering. hi one embodiment, agent is a hyaluronan antagonist. The hyaluronan antagonist can be a small molecule, a peptide, a carbohydrate, a peptide-carbohydrate complex, or a nucleic acid. The hyaluronan antagonist can be a carbohydrate selected from the group consisting of: hyaluronan hexasaccharides, chondroitin, or high molecular weight hyaluronan.

In another embodiment, the agent increases sialylation of CD44. For example, the agent can be a sialidase inhibitor, such as 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid. 10286-012W01 / BWH 899

In another embodiment, the antagonist is a peptide, e.g., a peptide that inhibits binding between CD44 and HA. In another embodiment, the antagonist is an antibody or antigen- binding fragment thereof. The antibody can be an anti-hyaluronan antibody.

In one embodiment, the administering is performed in vivo, h other embodiments, the administering is performed in vitro.

The method can further include the step of determining a change in CD44-hyaluronan interaction in the subject. hi one embodiment, the agent is administered topically, subcutaneously, intradermally, intravenously, orally, transmucosally, or rectally. The agent can be administered locally (e.g., to a specific lesion). Alternatively, the agent can be administered systemically.

In one embodiment, the method further includes the step of determining a change in CD44-hyaluronan interaction in the subject. In another embodiment, the method further includes the step of evaluating the presence of hyaluronan after administration, e.g., to evaluate further dosing.

In one embodiment, the CD44-HA mediated disorder is a cancer. The cancer can be, for example, a cutaneous cancer such as melanoma, basal cell and squamous cell carcinoma, or Kaposi's sarcoma. The cancer can be a leukemia or a lymphoma (e.g., chronic myeloid leukemia, chronic lymphatic leukemia, chronic granulocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myelocytic leukemia, Hodgkin's disease, non- hodgkin's lymphomas, Burkitt's lymphomas, and mycosis fungoides). The cancer can be a neoplastic disorder or a disorder of solid tumors such as an adenocarcinoma of the lung, kidney, uterus, prostate, bladder, ovary, colon; a sarcoma such as liposarcoma, synovial sarcoma, rhabdomyosarcoma, Ewing's tumor, neuroepithelioma; or another tumor such as retinoblastoma, Wilm's tumor, and mesothelioma. In a preferred embodiment, the cancer is an ovarian cancer.

In one embodiment, the CD44-HA mediated disorder is an inflammatory disorder, e.g., an autoimmune, allergic, transplantation-associated, or pathogen-associated disorder. The inflammatory disorder can be a cutaneous inflammatory disorder, e.g., contact dermatitis, atopic dermatitis, a cutaneous infection (e.g., a bacterial, viral, fungal, or parasitic infection of the skin), psoriasis, acute cutaneous graft versus host disease, acne, a drug-related hypersensitivity reaction, pemphigus vulgaris). The inflammatory disorder can be a disorder of the pancreas, e.g., insulin-dependent diabetes mellitus. In another embodiment, the inflammatory disorder is an 10286-012W01 / BWH 899

inflammatory disorder of bones or joints (e.g., rheumatoid arthritis, ankylosing spondylitis), of the nervous system (e.g., multiple sclerosis), of the gut (e.g., inflammatory bowel disease, Crohn's disease, ulcerative colitis), of the endocrine system (e.g., Grave's disease, Hashimoto's thyroiditis), or of the renal and hepatic system, or a multi-organ inflammatory disorder (e.g., lupus erythematosus). In one embodiment, the inflammatory disorder is sarcoidosis. In one embodiment, the disorder is a disorder caused by a pathogen, e.g., a bacterial, viral, fungal, or parasitic pathogen.

In one embodiment, the disorder is a vascular disorder.

In another aspect, the invention features a method for treating GVHD. In one embodiment, the method for treating GVHD includes administering an agent which inhibits expression or synthesis of HA.

In one embodiment, the method includes administering the agent at a dose sufficient to reduce the expression or synthesis of hyaluronan, and/or inhibits interaction of CD44 with hyaluronan. In one embodiment, the agent is administered at a dose that does not alter the interaction of CD44 with another ligand, e.g., E-selectin, L-selectin, or both. In a related embodiment, the agent is administered at a dose that does not alter expression of a specific glycoform of CD44, e.g., a glycoform having sialylated epitopes, e.g., an HCELL glycoform. h one embodiment, the agent degrades hyaluronan. The agent can be a glycosidase, e.g., hyaluronidase.

In another embodiment, the agent is a sugar analog, e.g., a fluorinated sugar analog, e.g., 3-F-GlcNAc or 4-F-GlcNAc.

In yet another embodiment, the agent inhibits a hyaluronan synthase (e.g., HAS1, HAS2, or HAS3). The agent can be a small inhibitory RNA (siRNA) that inhibits a hyaluronan synthase.

In one embodiment, the agent decreases interaction of hyaluronan with a CD44- expressing cell. The CD44-expressing cell can be a leukocyte, e.g., a mature leukocyte. The leukocyte can be a lymphocyte, e.g., a T cell.

In one embodiment, the agent is administered topically, subcutaneously, intradermally, intravenously, orally, transmucosally, or rectally. In one embodiment, the agent is administered locally (e.g., to a specific lesion). In another embodiment, the agent is administered systemically. 10286-012W01 / BWH 899

In another embodiment, the method for treating GVHD includes decreasing interaction of HA with CD44. i one embodiment, agent is a hyaluronan antagonist. The hyaluronan antagonist can be a small molecule, a peptide, a carbohydrate, a peptide-carbohydrate complex, or a nucleic acid. The hyaluronan antagonist can be a carbohydrate selected from the group consisting of: hyaluronan hexasaccharides, chondroitin, or high molecular weight hyaluronan.

In another embodiment, the agent increases sialylation of CD44. For example, the agent can be a sialidase inhibitor, such as 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid.

In another embodiment, the antagonist is a peptide, e.g., a peptide that inhibits binding between CD44 and HA. In another embodiment, the antagonist is an antibody or antigen- binding fragment thereof. The antibody can be an anti-hyaluronan antibody.

In one embodiment, the administering is performed in vivo. In other embodiments, the administering is performed in vitro.

The method can further include the step of determining a change in CD44-hyaluronan interaction in the subject.

In one embodiment, the agent is administered topically, subcutaneously, intradermally, intravenously, orally, transmucosally, or rectally. The agent can be administered locally (e.g., to a specific lesion). Alternatively, the agent can be administered systemically.

In another aspect, the invention features a method for inhibiting interaction of CD44 with hyaluronan. The method includes contacting a hyaluronan-expressing cell or hyaluronan with an agent that decreases synthesis or expression of hyaluronan, thereby decreasing interaction of CD44 with hyaluronan.

In one embodiment, the agent is a hyaluronan antagonist. A hyaluronan antagonist can be a nucleic acid, a peptide, a carbohydrate, a peptide-carbohydrate complex, or a small molecule.

In one embodiment, the agent degrades hyaluronan, e.g., the agent is a glycosidase, e.g., hyaluronidase.

In one embodiment, the agent is a sugar analog, any sugar analog that disrupts HA synthesis, e.g., a fluorinated analog, e.g., 3-F-GlcNAc or 4-F-GlcNAc. 10286-012W01 / BWH 899

hi another embodiment, the agent inhibits a hyaluronan synthase (e.g., hyaluronan synthase 1 (HASl), hyaluronan synthase 2 (HAS2), or hyaluronan synthase 3 (HAS3). An agent that inhibits a hyaluronan synthase can be a small inhibitory RNA (siRNA) of a hyaluronan synthase.

In other embodiments, the agent is any available agent that interferes with the synthesis and expression of HA. For example, butyrate is an agent that interferes with synthesis of HA.

In some embodiments, the contacting is performed in vivo. In other embodiments, the contacting is performed in vitro.

In one embodiment, the hyaluronan-expressing cell or hyaluronan is contacted in the presence of CD44 or a CD44-expressing cell. The CD44-expressing cell is a leukocyte, e.g., a mature leukocyte. The leukocyte can be a lymphocyte, e.g., a T cell.

In another aspect, the invention features a method for inhibiting interaction of CD44 with hyaluronan. The method includes contacting a hyaluronan-expressing cell or hyaluronan with an agent that decreases binding of hyaluronan to CD44, thereby decreasing interaction of CD44 with hyaluronan.

The agent can be a hyaluronan antagonist, e.g., a small molecule, a peptide, a carbohydrate, a peptide-carbohydrate complex, or a nucleic acid.

In one embodiment, the hyaluronan antagonist is a carbohydrate selected from the group consisting of: hyaluronan hexasaccharides, chondroitin, or high molecular weight hyaluronan.

In one embodiment, the HA antagonist is an agent that increases sialylation of CD44. For example, the antagonist is an agent that inhibits a sialidase, e.g., 2-deoxy-2,3-dehydro-N- acetyl-neuraminic acid.

In one embodiment, the hyaluronan expressing cell or hyaluronan is contacted in the presence of CD44 or a CD44-expressing cell.

In another embodiment, the antagonist is a peptide, an antibody, or an antigen-binding fragment thereof. The antibody can be an anti-hyaluronan antibody. In some embodiments, the contacting is performed in vivo. In other embodiments, the contacting is performed in vitro.

In one embodiment, the CD44-expressing cell is a leukocyte, e.g., a mature leukocyte. The leukocyte can be a lymphocyte, e.g., a T cell. 10286-012W01 / BWH 899

In another aspect, the invention features a kit for treating a disorder characterized by CD44-hyaluronan interactions. The kit includes an agent that decreases the expression or synthesis of hyaluronan and instructions for administering the agent to a subject having or at risk for having a disorder characterized by CD44-hyaluronan interactions.

In one embodiment, the agent degrades hyaluronan. The agent can be a glycosidase, e.g., hyaluronidase.

In another embodiment, the agent is a sugar analog, e.g., a fluorinated sugar analog, e.g., 3-F-GlcNAc or 4-F-GlcNAc.

In yet another embodiment, the agent inhibits a hyaluronan synthase (e.g., HASl, HAS2, or HAS3). The agent can be a small inhibitory RNA (siRNA) that inhibits a hyaluronan synthase.

The instructions can include information regarding doses of the agent effective to inhibit synthesis or expression of HA. The kit can also include instructions with information regarding doses of the agent effective to inhibit synthesis or expression of HA such that interaction of CD44 with another ligand (e.g., E-selectin or L-selectin, or both) is not altered. For example, the dosage can be a dosage that does not alter expression of a specific glycoform of CD44, e.g., a sialylated glycoform, e.g., HCELL.

In one embodiment, the instructions include information for treating an inflammatory disorder of the skin by topical administration of the agent. In one embodiment, the disorder is GVHD.

In another aspect, the invention features a kit for treating a disorder characterized by CD44-hyaluronan interactions. The kit includes an agent that decreases binding of hyaluronan to CD44; and instructions for administering the agent to a subject suffering from or at risk for a disorder characterized by CD44-hyaluronan interactions.

In one embodiment, the agent is a hyaluronan antagonist, e.g., a small molecule, a peptide, a carbohydrate, a peptide-carbohydrate complex, or a nucleic acid. h one embodiment, the antagonist increases sialylation of CD44, e.g., the agent inhibits a sialidase. For example, the agent is 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid.

In another embodiment, the antagonist is a peptide, an antibody, or an antigen-binding fragment thereof. In one embodiment, the antibody is an anti-hyaluronan antibody 10286-012W01 / BWH 899

In another aspect, the invention features a method for identifying an agent useful in the treatment of a CD44-hyaluronan mediated disorder. The method includes contacting a reaction mixture which includes HA and CD44 and with a test compound; and evaluating the ability of the test compound to reduce a CD44-hyaluronan interaction, wherein a reduction in a CD44- hyaluronan interaction is an indication that the test compound is a candidate for the treatment of a CD44-hyaluronan mediated disorder.

In one embodiment, the test compound is a small molecule, a peptide, a carbohydrate, a peptide-carbohydrate complex, or a nucleic acid. hi one embodiment, the reaction mixture includes an HA-expressing cell, and/or a CD44- expressing cell. The CD44-expressing cell can be a leukocyte.

In one embodiment, a binding assay is used to evaluate the ability of a test compound to inhibit a CD44-HA interaction.

In one embodiment, the reaction mixture further includes a tissue section, e.g., a tissue section which expresses HA (or CD44).

In one embodiment, a Stamper- Woodruff assay is used to evaluate the ability of a test compound to inhibit a CD44-HA interaction.

In one embodiment, method is used to identify an agent useful in the treatment of a cutaneous disorder. The cutaneous disorder can be acute cutaneous graft versus host disease.

In some embodiments, the contacting step of the method is performed in vivo. In other embodiments, the contacting is performed in vitro. In some embodiments, the cells are incubated with the test compound prior to the evaluating. In some embodiments, the method is repeated one or more times to evaluate a plurality of test compounds as candidate compounds.

The method can further include selecting a test compound from the plurality which reduces CD44-hyaluronan interactions.

The method for identifying an agent useful in the treatment of a CD44-hyaluronan mediated disorder can further include the step of evaluating the ability of the candidate compound to reduce a CD44-hyaluronan interaction in vivo.

The method can further include the step of administering the candidate compound to a subject, e.g., a subject having a CD44-hyaluronan mediated disorder, e.g., an animal for an animal model of a CD44-HA mediated disorder. 10286-012W01 / BWH 899

In another aspect, the invention features method for reducing contraception, the method including: administering to a subject an agent that reduces binding of L-selectin (e.g., on an embryo) to L-selectin ligands (e.g., on the endometrium).

The agent can be, for example, a small molecule, a peptide, a carbohydrate, a peptide- carbohydrate complex, or a nucleic acid.

In one embodiment, the agent is a sugar analog (e.g., a fluorinated sugar analog, e.g., 3-F- GlcNAc or 4-F-GlcNAc), or an antibody or antigen binding fragment thereof.

In another aspect, the invention features a kit for reducing contraception. The kit includes, for example: an agent that reduces binding of L-selectin (e.g., on an embryo) to L- selectin ligands (e.g., on the endometrium); and instructions for use.

In another aspect, the invention features a method for identifying an agent useful in the treatment of a CD44-hyaluronan mediated disorder. The method includes the steps of: providing a compound that binds either CD44 or hyaluronan; and evaluating the ability of the compound to inhibit a CD44-hyaluronan interaction.

The compound can be a hyaluronan antagonist, e.g., a small molecule, a peptide, a carbohydrate, a peptide-carbohydrate complex, or a nucleic acid.

In one embodiment, the hyaluronan antagonist is a carbohydrate selected from the group consisting of: hyaluronan hexasaccharides, chondroitin, or high molecular weight hyaluronan.

In one embodiment, the HA antagonist is an agent that increases sialylation of CD44. For example, the antagonist is an agent that inhibits a sialidase, e.g., 2-deoxy-2,3-dehydro-N- acetyl-neuraminic acid.

In one embodiment, the hyaluronan expressing cell or hyaluronan is contacted in the presence of CD44 or a CD44-expressing cell.

In another embodiment, the antagonist is a peptide, an antibody, or an antigen-binding fragment thereof. The antibody can be an anti-hyaluronan antibody.

In some embodiments, the contacting is performed in vivo. In other embodiments, the contacting is performed in vitro. h one embodiment, the CD44-expressing cell is a leukocyte, e.g., a mature leukocyte. The leukocyte can be a lymphocyte, e.g., a T cell. 10286-012W01 / BWH 899

The method can further include the steps of administering the HA antagonist to a subject or animal and determining a change in a CD44-hyaluronan interaction in the subject or animal.

In another aspect, the invention features a method for identifying an agent useful in the treatment of a CD44-hyaluronan mediated disorder. The method can include the steps of: providing a compound that decreases synthesis or expression of hyaluronan; and evaluating the ability of the compound to decrease a CD44-hyaluronan interaction.

In one embodiment, the agent is a hyaluronan antagonist. A hyaluronan antagonist can be a nucleic acid, a peptide, a carbohydrate, a peptide-carbohydrate complex, or a small molecule.

In one embodiment, the compound degrades hyaluronan, e.g., the agent is a glycosidase, e.g., hyaluronidase.

In one embodiment, the compound is a sugar analog, e.g., a fluorinated analog, e.g., 3-F- GlcNAc or 4-F-GlcNAc.

In another embodiment, the compound inhibits a hyaluronan synthase (e.g., hyaluronan synthase 1 (HASl), hyaluronan synthase 2 (HAS2), or hyaluronan synthase 3 (HAS3). An agent that inhibits a hyaluronan synthase can be a small inhibitory RNA (siRNA) of a hyaluronan synthase. h some embodiments, the contacting is performed in vivo. In other embodiments, the contacting is performed in vitro.

In one embodiment, the hyaluronan-expressing cell or hyaluronan is contacted in the presence of CD44 or a CD44-expressing cell. The CD44-expressing cell is a leukocyte, e.g., a mature leukocyte. The leukocyte can be a lymphocyte, e.g., a T cell.

The method can further include the steps of administering the compound to a subject or animal and determining a change in a CD44-hyaluronan interaction in the subject or animal.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. All cited patents, patent applications, and references (including references to public sequence database entries) are incorporated by reference in their entireties for all purposes. U.S.S.N. 60/454,719 is incorporated by reference in its entireties for all purposes. 10286-012W01 / BWH 899

DETAILED DESCRIPTION

The invention is based, in part, on the observation that binding of CD44 and hyaluronan mediates pathologic cell adhesion interactions, h particular, it was found that by reducing synthesis or expression of hyaluronan or the ability of HA to interact with CD44, various disorders mediated by CD44-HA interaction can be treated. It was also found that graft versus host disease (GVHD) is a disorder mediated by CD44-HA interaction and that the methods described herein can be used to teach GVHD in a subject.

CD44 and hyaluronan

CD44 is a glycoprotein receptor which can interact with several ligands including L- selectin, E-selectin and hyaluronan. The interaction of CD44 with its ligands plays a role in various pathologic cell adhesion interactions.

Various disorders have been found to be mediated by the interaction of CD44 and hyaluronan. For example, it was found that hyaluronan deposited on endothelial cells mediate the adhesion of CD44 expressing lymphocytes in various disorders of the skin such as GVHD.

Accordingly, the invention features methods of modulating the synthesis and expression of hyaluronan and modulating the interaction of hyaluronan with CD44. Hyaluronan is a high molecular weight glycosaminoglycan of repeating disaccharide structure synthesized by hyaluronan by alternating addition of glucuronic acid and N-acetylglucosamine to the growing chain using their activated nucleotide sugars (UDP - glucuronic acid and UDP-N- acetlyglucosamine) as substrates. The number of repeat disaccharides in a completed hyaluronan molecule can reach 10,000 or more, a molecular mass of ~4 million daltons (each disaccharide is -400 daltons). The average length of a disaccharide is ~1 nm.

The term "hyaluronan" as used herein refers to a family of molecules having hyaluron- like structure (e.g., containing hyaluronan disaccharide repeats) and properties such as the ability to interact with, e.g., bind to, CD44.

As discussed above, CD44 is a broadly distributed cell surface glycoprotein receptor for the glycosamino glycan hyaluronan (HA). CD44 is expressed on a diverse variety of cell types including most hematopoietic cells, keratinocytes, chondrocytes, many epithelial cell types, and some endothelial and neural cells. CD44 is known to participate in a wide variety of cellular functions, including cell-cell aggregation, retention of pericellular matrix, matrix-cell and cell- 10286-012W01 / BWH 899

matrix signaling, receptor-mediated interaalization/degradation of hyaluronan, and cell migration. All of these functions are dependent upon CD44-hyaluronan interactions. Such functions can also be sulfation dependent.

The term "CD44" as used herein refers to mammalian CD44, preferably human CD44. Human CD44 includes several protein products, including CD44S, CD44H, HCELL, CD44R1, CD44R2, which are encoded by alternating spliced MRNA variants of the CD44 nucleic acid disclosed in Screaton,G.R., et al., Proc. Natl. Acad. Sci. U.S.A. 89 (24), 12160-12164 (1992) (genomic sequence). The term also refers to the various glycoforms of CD44.

The gene encoding human CD44 consists of 20 exons (19 exons in earlier literature, exons 6a and 6b have been reclassified as exons 6 and 7, to make 20 exons total). Although a single gene located on the short arm of human chromosome 11 encodes CD44, multiple mRNA transcripts that arise from the alternative splicing of 12 of the 20 exons have been identified. The standard and most prevalent form of CD44 (termed CD44s) consists of a protein encoded by exons 1-5,16-18, and 20. This form is the most predominant form on hematopoetic cells, and is also lαiown as CD44H. CD44s exhibits the extracellular domains (exons 1-5 and 16), the highly conserved transmembrane domain (exon 18), and the cytoplasmic domain (exon 20). The 1482 bp of open reading frame mRNA for human CD44s results in translation of a polypeptide chain of -37 kDa. Post-translational addition of N-linked and O-linked oligosaccharides contribute to the ~85-kDa molecular mass of the final CD44 protein as estimated by SDS-PAGE.

The standard or hematopoietic isoform of CD44 (CD44H ) is a type 1 transmembrane molecule consisting of- 270 amino acids (aa) of extracellular domain (including 20 aa of leader sequence, a 21 aa transmembrane domain and a 72 aa cytoplasmic domain. The amino terminal -180 aa are conserved among mammalian species (-85% homo logy). This region contains six conserved cysteines, and six conserved consensus sites for N glycosylation. Five conserved consensus sites for N-glycosylation are located in the amino terminal 120 aa of CD44. All five sites appear to be utilized in the murine and human cell lines. Several studies have shown that cell specific N-glycosylation can modulate the HA binding function of CD44. Cell lines and normal B-cells showed differenced in N-glycosylation associated with different HA binding states. In particular, CD44 from HA binding cells had less glycosylation than from non-HA binding cells. Additionally, removal of sialic acids (both from the cell surface and from CD44- Ig fusion proteins) enhances HA binding. 10286-012W01 / BWH 899

In contrast, the non-conserved region (~aa 183 to 256) shows only -35% similarity between mammalian species. This region contains potential sites for numerous carbohydrate modifications of CD44 and the site of alternative splicing which allows for the insertion of extra amino acid sequence from variable exons of the CD44 gene.

A HCELL polypeptide comprises an amino acid sequence of CD44 that interacts with an antibody having the binding specificity of monoclonal antibody HECA-452 (ATCC Number: HB-11485). HECA-452 recognizes cutaneous lymphocyte associated antigen. HECA-452 binding of HCELL decrease after N-glycosidase-F, sialidase or fucosidase treatment. Furthermore, HCELL activity, e.g., E-selectin and L-selectin binding, also decreases upon N- glycosidase-F, sialidase, or fucosidase treatment demonstrating the importance of the sialofucosylated N-linked glycans in HCELL function. In contrast, sialylation of CD44 inhibits binding of CD44 to hyaluronic acid. Moreover, CD44 binding to hyaluronate is increased by sulfation, but sulfation is not necessary for the E- and L-selectin activity of HCELL.

Preferably, the CD44 polypeptide is the standard or hematopoietic isoform of CD44 (CD44H). Alternately, the CD44 polypeptide is the RI (CD44R1) or R2 isoform (CD44R2). In other embodiments, the CD44 can be an HCELL polypeptide, e.g., a polypeptide described in PCT Publication No. WO 02/44342.

Inhibition of HA synthesis and expression

The invention features methods of inl ibiting the interaction of HA with CD44 which include inhibiting the synthesis for expression of HA. Hyaluronan antagonists which directly or indirectly inhibit synthesis and/or expression of HA are used as agents in the methods described herein. Such HA antagonists include, but not limited to, small molecules, peptides, carbohydrates, peptide-carbohydrate complexes or nucleic acids which directly or indirectly inhibit synthesis or expression of HA. HA antagonist small molecules, as used herein, include: peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds having a molecular weight of less than about 2,000 grams per mole, 1,000 grams per mole, or 500 grams per mole. Preferably, the HA antagonist is specific in that it modulates the interaction HA and CD44 without broad implications, e.g., on the immune system. Non-limiting examples of HA antagonists are described herein. 10286-012W01 / BWH 899

Hyaluronan Antagonists which Directly Inhibit Synthesis of Hyaluronan

Glycosidases

Glycosidases which degrade hyaluronan can be used as hyaluronan antagonists, e.g., in the methods described herein. Such glycosidases include hyaluronidases. Hyaluronidases (HAses) are neutral- and acid-active enzymes found in diverse organisms. Hyaluronidases degrade hyaluronan, and, to a lesser extent, chondroitin sulfates (for a review, see Kreil et al. Protein Sci. 4:1666-9, 1995). Mammalian hyaluronidases such as bovine or human hyaluronidases can be used (see U.S. Patent No. 6,123,938 for an example of a human hyaluronidase). In addition, hyaluronidases are commercially available. For example, a hyaluronidase isolated from a testicular extract from cattle is available for clinical use (Wydase™, Wyeth-Ayerst). Methods and doses of administration for treating disorders described herein can be based upon methods of administration of, for example, bovine testicular hyaluronidase to treat myocardial infarction (see, e.g., Wolf et al. J. Pharmacol. Exper. Therap. 222:331-7, 1982; Braunwald et al. Am. J. Cardiol., 1976). Hyaluronidase can also be administered via alternative routes (e.g., topically, dermally, subcutaneously). It will be appreciated by a skilled artisan that the route and mode of administration may vary depending on the desired results. In addition, dose and dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). In some embodiments, the hyaluronidase is administered as part of a pharmaceutical composition which includes one or more agents for topical or dermal administration of the composition. Such compositions can be used, e.g., to treat skin disorders mediated by CD44 and hyaluronan interaction. The dose of hyaluronidase in such compositions may vary from the dosages of hyaluronidase administered via intervenous administration.

Sugar analogs

Sugar analogs can be used as hyaluronan antagonists, e.g., to inhibit synthesis of hyaluronan. Such analogs can be used, e.g., in the methods described herein. Any sugar analog that inhibits the synthesis of HA can be used in the methods described herein. Preferably, the sugar analog is a fluorinated N-acetylglucosamine (F-GlcNAc). Analogs of the naturally occurring cell surface carbohydrate N-acetylglucosamine (GlcNAc) have been synthesized that are fully-acetylated and possess an isosteric substitution of a fluorine for a hydroxyl group at the 10286-012W01 / BWH 899

carbon 3- and 4-positions, 2-acetamido-2-deoxy-l,4,6-tri-O-acetyl-3-deoxy-3-fluoro-D- glucopyranose (3-F-GlcNAc) and 2-acetamido-2-deoxy-l,3,6-tri-O-acetyl-4-deoxy-4-flύoro-D- glucopyranose (4-F-GlcNAc) (Bemacki, R.J. et al. (1977) J. Supra. Stru., 7:235-250; Sharma, M. and W. Korytnyk (1980) Carbohyd. Res., 79:39-51; Sharma, M. et al. (1990). Carbohyd. Res., 198:205-22):

4-F-GlcNAc 3-F-GlcNAc

This structural modification has been postulated to cause the termination of poly-N- acetyllactosamine chain elongation or to inhibit the enzymatic processes of glycoconjugate metabolism involved in oligosaccharide biosynthesis (Bemacki, R.J. et al. (1977) J. Supra. Stru., 7:235-250). There is strong evidence that these compounds enter the cell by passive diffusion, rapidly de-O-acetylate and form UDP-fluorinated-N-acetylglucosamine, and incorporate into tumor cellular glycoproteins (Bemacki, R.J. et al. (1977) J. Supra. Stru., 7:235-250).

Methods of preparing fluorinated N- acetylglucosaim.es are known in the art. For example, fully acetylated 3-fluoro- or 4-fluoro-N-acetyl-D-glucosamine (3-F-GlcNAc or 4-F- GlcNAc), can be prepared as described in Sharma et al., Carbohydrate Research (1990) Vol. 198 pp. 205-221, and in Thomas et al., Carbohydrate Research (1988) Vo. 175, pp. 153-157, the contents of which are hereby incorporated by reference in their entirety.

Preferably, the sugar analog, e.g., the fluorinated sugar analog, is administered at an optimal dose for desired effect (e.g., therapeutic effect). In some embodiments, the dose of the fluorinated sugar analog is a dose effective to inhibit the interaction of CD44 with hyaluronan, but does not inhibit interaction of CD44 with other ligands such as E-selectin or L-selectin. For example, an effective dose can be a dose which results in inhibition of CD44-HA interaction, but does not substantially inhibit interaction of CD44 with E-selectin or L-selectin. hi other embodiments, the dose of the fluorinated sugar analog is a dose effective to inhibit synthesis of 10286-012W01 / BWH 899

hyaluronan but does not substantially effect sialyation of certain CD44 glycoforms such as HCELL.

Hyaluronan Antagonist which Indirectly Inhibit Synthesis or Expression of Hyaluronan The expression of hyaluronan can also be inhibited by inhibiting the expression of a gene involved in hyaluronan synthesis to decrease expression of hyaluronan and thereby decrease interactions between CD44 and hyaluronan. Examples of proteins involved in hyaluronan synthesis include, e.g., hyaluronan synthases.

RNA Interference and antisense agents

In the present invention, RNAi can be used to inhibit the expression of a gene involved in hyaluronan synthesis, such as hyaluronan synthase, to decrease expression of hyaluronan, to thereby decrease interactions between CD44 and hyaluronan. Various inhibitory RNAi molecules can be identified and those that inhibit expression of a hyaluronan synthase or other gene can be formulated as pharmaceutical compositions to be administered in the methods of treatment described herein.

"RNA interference" (RNAi) is a term used to refer to the mechanism by which a particular mRNA is degraded in host cells. To inhibit an mRNA, double-stranded RNA (dsRNA) corresponding to a portion of the gene to be silenced (e.g., a hyaluronan synthase, e.g., Hasl, Has2, Has3) is introduced into a cell. The dsRNA is digested into 21-23 nucleotide-long duplexes called short interfering RNAs (or siRNAs), which bind to a nuclease complex to fomi what is known as the RNA-induced silencing complex (or RISC). The RISC targets the homologous transcript by base pairing interactions between one of the siRNA strands and the endogenous mRNA. It then cleaves the mRNA about 12 nucleotides from the 3' terminus of the siRNA (see Sharp et al., Genes Dev. 15:485-490, 2001, and Hammond et al., Nature Rev. Gen. 2: 110-119, 2001). RNAi has proven successful in human cells, including human embryonic kidney and HeLa cells (see, e.g., Elbashir et al, Nature 411:494-498, 2001). Gene silencing can be induced in mammalian cells by enforcing endogenous expression of RNA hairpins (see Paddison et al, Proc. Natl Acad. Sci. USA 99:1443-1448, 2002) or, as noted above, by fransfection of small (21-23 nt) dsRNA (reviewed in Caplen, Trends in Biotech. 20:49-51, 2002). 10286-012W01 / BWH 899

RNAi technology utilizes standard molecular biology methods. The dsRNA (which, here, for example, would correspond to the sequence encoding an HA synthase) can be produced by standard methods (e.g., by simultaneously transcribing both strands of a template DNA corresponding to an hyaluronan synthase sequence with T7 RNA polymerase; the RNA can also be chemically synthesized or recombinantly produced). The nucleotide sequences of HA synthase genes are known. See, for example, Shyjan, et al., J. Biol. Chem. 271(38):23395- 23399; 1996; Itano, and Kimata, Biochem. Biophys. Res. Commun. 222(3):816-820 (1996); Spicer, et al., J Biol. Chem. 272 (14):8957-8961 (1997); Watanabe and Yamaguchi, J. Biol. Chem. 271(38): 22945-22948 (1996)). Kits for producing dsRNA are available commercially (from, e.g., New England Biolabs, Inc). The RNA used to mediate RNAi can include synthetic or modified nucleotides, such as phosphorothioate nucleotides. Methods of transfecting cells with dsRNA or with plasmids engineered to make dsRNA are also routine in the art.

Gene silencing effects similar to those observed with RNAi have been reported in mammalian cells transfected with an mRNA-cDNA hybrid construct (Lin et al., Biochem. Biophys. Res. Comm. 281:639-644, 2001). Accordingly, mRNA-cDNA hybrids containing hyaluronan synthase sequence, as well as duplexes that contain hyaluronan synthase sequence (e.g., duplexes containing 21-23 bp monomers), are within the scope of the present invention. The hybrids and duplexes can be tested for activity according to the assays described herein (i.e., they can serve as the test agents), and those that exhibit inhibitory activity can be used to treat patients who have, or who may develop, a disease or condition associated with CD44-hyaluronan interactions.

The dsRNA molecules of the invention (double-stranded RNA molecules corresponding to portions of an hyaluronan synthase gene) can vary in a number of ways. For example, they can include a 3' hydroxyl group and, as noted above, can contain strands of 21, 22, or 23 consecutive nucleotides. Moreover, they can be blunt ended or include an overhanging end at either the 3' end, the 5' end, or both ends. For example, at least one strand of the RNA molecule can have a 3' overhang from about 1 to about 6 nucleotides (e.g., 1-5, 1-3, 2-4 or 3-5 nucleotides (whether pyrimidine or purine nucleotides) in length. Where both strands include an overhang, the length of the overhangs may be the same or different for each strand. To further enhance the stability of the RNA duplexes, the 3' overhangs can be stabilized against degradation (by, e.g., including purine nucleotides, such as adenosine or guanosine nucleotides or replacing pyrimidine 10286-012W01 / BWH 899

nucleotides by modified analogues (e.g., substitution of uridine 2 nucleotide 3' overhangs by 2'- deoxythymidine is tolerated and does not affect the efficiency of RNAi). The single stranded hyaluronan synthase RNA molecules that make up the duplex or hybrid inhibitor, or that act simply as antisense RNA oligonucleotides, are also within the scope of the invention. Any dsRNA can be used in the methods of the present invention, provided it has sufficient homology to a target gene of interest, e.g., a gene involved in the expression of hyaluronan, e.g., a hyaluronan synthase gene, to mediate RNAi. While duplexes having 21-23 nucleotides are described above, there is no upper limit on the length of the dsRNA that can be used in the methods described herein (e.g., the dsRNA can range from about 21 base pairs of the gene to the full length of the gene or more (e.g., 50-100, 100-250, 250-500, 500-1000, or over 1000 base pairs).

When these nucleic acids are administered to a human, they can reduce hyaluronan synthase mRNA levels, thereby inhibiting expression of hyaluronan and interaction of hyaluronan with CD44. The cell or organism is maintained under conditions in which hyaluronan synthase mRNA is degraded, thereby mediating RNAi in the cell or organism. Alternatively, cells can be obtained from the individual, treated ex vivo, and re-introduced into the individual.

Antisense agents can also be used to inhibit expression of molecules that contribute to hyaluronan synthesis and expression, such as HA synthases. An "antisense" nucleic acid can include a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire HA synthase coding strand, or to only a portion thereof (e.g., the coding region of human Hasl corresponding). In another embodiment, the antisense nucleic acid molecule is antisense to a "non-coding region" of the coding strand of a nucleotide sequence encoding human Hasl (e.g., the 5'- and 3 '-untranslated regions).

An antisense nucleic acid can be designed such that it is complementary to the entire coding region of HA synthase mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or non-coding region of HA synthase mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of HA synthase mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide 10286-012W01 / BWH 899

sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 or more nucleotide residues in length.

An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been sub-cloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules which can be used in methods described herein are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a HA synthase protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred. h yet another embodiment, the antisense nucleic acid molecule is an alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier et al. (1987) Nucl. Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucl. 10286-012W01 / BWH 899

Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

Inhibition of CD44-HA binding interactions

Agents useful for inhibiting the interaction of CD44 and hyaluronan include hyaluronan antagonists which interact with hyaluronan and inhibit the binding of hyaluronan with CD44. hi addition, agents which interact with CD44 and inhibit the interaction of CD44 with hyaluronan can be used. For example, agents which interact with CD44 such as carbohydrates, peptides and antibodies which bind to portions of CD44 which are involved in the binding of CD44 to hyaluronan are useful, e.g., in the methods described herein.

Carbohydrates

A variety of carbohydrates can be used to inhibit interactions between hyaluronan and CD44. Excess carbohydrate can prevent binding between CD44 and hyaluronan deposits on a cell or tissue, thereby abrogating inflammatory or unwanted cellular interactions. Useful carbohydrates include hyaluronan itself, e.g., sodium hyaluronate (NaHA), in commercially available products such as Artz®, Seikagaku (Japan); Hyalgan®, Fidia (Italy); Synvisc®, Biomatrix (USA); Opegan®, Seikagaku (Japan); OpeganHi®, Seikagaku (Japan); Healon®, Pharmacia-Upjohn (Sweden); and Opelead®, Shiseido (Japan). Related carbohydrates that can be used include hyaluronan hexasaccharides and chondroitin.

It will be appreciated by a skilled artisan that the route and mode of administration may vary depending on the desired results. In addition, dose and dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). In some embodiments, the carbohydrate, e.g., hyaluronan, can be administered as part of a pharmaceutical composition which includes one or more agents for topical or demial administration of the composition. Such compositions can be used, e.g., to treat skin disorders mediated by CD44 and hyaluronan interaction. The dose of hyaluronan in such compositions may vary from the dosages of hyaluronan administered via oral administration, e.g., the dose may be less than that recommended for oral administration to achieve a therapeutic effect. 10286-012W01 / BWH 899

Peptides

One class of agents that can be used in the invention are peptides. Peptides that are hyaluronan antagonists can be small (e.g., about 3 to 30 amino acids in length), single polypeptide chains, or multimeric polypeptides (such as antibodies, discussed below).

A peptide antagonist can be modified, e.g., glycosylated, phosphorylated, ubiquitinated, methylated, cleaved, disulfide bonded and so forth. A peptide antagonist can contain non-natural amino acids and/or synthetic modifications to enhance, for example, stability or solubility. The polypeptide can have a specific conformation, e.g., a native state or a non-native state. In some cases, however, the polypeptide is unstructured, e.g., adopts a random coil conformation or lacks a single stable conformation. Exemplary peptide antagonists include: cell surface proteins that are substrates for hyaluronan, and fragments thereof (e.g., CD44; TSG-6, also lαiown as stabilin- 1; and Link proteins, such as aggrecan, versican, neurocan, brevican). Peptide antagonists can be glycosylated surface proteins or hypoglycosylated variants, or fragments thereof, of hyaluronan- or CD44- associated proteins and carbohydrates (e.g., cell surface receptors, extracellular matrix binding proteins such as integrins, cell-binding proteins such as cell attachment molecules or "CAMs" such as cadherins, selectins, and so forth). In some embodiments, the molecule or polypeptide is associated with a disease, e.g., a CD44-hyaluronan mediated disease.

The antagonist peptide is preferably soluble. For example, soluble domains or fragments of a protein can be used.

Hyaluronan binding peptides can be modified with, for example, detectable substances such as the detectable substances described for antibodies, below. Likewise, hyaluronan binding peptides can be used for any diagnostic, prognostic, and therapeutic procedure, including the procedures described for antibodies, below.

Antibodies

The invention encompasses antibodies and antibody fragments, such as F_a or (F_ab)_2, that bind immunospecifically to hyaluronan. Hyaluronan, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. As disclosed herein, hyaluronan, hyaluronan derivatives, analogs, fragments, or homologs thereof may be utilized as immunogens in the generation of anti-hyaluronan antibodies. Hyaluronan and 10286-012W01 / BWH 899

hyaluronan derivatives may be crosslinked to a carrier protein, e.g., keyhole limpet hemocyanin, prior to immunization, in order to enhance antibody production.

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen, such as hyaluronan. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F_ab and F^v fragments, and an F_ab expression library. In a specific embodiment, antibodies to human hyaluronan molecules are disclosed. Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies to hyaluronan or a derivative, fragment, analog or homolog thereof.

For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by injection with the native protein, or a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, hyaluronan. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. If desired, the antibody molecules directed against hyaluronan can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.

The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope hyaluronan. A monoclonal antibody composition thus typically displays a single binding affinity for a particular moiety, e.g., sugar moiety, with which it immunoreacts. For preparation of monoclonal antibodies directed towards a particular compound, e.g., carbohydrate, e.g., hyaluronan, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized. Such techniques include, but are not limited to, the hybridoma technique (see Kohler & Milstein, 1975 Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the 10286-012W01 / BWH 899

EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc NatlAcad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Each of the above citations are incorporated herein by reference in their entirety.

According to the invention, techniques can be adapted for the production of single-chain antibodies specific to hyaluronan (see e.g., U.S. Patent No. 4,946,778). In addition, methodologies can be adapted for the construction of F_ab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F_ab fragments with the desired specificity for hyaluronan or derivatives, fragments, analogs or homologs thereof. Non-human antibodies can be "humanized" by techniques well known in the art. See e.g., U.S. Patent No. 5,225,539. Antibody fragments that contain the idiotypes to hyaluronan may be produced by techniques known in the art including, but not limited to: ( ) an F_{(a ')2} fragment produced by pepsin digestion of an antibody molecule; (z^'z) an F_a fragment generated by reducing the disulfide bridges of an F₍_ _')2 fragment; (iii) an F_a fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F_v fragments.

Additionally, recombinant anti-hyaluronan antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT hitemational Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; U.S. Pat. No. 5,225,539; European Patent Application No. 125,023; Better et /.(1988) Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443; Liu et al. (1987) J Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al. (1987) Cancer Res 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J Natl Cancer Inst 80:1553-1559); Morrison(1985) Science 229:1202-1207; Oi et al. (1986) 10286-012W01 / BWH 899

BioTechniques 4:214; Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J Immunol 141 :4053-4060. Each of the above citations are incorporated herein by reference in their entirety. Antibodies may be selected by phage display technology.

In one embodiment, methodologies for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to hyaluronan is facilitated by generation of hybridomas that bind to hyaluronan. Antibodies that are specific for derivatives, fragments, analogs or homologs thereof, are also provided herein.

Anti- hyaluronan antibodies may be used in methods known within the art relating to the localization and/or quantitation of hyaluronan (e.g., for use in measuring levels of HA within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like), h a given embodiment, antibodies for hyaluronan, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, can be used as therapeutic agents.

An anti-hyaluronan antibody (e.g., monoclonal antibody) can be used to isolate hyaluronan by standard techniques, such as affinity chromatography or immunoprecipitation.

An anti-hyaluronan antibody can facilitate the purification of natural hyaluronan from cells.

Moreover, an anti-hyaluronan antibody can be used to detect hyaluronan (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the molecule.

Anti-hyaluronan antibodies can be used diagnostically to monitor hyaluronan or protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Alternatively, the antibodies are used to treat or diagnose a disorder, e.g., a leukemia. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine 10286-012W01 / BWH 899

fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, I, S or H. Anti-hyaluronan antibodies can by administered in unconjugated form, e.g., for mediating interaction between hyaluronan and CD44, or can be conjugated, e.g., to a therapeutic modality, e.g., a therapeutic modality which can be used to treat the disorder mediated by CD44 and hyaluronan interaction.

In addition to the agents described above, other classes of agents can be used to inhibit interactions between CD44 and HA. For example, increasing sulfation of CD44 can increase the binding of CD44 to HA . Therefore, inhibition of sulfation can be used to inhibit CD44-HA interaction. Sulfation can be inhibited by, for example, decreasing the activity of sulfotransferases. RNA interference can be used to reduce the expression of sulfotransferases (e.g., a sulfotransferase described in Fukuda M, et al, JBiol Chem. 276(51):47747-50, 2001). For methods of performing RNA interference, see the section on RNA interference above. Other methods that modulate post-translational modification of CD44 can be used to inhibit CD44-HA interactions. For example, increasing sialylation of CD44 can decrease CD44-HA interactions. Agents that increase sialylation include inhibitors of sialidases (e.g., as described elsewhere herein), and agents that stimulate expression of sialyltransferases, such as nucleic acids encoding sialyltransferases, and compounds that stimulate the activity or transcription of sialyltransferases.

To express nucleic acid molecules in order to stimulate expression of a protein that will (directly or indirectly) lead to inhibition of CD44-HA interactions, nucleic acids encoding the protein (e.g., a sialyltransferase) can be delivered to cells. Transfection of cells in vitro can be performed using methods known in the art, such as calcium-phosphate transfection, or lipofection. Nucleic acids, e.g., encoding a sialyltransferase, can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Nail. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system. 10286-012W01 / BWH 899

Methods of Treatment

Disorders

The compositions and methods described herein can be used to treat or prevent CD44- hyaluronan mediated disorders. These disorders include cancers and inflammatory disorders, e.g., cancers and inflammatory disorders of the skin. CD44-hyaluronan mediated inflammatory disorders include, for example, autoimmune, allergic, transplantation-associated, and pathogen- associated disorders. Cutaneous inflammatory disorders that can be treated according to the methods described herein include contact dermatitis, atopic dermatitis, cutaneous infections (e.g., bacterial, viral, fungal, or parasitic infections of the skin), psoriasis, acute cutaneous graft versus host disease, acne, drug-related hypersensitivity reactions, pemphigus vulgaris, etc. Pancreatic inflammatory disorders include insulin-dependent diabetes mellitus. Also included are inflammatory disorders of joints (e.g., rheumatoid arthritis), of the nervous system (e.g., multiple sclerosis), of the gut (e.g., inflammatory bowel disease, Crohn's disease, ulcerative colitis), of the endocrine system (e.g., Grave's disease, Hashimoto's thyroiditis), renal and hepatic inflammatory disorders, and others.

Cancerous CD44-hyaluronan mediated disorders which can be treated by the methods described herein include cutaneous cancers such as melanomas, basal cell and squamous cell carcinomas, and Kaposi's sarcoma. Leukemias and lymphomas (e.g., chronic myeloid leukemia, chronic lymphatic leukemia, chronic granulocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myelocytic leukemia, Hodgkin's disease, non-hodgkin's lymphomas, Burkitt's lymphomas, and mycosis fungoides), can be treated according to the methods described herein. Also included are neoplastic disorders and disorders of solid tumors such as adenocarcinomas of the lung, kidney, uterus, prostate, bladder, ovary, colon; sarcomas such as liposarcoma, synovial sarcoma, rhabdomyosarcoma, Ewing's tumor, neuroepithelioma; and other tumors such as retinoblastoma, Wilm's tumor, and mesothelioma.

Pharmaceutical compositions

The agents and compounds, (also referred to herein as "Therapeutics" or "active compounds") of the invention, and pharmaceutically acceptable derivatives or salts thereof, can 10286-012W01 / BWH 899

be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the active compound and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The active compounds disclosed herein can also be formulated as liposomes. Liposomes are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 11: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous), intradermal, subcutaneous, oral, respiratory, (e.g., inhalation), transdermal (i.e., topical), transmucosal, and vaginal or rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or 10286-012W01 / BWH 899

phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, h many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., 3-F-GlcNAc or 4-F-GlcNAc, an HA-binding peptide, hyaluronidase) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. 10286-012W01 / BWH 899

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally lαiown in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. hi one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as 10286-012W01 / BWH 899

pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

In some embodiments, oral or parenteral compositions are formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

Sustained-release preparations can be prepared, if desired. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the active compound, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and j ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

The pharmaceutical compositions can be administered in combination with other agents. For example, the pharmaceutical compositions can be administered in combination with other anti-inflammatory agents or anti-cancer agents.

Nonlimiting examples of anti-cancer agents include, e.g., antimicrotubule agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, intercalating agents, agents capable of interfering with a signal transduction pathway, agents that promote apoptosis, radiation, and antibodies against other tumor-associated antigens (including naked 10286-012W01 / BWH 899

antibodies, immunotoxins and radioconjugates). Examples of the particular classes of anti- cancer agents are provided as follows: antitubulin/antimicrotubule, e.g., paclitaxel, vincristine, vinblastine, vindesine, vinorelbin, taxotere; doxorubicin, etoposide, mitoxantrone, daunorabicin; antimetabolites, e.g., 5-fluorouracil (5-FU), methotrexate, cytarabine/Ara-C, trimetrexate, fluorouridine; alkylating agents, e.g., cisplatin, carboplatin, mitomycin C, cyclophosphamide, pipobroman, 4-ipomeanol; actinomycin D; and anti-hormones, for example anti-estrogens such as tamoxifen.

Kits

Also within the scope of the invention are kits comprising a hyaluronan antagonist, e.g., a hyaluronan antagonist described herein. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, an antibody or peptide to a label or therapeutic agent, or a radioprotective composition; devices or other materials for preparing the hyaluronan antagonist for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject. Instructions for use can include instructions for therapeutic application including suggested dosages and/or modes of administration, e.g., in a patient with a an inflammatory disorder or cancer. Instructions for use can include instructions for diagnostic applications, e.g., of anti-hyaluronan antibodies (or antigen-binding fragment thereof) to detect hyaluronan, in vitro, e.g., in a sample, e.g., a biopsy or cells from a patient having a cancer or inflammatory disorder, or in vivo.

The kit can include a therapeutic agent, e.g., a therapeutic agent described herein. The kit can include a label, e.g., any of the labels described herein. The kit can include a reagent useful for chelating or otherwise coupling a label or therapeutic agent to the agent, e.g., a reagent discussed herein, e.g., peptide or antibody.

The kit can further contain at least one additional reagent, such as a diagnostic or therapeutic agent, e.g., a diagnostic or therapeutic agent as described herein, and/or one or more additional hyaluronan antagonists or other agents described herein formulated as appropriate, in one or more separate pharmaceutical preparations. 10286-012W01 / BWH 899

Methods of Screening

Also provided herein are methods, which may be referred to herein as "screening assays," for identifying modulators (e.g., antagonists or inhibitors) that bind to hyaluronan. Useful agents may also interact with CD44. The antagonists may also interact with hyaluronan by way of binding to, or otherwise interfering with, molecules that act either upstream or downstream of hyaluronan synthesis or expression (e.g., molecules that participate in the biochemical pathway(s) that involve the synthesis and expression of hyaluronan at sites of inflammation).

The agent can be essentially any physiologically acceptable (i.e., non-lethal) substance. For example, an inhibitor can be a protein, peptide, or polypeptide (all of these terms refer to linear polymers of amino acid residues;^' the term "protein" being commonly used to refer to full- length, naturally occurring proteins and the terms "peptide" or "polypeptide" being commonly used to refer to fragments thereof). The hyaluronan antagonist can also be a peptidomimetic, a peptoid, another small molecule (e.g., a small synthetic molecule), a nucleic acid, or another drag. While the invention is not limited to inhibitors that act by any particular mechanism, some of these inhibitors (e.g., anti-hyaluronan antibodies or fragments thereof) may inhibit the activity of hyaluronan, while others (e.g., an antisense oligonucleotide or a siRNA) can have an inhibitory effect on hyaluronan expression. Likewise, an inhibitor can affect the expression or activity of a molecule that acts on hyaluronan (e.g., a molecule involved in the post-synthetic processing of hyaluronan) or upon which hyaluronan acts (e.g., CD44). Agents identified as antagonists can be used to modulate the expression or activity of hyaluronan in a therapeutic protocol. They can, for example, disrupt the events that normally occur when hyaluronan interacts with an adhesion molecule, e.g., CD44.

The assays used to identify hyaluronan antagonists can be carried out variously in vitro, in cell culture, or in vivo, and they can reveal the presence or absence of hyaluronan (i.e., they can be qualitative) or the level of its expression or activity (i.e., they can be quantitative). Moreover, the assays can be conducted in a heterogeneous format (where hyaluronan or a molecule to which it binds is anchored to a solid phase) or a homogeneous format (where the entire reaction is carried out in a liquid phase). In either approach, the order in which the reactants are added can be varied to obtain different information about the agents being tested. For example, exposing hyaluronan to the test agent and a binding partner at the same time identifies agents that interfere with binding (by, e.g., competition), whereas adding the test agent 10286-012W01 / BWH 899

after binding has occurred identifies agents capable of disrupting preformed complexes (such agents may have higher binding constants and thereby displace one of the components from the complex).

Whether the methods are carried out in vitro or in vivo, they can employ biological samples. Generally, the biological sample can be provided or obtained from a test subject and can be (or can include) an organ, tissue, cell or biological fluid (e.g., a blood or serum sample) in which hyaluronan is normally expressed. The sample can be tested for hyaluronan expression (e.g., hyaluronan synthase mRNA or protein expression) or for binding activity (e.g., a Stamper- Woodruff assay). In vitro techniques for detecting hyaluronan include enzyme linked immunosorbent assays (ELISAs), immuno-precipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques can be carried out with labeled probes, such as anti-hyaluronan antibodies, which can be detected by standard imaging techniques. Regardless of the precise context in which hyaluronan expression is assessed, the antibodies used can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., an Fab or F(ab'). fragment) can be used. The term "labeled" is intended to encompass entities (e.g., probes such as antibodies) that are directly labeled by being linked or coupled (i.e., physically linked) to a detectable substance as well as entities that are indirectly labeled by virtue of being capable of reacting with a detectable substance or participating in a reaction that gives rise to a detectable signal.

To determine the activity of hyaluronan, any standard assay for hyaluronan-CD44 binding or cell binding can be carried out. A screen (e.g., a high throughput screen) for hyaluronan antagonists can be carried out by: (a) binding one or more types of hyaluronan substrate proteins (e.g., CD44) or peptides to a solid support (e.g., the wells of microtiter plates);

(b) exposing the substrate to a blocking agent (standard blocking agents are known); and

(c) exposing the substrate to hyaluronan and a test compound (i.e., a potential hyaluronan antagonist). Alternatively, hyaluronan can be bound to a solid support, and an hyaluronan ligand (e.g., CD44) can be added and tested for binding to hyaluronan.

The components of the reaction (e.g., the hyaluronan and test compound, or CD44 and test compound) are typically supplied in a buffered solution and the reaction is allowed to proceed at a temperature (the temperature can vary from, for example, room temperature (about 23 °C) to a physiological temperature (about 37°C)) and for a period of time that is in the linear 10286-012W01 / BWH 899

range of the assay. The reaction can be terminated in a number of ways (by, for example, rinsing the support several times with a buffered solution), and binding can be determined (standard techniques are available to measure, for example, radioactive tags). Antagonists are identified as the agents that reduce the extent to which the hyaluronan was able to bind the substrate.

Appropriate controls can be carried out in connection with any of the methods of the invention. For example, the method described above (and others aimed at identifying hyaluronan antagonists) can be carried out in the presence and absence of a test compound (representing experimental and control paradigms, respectively). Alternatively, test compounds and placebos (e.g., biologically inactive test compounds, such as denatured or mutant proteins or nucleic acids) can be used.

The agents tested for inhibitory activity can be those within a library, and the screen can be carried out using any of the numerous approaches used with combinatorial libraries. One can use, for example, biological libraries or peptoid libraries, which contain molecules having the functionalities of peptides, but with novel, non-peptide backbones that are resistant to enzymatic degradation but which nevertheless remain bioactive (see, e.g., Zuckermann et al, J. Med. Chem. 37:2678-85, 1994). One can also use spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, An ticancer Drug Des. 12:145, 1997). Molecular libraries can be synthesized according to methods known in the art (see, e.g., DeWitt et al, Proc. Natl. Acad. Sci. USA 90:6909, 1993; Erb et al, Proc. Natl Acad. Sci. USA 91:11422, 1994; Zuckermann et al, J. Med. Chem. 37:2678, 1994; Cho et al, Science 261:1303, 1993; Carrell et al, Angew. Chem. Int. Ed. Engl. 33:2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al, J Med. Chem. 37:1233, 1994).

Libraries of compounds maybe presented in solution (e.g., Houghten, Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555- 556, 1993), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. USA 89:1865-1869, 1992) or on phage (Scott and Smith, Science 249:386-390, 1990; Cwirla et al, Proc. Natl. Acad. Sci. 10286-012W01 / BWH 899

87:6378-6382, 1990; Felici, J Mol Biol 222:301-310, 1991; Ladner supra.). Regardless of the precise mode of presentation, the agents in the libraries are exposed to hyaluronan and a substrate; here, as above, agents within the libraries can be identified as inhibitors by virtue of their ability to prevent, to any extent, the ability of hyaluronan to bind its substrate.

Hyaluronan-CD44 interactions can also be assayed in cell-based systems. These methods can be carried out by, for example, contacting a cell that expresses a CD44 protein with a test agent and assessing the ability of the test agent to inhibit binding to hyaluronan. The antagonist can affect hyaluronan directly or indirectly (by inhibiting a molecule that acts on, or that is acted on by, hyaluronan). Cell-based systems can also be used to identify agents that inhibit hyaluronan by inhibiting its expression (in that event, it is expected that the test agents will be nucleic acids (e.g., siRNA or antisense oligonucleotides) or transcription factor-binding factors, although the invention is not so limited). The cell can be any biological cell that expresses or binds (e.g., an presents) hyaluronan, whether naturally or as a result of genetic engineering. For example, the cell can be a mammalian cell, such as a murine, canine, ovine, porcine, or human cell. The cell can also be non-mammalian (e.g., a Drosophila cell). The cell can be compared to a cell that expresses a small-interfering RNA (siRNA) that inhibits hyaluronan expression (e.g., the cell can express an "irrelevant" siRNA, that does not interfere with hyaluronan expression or activity).

As discussed above, the assays performed in the methods of the invention can reveal whether a test agent interferes with the ability of hyaluronan to simply bind to, or otherwise associate with, another molecule or moiety, e.g., CD44. For example, one can determine whether an antagonist inhibits the ability of hyaluronan to bind to a substrate or a component of a cell. These methods can be carried out by, for example, labeling either the hyaluronan or its binding partner (e.g., CD44) with a marker, such as a radioisotope or enzymatic label, so that hyaluronan-containing moieties can be detected. Suitable labels are known in the art and include, for example, ¹²⁵I, ³⁵S, ¹ C, or ³H (which are detectable by direct counting of radioemmissions or by scintillation counting). Enzymatic labels include horseradish peroxidase, alkaline phosphatase, and luciferase, which are detected by determining whether an appropriate substrate of the labeling enzyme has been converted to product. Fluorescent labels can also be used. Another way to detect interaction (between any two molecules) using a fluorophore is by fluorescence energy transfer (FET) (see, e.g., Lakowicz et al, U.S. Patent No. 5,631,169 and 10286-012W01 / BWH 899

Stavrianopoulos et al., U.S. Patent No. 4,868,103). A fluorophore label on the first, or "donor," molecule emits fluorescent energy that is absorbed by a fluorescent label on the second, or "acceptor," molecule, which fluoresces due to the absorbed energy (the labels on the two molecules emitting different, and therefore distinguishable, wavelengths of light). Alternately, the "donor" protein can simply utilize the natural fluorescent energy of tryptophan residues. Since the efficiency of energy transfer between the labels is related to the distance separating them, the spatial relationship between the molecules can be assessed. Where the two molecules bind one another, emission from the acceptor molecule is maximal; emission can be measured readily (with, for example, a fluorimeter).

Binding can also be detected without using a labeled binding partner. For example, a microphysiometer can be used to detect the interaction of a protein with hyaluronan without the labeling the protein or carbohydrate (McConnell et al, Science 257:1906-1912, 1992). Another label-free option is to assess interaction between hyaluronan and a target molecule with real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky, Anal Chem. 63:2338-2345, 1991 and Szabo et al, Curr. Opin. Struct. Biol 5:699-705, 1995). BIA detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal that indicates real-time reactions between biological molecules.

As noted above, hyaluronan antagonists can be detected in assays where a hyaluronan substrate is bound to a solid support. More generally, wherever hyaluronan-related binding is assessed (whether between hyaluronan and a substrate or other entity), one of the binding partners can be anchored to a solid phase (e.g., a microtiter plate, a test tube (e.g., a microcentrifuge tube) or a column). The non-anchored binding partner can be labeled, either directly or indirectly, with a detectable label (including any of those discussed herein), and binding can be assessed by detecting the label. If desired, the hyaluronan can be linked to a moiety (e.g., protein) that binds a matrix. For example, one can identify a hyaluronan antagonist by crosslinking hyaluronan to a protein, or by fusing a hyaluronan substrate (e.g., CD44) to glutathione-S-transferase; absorbing the fusion protein to a support (e.g., glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione-derivatized microtiter plates); exposing 10286-012W01 / BWH 899

the immobilized fusion protein to a potential binding partner (e.g., a potential hyaluronan antagonist) washing away unbound material; and detecting bound material. The exposure should take place under conditions conducive to complex formation (e.g., a physiologically acceptable condition). Alternatively, the complexes can be dissociated from the matrix, and the level of hyaluronan binding can be determined using standard techniques.

Hyaluronan or molecules with which they interact (e.g., hyaluronan substrates, e.g., CD44) or which with they may interact (e.g., potential antagonists) can also be immobilized on matrices using biotin and avidin or streptavidin. For example, biotinylated CD44 or molecules to which they bind can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., using the biotinylation kit sold by Pierce Chemicals, Rockford, IL), and immobilized in the wells of avidin- or streptavidin-coated 96 well plates (Pierce Chemical). Regardless of the precise way in which hyaluronan or a hyaluronan substrate is immobilized, the hyaluronan is exposed to a potential binding partner, any unreacted components are removed (e.g., by washing; under conditions that retain any complexes); and the remaining complexes are detected (by virtue of a label or with an antibody (e.g., an antibody that specifically binds hyaluronan, is used in the assay). Although somewhat more labor intensive, the step of detecting hyaluronan (or a hyaluronan-containing protein complex) can also be carried out by enzyme- linked assays, which rely on detecting an enzymatic activity associated with the hyaluronan or its target molecule.

Where the binding assay is carried out in a liquid phase, the reaction products (e.g., hyaluronan-containing complexes) can be separated from unreactive components by, for example: differential centrifugation (see, e.g., Rivas and Minton, Trends Biochem. Sci. 18:284- 287, 1997); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al, Eds. Current Protocols in Molecular Biology 1999, J. Wiley & Sons, New York.); and immunoprecipitation (as described, for example, in Ausubel, supra). Where FET is utilized (see above), further purification is not required.

Where hyaluronan expression is assessed, a cell or cell-free mixture is contacted with a candidate compound and the expression of hyaluronan synthase mRNA or protein is evaluated (the level can be compared to that of hyaluronan synthase mRNA or protein in the absence of the candidate compound or in the presence of another control substance (e.g., where the candidate compound is an antisense oligonucleotide, the "control" can include a "sense" oligonucleotide)). 10286-012W01 / BWH 899

Clearly, where mRNA or protein expression is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is an inhibitor of hyaluronan synthase mRNA or protein expression. The level of hyaluronan synthase mRNA or protein expression can be readily determined using methods well known in the art (e.g., Northern blot analysis, Western blot analysis or other immunoassay, by polymerase chain reaction analyses (e.g., rtPCR; see U.S. Patent No. 4,683,202), probe arrays, and by serial analysis of gene expression (see U.S. Patent No. 5,695,937)).

The level of mRNA corresponding to an hyaluronan synthase gene in a cell can be determined both by in situ and by in vitro formats. Where a nucleic acid molecule is used as a probe, the probe can be, or can include, an hyaluronan synthase sequence (e.g., Hasl, Has2, Has3), or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, or more nucleotides or ranges between (e.g., 8-14, 16-29, 31-49, or 51-99 nucleotides). The probe can be disposed on an address of an array (e.g., a two-dimensional gene chip array), which can be used in an assay to detect hyaluronan synthase inhibitors, which can, in turn, be used as therapeutic agents. For in situ methods, a cell or tissue sample can be prepared and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the hyaluronan synthase gene being analyzed.

Moreover, any of the methods described above can be carried out in concert with any other(s). For example, a hyaluronan antagonist can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a hyaluronan synthase protein can be confirmed in vivo (e.g., in an animal such as a mouse or a non-human primate). Moreover, as described further below, one can combine (or sequentially perform) assays to identify hyaluronan antagonists with those to identify anti-inflammatory or anti-cancer agents.

Adhesion Assays. The invention also provides methods for identifying agents, including those determined to be hyaluronan antagonists, that inhibit binding and/or activity of a CD44- expressing cell. For example, assays that measure cell adherence under conditions of shear stress can be used to identify agents that inhibit physiologic hyaluronan-CD44 interactions.

One type of assay that measures cell adherence can be performed as follows. A cell that expresses an hyaluronan ligand (e.g., CD44) can be identified by providing hyaluronan immobilized on a solid phase e.g., glass, plastic or membrane, and contacting the solid phase with a fluid sample containing a suspension of test cells. Alternatively, an hyaluronan- 10286-012W01 / BWH 899

expressing cell can be provided, and contacted to a solid^'phase containing immobilized CD44. In some aspects the fluid sample is moving. By a moving fluid sample under shear stress it is meant that the sample flows across the surface of the membrane. Interactions between fluid sample in flow and immobilized ligand can be examined under a wide range of defined flow conditions, ranging from static incubation through physiological levels of shear flow, static conditions and serial application of static and shear conditions, and into supraphysiologic shear levels. For example, shear flow conditions is a flow force greater than 0.6 dynes/cm². Alternatively, shear flow condition is a flow force at least 2.8 dynes/cm². Additionally, shear flow condition is a flow force of at least 9.0 dynes/cm². In some aspects, the fluid moves across the membrane such that physiological shear stress is achieved at the surface. The interaction between the solid phase and the cells is then determined. An interaction between the cells of the fluid sample and the solid phase can indicate that the cell expresses a ligand for the immobilized component. Such assays can further compare adherence in the presence of hyaluronan antagonists described herein.

The interaction between the cells and the solid phase can be, e.g., rolling, firm attachments or specific interaction. In some aspects, the specific interaction is determined by the affinity coefficient. For example a specific interaction is an interaction that has a K^ is in the range of 0.1 rnM to 7mM. Preferably, the K__d is greater than 1 mM.

A cell/agent interaction or alternately a cell/solid phase interaction can be determined for example, by visual inspection under a microscope, colormetrically, flourometrically, by flow cytometry or using a parrallel plate flow chamber assay. Alternatively, the interaction is analyzed by labeling the cells, CD44, hyaluronan, or the agent using florescent labels, biotin, enzymes such as alkaline phosphatase, horseradish peroxidase or beta-galactosidase, radioactive isotopes or other labels known in the art. The label can be added to the cells, hyaluronan, CD44 polypeptide or the agent prior or subsequent to contacting the test cell population with the agent. The membrane or solid phase can then be subject to spectrophotometic or radiographic analysis to quantify the number interacting with the selectin polypeptide of solid phase.

Gene Profiles and Arrays: Any of the samples used in the assays of the invention can be evaluated for more than just hyaluronan expression or activity (i.e., hyaluronan expression or binding activity can be evaluated in the context of the expression or activity of other genes such as hyaluronan synthase genes; in the context of a gene profile). The methods in which numerous 10286-012W01 / BWH 899

genes are evaluated can be carried out by providing a sample (e.g., a sample as described above (which may be supplied by the patient or a person who cares for the patient)) and determining the level of expression of two or more genes (e.g., 5, 10, 12, 15, 20, or 25 or more genes) in the sample, one of which is a gene that encodes a hyaluronan synthase (other candidate genes include those that encode molecules that act upstream or downstream of the hyaluronan synthase in the biosynthesis and presentation/deposition of hyaluronan). The levels of expression obtained from a particular sample or subject can be compared to a reference value or reference profile, which can be obtained by any of the methods described herein (i.e., by any of the assays for DNA or protein expression or activity). Methods in which hyaluronan expression or activity is measured (whether alone or in the context of a larger gene profile) can be used to monitor a treatment for a disorder, such as a disorder caused by a CD44-hyaluronan interaction, in a subject, and the information gained can be used to adjust the subject's treatment accordingly (to bring the subject's hyaluronan expression and gene profile closer to that of a healthy individual or an individual whose treatment has been successful in reducing the signs or symptoms of the disease; see, e.g., Golub et al, Science 286:531, 1999).

Accordingly, the invention features methods of evaluating a hyaluronan in a subject in order to assess the risk of, or the extent of, disease (e.g., a CD44-hyaluronan mediated disease) in the subject (when carried out over time, these methods can indicate the pace of the disease or the subject's responsiveness to a given treatment). The methods can be carried out by providing a biological sample from a subject and determining the level of hyaluronan expression or activity, optionally while determining the level of expression or activity of other genes. The sample can be processed (e.g., samples or cells can be lysed/extracted and mRNA or proteins or carbohydrates can be isolated (although absolute purity is not required); if desired, nucleic acids can be amplified) from other cellular components, and the processed sample can be applied to the array. One can then determine which addresses become occupied (by detecting array-bound nucleic acids, proteins, or carbohydrates). This reflects the nucleic acid, carbohydrate, or protein content of the sample. One can then, if desired, compare the subject's expression profile to one or more reference profiles and select the reference profile most similar to the subject reference profile (as the status of the patient providing the reference profile can be determined, a patient having a similar profile is likely to have a similar clinical status or expected course of disease). 10286-012W01 / BWH 899

Just as simple, assays for hyaluronan expression or binding activity can be carried out to identify hyaluronan antagonists, arrays can be used to determine the effect of a potential antagonist on a hyaluronan synthase and other genes or gene products. For example, one can treat a cell (in culture or in vivo (e.g., in an animal model)), process the cellular material to obtain mRNA or protein and apply that mRNA or protein to an array. The effect of the potential antagonist on the sample (as evidenced by detectable binding at particular addresses of the array) indicates whether the potential antagonist should be developed further as a therapeutic agent and, if so, what other measures should be considered. For example, if a potential antagonist has an undesirable effect on the treated cell or another cell type, one could co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be deteπnined at the molecular level. Thus, the effects of an agent on expression of genes other than the target gene can be ascertained and counteracted.

As noted above, hyaluronan expression or activity, alone or in the context of other molecules, can be tested in a variety of cell types to examine tissue specific expression. If a sufficient number of diverse samples are analyzed, clustering (e.g., hierarchical clustering, k- means clustering, Bayesian clustering and the like) can be used to identify other genes that are co-regulated with hyaluronan synthase. Thus, where the methods of the invention employ arrays, they can result in quantitation of the expression of multiple genes. Quantitative data can be used to group (e.g., cluster) genes on the basis of their tissue expression r se and on their level of expression in that tissue.

A variety of routine statistical measures can be used to compare two reference profiles. One possible metric is the length of the distance vector that is the difference between the two profiles. Each of the subject and reference profile is represented as a multi-dimensional vector, wherein each dimension is a value in the profile.

The methods described above, e.g., in which a hyaluronan synthase is assessed in the context of a gene profile, can be carried out with arrays, which include a substrate having a plurality of addresses, at least one of which includes a capture probe that specifically binds a hyaluronan molecule or hyaluronan synthase (e.g., Has nucleic acid or protein). The substrate can be a glass slide, a wafer (e.g., silica, plastic or other synthetic wafer), a mass spectroscopy plate, or a three-dimensional matrix, such as a gel pad. The substrate can be densely arrayed, having at least 10, 50, 100, 200, 500, 1,000, 2,000, 5,000 or 10,000 or more addresses/cm², or 10286-012W01 / BWH 899

any number ranging between these (e.g., 10-50, 50-100, 100-200, etc.). However, the array need not be so complex to yield useful information (i.e., fewer than a dozen or so molecules can be arrayed).

At least one address of the plurality (and, in some cases, a subset of the plurality) will include a nucleic acid capture probe that hybridizes specifically to a hyaluronan synthase nucleic acid (the sense or anti-sense strand). Where there are a subset of hyaluronan synthase probes, each address of the subset can include a capture probe that hybridizes to a different region of a hyaluronan synthase nucleic acid. Alternatively, each address of the array or a subset of the plurality can include a unique polypeptide (e.g., an antibody (e.g., a monoclonal antibody or a single-chain antibody) or substrate), at least one address being capable of specifically binding a hyaluronan synthase or a fragment (e.g., a biologically active fragment) thereof. Methods of producing polypeptide arrays are described in, for example, De Wildt et al, Nature Biotech. 18:989-994, 2000; Lueking et al, Anal. Biochem. 270:103-111, 1999; Ge, Nucleic Acids Res. 28:e3, 1-VII, 2000; MacBeath and Schreiber, Science 289:1760-1763, 2000; and WO 99/51773A1. See also U.S. Patent Nos. 5,143,854, 5,510,270, and 5,527,681, which describe arrays generated by photolithographic methods; U.S. Patent No. 5,384,261, which describes arrays generated by mechanical methods (e.g., directed-flow methods); U.S. Patent No. 5,228,514, which describes arrays generated by pin-based methods; and PCT application No. US/93/04145, which describes arrays generated by bead-based techniques.

Where the array includes a hyaluronan synthase, it can be used to detect a hyaluronan synthase-binding compound (e.g., an antibody or hyaluronan synthase-binding protein or substrate) in a sample from a subject. Where nucleic acids are arrayed, they can be identical to a hyaluronan synthase nucleic acid, but they need not be; they can also be homologous (having, for example, at least 60, 70, 80, 85, 90, 95 or 99 % identity to a hyaluronan synthase nucleic acid or fragment thereof (e.g., an allelic variant, site-directed mutant, random mutant, or combinatorial mutant)). Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished 10286-012W01 / BWH 899

using a mathematical algorithm. The percent identity between two nucleotide sequences can be determined using the algorithm of Needleman and Wunsch ((1970) J. Mol Biol 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix and a gap weight of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Generally, to determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes can be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein nucleic acid "identity" is equivalent to nucleic acid "homology").

The nucleic acid sequences described herein can be used as a "query sequence" to perform a search against hyaluronan synthase sequences, for example. Such searches can be perfom ed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to evaluate identity at the nucleic acid level. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to evaluate identity at the protein level. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Alignment of nucleotide sequences for comparison can also be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., 10286-012W01 / BWH 899

Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al, eds. 1995 supplement)).

Any of the methods of the invention in which hyaluronan expression or activity within any given tissue or cell is assessed can include a further step whereby the result is transmitted to a caregiver or other interested party (e.g., the patient). The result can be simply the level of hyaluronan expression or activity; the level of expression or activity within the context of an expression profile; the level of binding activity, e.g., as assayed by a Stamper- Woodruff assay or another shear-based assay; a result obtained by comparing the subject's hyaluronan, hyaluronan synthase, or a hyaluronan-inclusive expression profile with that of a reference profiles, a most similar reference profile, or a descriptor of any of the aforementioned. The result can be transmitted in any way information travels (e.g., across a computer network by way of, for example, a computer data signal embedded in a carrier wave).

Computer media: The invention also features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of hyaluronan in a sample, and a descriptor of the sample. The descriptor of the sample can be an identifier of the sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a preferred treatment). The data record can further include values representing the level of expression of genes other than hyaluronan synthase (e.g., other genes associated with a CD44-hyaluronan mediated disorder, or other genes on an array). The data record can be structured as a table (e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments)).

Also featured is a computer medium having executable code for effecting the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity of the subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of hyaluronan expression.

The following invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. 10286-012W01 / BWH 899

EXAMPLE

Perivascular lymphocytic infiltrates are the earliest histopathologic feature of aGVHD reactions, and the entry of donor-derived alloreactive effector lymphocytes in the relevant target organs leads to tissue injury. In general, tissue-specific migration of lymphocytes is controlled by discrete adhesive receptor-ligand interactions between surface structures of blood-bome lymphocytes and upregulated surface adhesive structures on endothelium of target tissues (Chin et al., Proc Soc Exp Bio Med., 196:374-380, 1991 ; Sackstein et al, J Invest Dermatol, 91 : :423- 428, 1988; Chin et al., J Invest Dermatol, 93:82S-87S, 1989). This homing process is initiated by lymphocyte "braking" interactions which consist of tethering and rolling attachments on endothelium with sufficient strength to overcome the shear forces of blood flow (Carlos and Harlan, Blood, 84:2068-2101, 1994; Sackstein et al., Blood, 101(2):771-8, 2003 (Aug 22, 2002; [epub ahead of print])). Thus, the capacity of tissue endothelium to support shear-resistant binding of lymphocytes is a key regulator of lymphocyte recruitment at sites of inflammation. In aGVHD, in particular, the endothelial adhesive system mediating this recraitment must be highly effective and efficient since mononuclear cell infiltrates are developing during periods of profound lymphopenia in the peri-engraftment period (Sackstein et al., Blood, 101(2):771-8, 2003).

Stamper- Woodruff assay conditions were used to analyze lymphocyte binding to dermal endothelium of skin eruptions following both allogeneic and autologous HSCT. These studies, performed under shear stress conditions that mimic blood flow, showed that papillary dermal vessels in acGVHD reactions, but not in most other post-HSCT skin eruptions, are specialized to support shear-resistant adherence of lymphocytes (Sackstein et al., Blood, 101(2):771-8, 2003). Using function-blocking antibodies, the findings demonstrated that several endothelial adhesion molecules that are known to recruit lymphocytes to the skin (Robert and Kupper, N Engl J Med., 341:1817-1828, 1999) - E-selectin, P-selectin and VCAM-1 - are not the primary effectors of this binding interaction in acGVHD (Sackstein et al., Blood, 101(2):771-8, 2003). Moreover, the findings indicated that lymphocyte L-selectin and LFA-1 also do not contribute to the observed binding interaction.

The present experiment was performed to identify the adhesion molecule(s) mediating the observed shear-resistant binding of lymphocytes to dermal endothelium in acGVHD. 10286-012W01 / BWH 899

Stamper- Woodruff assays of skin specimens of acGVHD were performed utilizing chemical and enzymatic treatments and function-blocking antibodies to define the relevant receptor-ligand adhesive interaction(s). It was found that lymphocyte binding to vascular endothelium in involved skin of aGVHD reactions is mediated by interactions between lymphocyte surface CD44 and dermal endothelial deposits of hyaluronic acid (HA). These data suggest that recraitment of lymphocytes to skin in acGVHD relies in part on upregulated expression of HA. Thus, the findings offer new perspectives on the molecular basis of acute cutaneous GVHD reactions and highlight a role for hyaluronic acid in the pathobiology of this condition.

MATERIAL AND METHODS

Patients

All patients gave written informed consent to participate in this study. Skin biopsy samples were obtained from 23 allo-HSCT recipients (12 male and 11 female, aged 20 to 63 (median, 43 years)) at DFCI/BWH who had clinicohistologic diagnosis of GVHD. Skin lesions developing post-transplant were typically generalized erythematous macular and papular eraptions which covered <50% surface area at the time of the biopsy. All specimens were obtained from skin eruptions occurring within 100 days post-transplant, over a range of 6 to 60 days (median, 15 days). Two 4 mm punch biopsies were obtained from involved skin (with exception efface, palms or soles). One specimen was snap frozen in liquid nitrogen for laboratory studies, and the second specimen was placed in buffered formalin for routine histopathology.

Histopathologic criteria for acGVHD on formalin- fixed, hematoxylin/eosin-stained sections were based on those established by Lemer at al (Lerner et al., Transplant Proc, 6:361- 371, 1974) with minor modifications (Horn et al., J Invest Dermatol, 103:206-210, 1994). All histologic samples consist of superficial dermal perivascular mononuclear infiltrates and other additional characteristics depending on the histologic grade of the disease (Sackstein et al., Blood, 101(2):771-8, 2003; Horn et al., J Invest Dermatol, 103:206-210, 1994). Diagnosis of acGVHD was made by histologic analysis and clinical correlation.

Cell preparation 10286-012W01 /BWH 899

Lymphocyte suspension for lymphocyte-skin adherence assays consisted of human peripheral blood mononuclear cells (PBMC) or rat thoracic duct lymphocytes (TDL) (Sackstein et al., Immunology, 85:198-204, 1995; Sackstein et al., Blood., 89:2773-2781,1997; Dimitroff et al., J Cell Biol, 153:1277-1286, 2001). Human PBMC were isolated by Histopaque-1077 (Sigma Diagnostics, Inc, St. Louis, MO) density gradient centrifugation of venous blood collected in sodium citrate. The interface cells were collected, washed 4 times in RPMI-1640 medium (Gibco-BRL, Grand Island, NY), and suspended at 10⁷ cells/mL in RPMI-1640 containing 5% fetal bovine serum (FBS) for adherence assays. To obtain TDL, the thoracic duct of Sprague- Dawley rats was cannulated and draining lymph was collected at room temperature in phosphate- buffered saline (PBS) containing 5 units/mL heparin over 4 hour periods, beginning 12 hours following cannulation. Collected TDL were washed 3 times in RPMI-1640 and resuspended at 10⁷ cells/mL in RPMI-1640 containing 5% FBS for adherence assays (13). Human PBMC and rat TDL both adhered to dermal endothelium with equal efficacy.

Lymphocyte-skin adherence assay

The lymphocyte-skin adherence assay, previously described in detail (Sackstein et al., Blood, 101(2):771-8, 2003), utilizes Stamper-Woodruff assay conditions originally performed for investigating lymphocyte binding to lymph node high endothelial venules (HEV) (Stamper and Woodruff, JExp Med., 144:828-833, 1976), with substitution of skin frozen sections for lymph node sections (Sackstein et al., J Invest Dermatol, 91 :423-428, 1988). For all adherence assays, frozen skin punch biopsy specimens were embedded in Tissue-Tec OCT compound for cryostat sectioning (6 micron thick). Sections were fixed in 3% glutaraldehyde in PBS, rinsed in PBS and incubated in 0.2 M lysine monohydrochloride to block reactive carboxyl groups. Slides were washed in RPMI-1640 medium containing 2% FBS and placed on trays. Cell suspensions (PBMC or TDL) were deposited as 0.2 mL aliquots (10⁷ cells/mL) onto sections and trays were placed on a rotating platform (80 rpm on a flat orbital shaker) at 4°C for 30 minutes. Sections were rinsed in cold PBS, fixed in 3% glutaraldehyde in PBS, stained with methyl green-thionin and analyzed by light microscopy. A minimum often frozen sections from each patient were analyzed for adherence. Sections which contained lymphocytes adherent as chains and small clusters within areas of papillary dermis were scored as positive. Sections of rat lymph node 10286-012W01 / BWH 899

served as control to confirm the capacity of input lymphocytes to bind HEV structures under Stamper- Woodruff assay conditions.

Treatment of lymphocytes.

To assess divalent cation-dependent binding interactions, lymphocytes were suspended in RPMI-1640 / 5% FBS containing 5 mM ethylenediamine tetra-acetic acid (EDTA). To analyze effect of lymphocyte activation on adherence, lymphocytes (10 cells/mL) were suspended in cell culture medium for 1 hour at 37 °C, containing 10 ng/mL phorbol myristate acetate (PMA) (GIBCO-BRL). Cells were then washed twice in PBS, resuspended in RPMI-1640 / 5% FBS and used in the binding assay.

Enzyme treatment of tissue sections

For enzyme digestion studies, adherence assays were performed on alternating sequential sections of enzyme treated or untreated sections (buffer matched control). Results of enzyme incubation were examined semi-quantitatively using an ocular grid to count bound lymphocytes within comparable consecutive areas of the papillary dermis. Two grids per slide were examined, minimum of two slides per experiment, 2 separate experiments for each skin specimen. Tissue sections were incubated with the enzyme solution for 30 min at 37 °C. After washing in PBS, sections were overlaid with lymphocytes, as described above. The enzymes and conditions used were as follows: Heparitinase II from Flavobacterium heparinum (Calbiochem, La JoUa, CA), 5 mU/mL in buffer (100 mmol/L NaAcetate, 10 mmol/L CaAcetate, pH=7); Keratanase from Pseudomonas species (Calbiochem, La Jolla, CA), 0.5 U/mL in buffer (50 mmol/L Tris/HCl, pH=8); Hyaluronidase from Streptomyces hyalurolyticus (Sigma, St.Louis, MO), 20 U/mL in PBS buffer. Sections from all acGVHD patients were preincubated with all three enzymes sequentially. For treatment with neuraminidase (sialidase) from Vibrio cholerae (Roche Diagnostics GmbH, Mannheim, Germany), slides were rinsed twice after the fixation with enzyme buffer (50 mmol/L NaAcetate, 154 mmol/L NaCl, 9 mmol/L CaCl₂, pH 5.5) and then incubated at 37°C for 1 hour with 50μL buffer (control) or 0.1 U/mL neuraminidase in buffer. For protease digestion, slides were incubated with RPMI 1640 alone or RPMI 1640 containing enzymes: 100 U/mL chymotrypsin (Sigma; 15 minutes at 37°C) or 0.1% bromelain (Sigma; 30 minutes at 37°C). To assess specificity, the protease inhibitors phenylmethylsulfonyl fluoride 10286-012W01 / BWH 899

(PMSF; 1.0 mg/mL; Sigma) and chymostatin (900 μg/mL; Boehringer Mannheim) were coincubated with chymotrypsin (100 U/mL) for 15 minutes at 37 C. After enzyme treatments, slides were washed three times with RPMI 1640 and placed in RPMI 1640 with 2% FBS until use in the binding assay.

Anti-CD44-antibody treatment of PBMC

Adherence assays were performed on alternating sequential sections in presence of Hermes- 1, a rat anti-human CD44 mAb (Endogen, Cambridge, MA) which blocks CD44 binding to HA (Culty M, et al., J Cell Biol, 1990) or with rat IgG isotype control. Cell suspensions of PBMC were incubated with 10 μg/mL of Hermes- 1 or rat IgG isotype antibody, for 60 min, at 4 °C and lymphocyte-skin adherence assay was then performed. As an additional control, lymphocytes were preincubated with 0.3 mg/mL HA (Hyaluronic Acid, Human Umbilical Cord, Potassium Salt, Calbiochem, La Jolla, CA) for one hour at 4 °C, prior to overlay the skin sections.

Immunohistochemistry techniques

For all adherence assays, frozen tissue sections (6 micron) were cut sequentially and alternating sections were fixed in acetone for immunohistochemical staining of endothelial structures and for histochemical staining of hyaluronan. hnmunohistochemical staining of endothelial structures was performed using a three-step method: sections were incubated with primary antibodies, anti-CD34 monoclonal antibody (QBEndlO, hnmunotech, Marseille, France), anti-CD31 monoclonal antibody (Southern Biotechnology Associates, Inc., Birmingham, AL) and/or mouse IgGl isotype, at the concentration of 10 μg/mL in PBS / 10% FBS, for 1 hour at room temperature, followed by biotinylated secondary andtibody (rabbit anti- mouse immunoglobulin, Dako, Copenhagen, Denmark) 1:500 in PBS / 10% FBS for 30 min, and then streptavidin coupled to horse radish peroxidase (Dako, Copenhagen, Denmark) 1 :500 in PBS / 10% FBS for 30 min. After rinsing in PBS, sections were incubated for 3 to 5 minutes in NovaRed substrate kit for peroxidase (Vector Laboratories, Burlingame, CA). Sections were lightly counterstained with hematoxylin. Prior to the incubation with primary antibody, sections were incubated in 10% H₂O₂ to inactivate endogenous peroxidases. 10286-012W01 / BWH 899

Deposition of hyaluronan was visualized using a biotinylated bovine HA-binding proteoglycan (bPG), extracted from bovine nasal cartilage (Underhill and Zhang, Methods Mol Biol, 137:441-447, 2000). The sections were incubated for 10 min in 10% H₂O₂ (to inactivate endogenous peroxidases), rinsed in PBS three times and incubated with lOμg/mL bPG dissolved in PBS / 10% FBS, for 12 hours at 4 °C. After washing in PBS, sections were incubated for 15 min with 1 :500 dilution of streptavidin coupled to horse radish peroxidase in PBS / 10% FBS, washed in PBS, and incubated for 20 to 30 min with a peroxidase substrate consisting of 3- amino-9-ethyl carbazole (AEC) and H₂O₂ (Underhill and Zhang, Methods Mol Biol, 137:441- 447, 2000). Sections were then rinsed in PBS and counterstained with hematoxylin. For permanent preservation of the stain, sections were coated with Crystal/Mount (Biomedia, Foster City, CA) before attaching a cover slip. Control sections were treated with hyaluronidase from Streptomyces hyalurolyticus (Sigma, St.Louis, MO), 40 U/mL in PBS buffer, for 30 minutes at 37 °C, prior to the incubation with bPG. As an additional control, bPG was preincubated with 0.3 mg/mL HA (Hyaluronic Acid, Human Umbilical Cord, Potassium Salt, Calbiochem, La Jolla, CA) for one hour at 4 °C, prior to the treatment of the skin sections.

HA-binding assay

Adherence of lymphocytes and of KGla cells (a human primitive hematopoietic cell line) (Furley AJ, et al., Blood 1986) to immobilized HA was performed in multiwell plates (Multiwell, 24 well, tissue culture plate, Falcon 3047, Becton Dickinson) coated with HA (Hyaluronic Acid, Human Umbilical Cord, Potassium Salt, Calbiochem, La Jolla, CA). HA-coated wells were prepared by incubation in a solution of HA (5 mg/mL in H₂O), followed by drying for 12 hours at 37 °C . HA-coated plates were then treated with 3% bovine serum albumin (BSA) in RPMI- 1640 for 2 h at 37 °C and washed three times with Cell Adhesion Medium (CAM) (100 mL RPMI-1640, 1 mL of lmol/L Hepes, 0.2 g BSA, lmL of 100 mmol/L sodium pyravate). Cells were added to each well (10⁶ cells/mL in CAM) and the plate was centrifuged at 50 G at 4 °C for 2 minutes to bring cells in contact with substrate. The plate was then placed at 37 °C for 2 h. Binding was performed under both static and shear conditions. Plates were placed on rotatory (80 rpm) and rocker (range 5-30 cycles/min) platforms. Following incubations, non-adherent cells were removed by vigorous washing with CAM. Bound cells were then analyzed by light microscopy. Controls consisted of pretretment of input lymphocytes with Hermes- 1 mAb (10 10286-012W01 / BWH 899

μg/mL), or treatment of wells with hyaluronidase (20 U/mL) prior to addition of cells, or omission of HA in the preparation of wells.

RESULTS

Lymphocytes bind to acGVHD skin sections.

As previously reported (Sackstein et al., Blood, 101(2):771-8, 2003), rat TDL or human PBMC adhered to discrete areas of the papillary and upper reticular dermis in punch biopsy skin specimens of acGVHD patients. Lymphocytes did not attach to epidermis or to stractures within the deep reticular dermis, however, binding of lymphocytes to the hair follicular bulge area was occasionally observed (data not shown). The number of cells adherent and the patterns of adherence were similar among both rat TDL and human PBMC. The adherent lymphocytes appeared as clumps or chains, and the localization of the adherent lymphocytes was traceable through consecutive sections of the skin, indicating that binding was to specific stractures. There was no lymphocyte adherence to the human skin specimens obtained from healthy volunteers, performed under the same shear stress conditions (data not shown). Sections of rat lymph node showed lymphocyte binding to HEV and served as control to confirm the capacity of input lymphocytes to bind vascular structures under Stamper- Woodruff assay conditions.

Lymphocyte binding to acGVHD skin sections is not divalent-cation dependent.

To assess the role of divalent cations in the binding interactions observed, assays were performed in the presence of 5 mM EDTA. EDTA treatment did not affect lymphocyte binding to dermal endothelium, indicating that lymphocyte binding was not mediated by the selectin, nor integrin classes of adhesion molecules.

PMA treatment of lymphocytes does not alter binding to acGVHD skin sections.

PBMC were incubated for 1 hour at 37 °C with 10 ng/mL PMA and then used in the lymphocyte binding assay. PMA-treated PBMC were not able to bind HEV on rat lymph node sections, as previously shown (Oxley and Sackstein, 1994), because of activation-induced shedding of L-selectin, but they readily adhered to skin tissue sections from acGVHD patients. Together with results of EDTA incubations, this finding confirmed that lymphocyte binding was not L-selectin-mediated. 10286-012W01 / BWH 899

The effect of enzyme treatment of acGVHD skin sections on lymphocyte binding.

Pre-treatment of skin sections with neuraminidase (0.1 U/mL), chymotrypsin (100 U/mL) or bromelain (0.1%) before the binding assay had no effect on lymphocyte binding to the skin endothelium, but abrogated binding of lymphocytes to HEV of rat lymph node sections (data not shown). These findings indicated that the relevant binding determinants in endothelium do not comprise sialic acid residues and are protease- insensitive.

To explore the role of extracellular matrix elements, skin sections were treated with heparitinase, hyaluronidase, and keratanase. Treatment with heparitinase and keratanase prior to the adherence assay had no effect on binding of lymphocytes to endothelium. On the other hand, hyaluronidase digestion of skin sections completely blocked lymphocyte adherence to endothelium in all cases. The number of lymphocytes adherent to papillary dermis were quantified and compared with corresponding sequential sections incubated with buffer matched control. The results of enzyme digestion studies indicated that the molecule responsible for lymphocyte adherence to endothelium in the skin was hyaluronan. In contrast to acGVHD skin, none of the three enzymes (hyaluronidase, heparitinase or keratanase) influenced lymphocyte binding to the HEV of the rat lymph nodes comparing to the buffer treated control (data not shown).

Lymphocyte binding to acGVHD skin sections is mediated by CD44.

To directly examine whether lymphocyte adherence was mediated by lymphocyte CD44, PBMC were pre-incubated with the anti-CD44 mAb Hermes-1 or rat IgG isotype control Ab. Hermes- 1 completely inhibited lymphocyte binding to acGVHD skin sections, whereas isotype control mAb did not affect binding. Consistent with these findings, lymphocyte adherence to dermal vessels under shear was abrogated by pre-incubation of lymphocytes with soluble HA (0.3 mg/mL). This effect was not due to a non-specific alteration of lymphocyte binding capabilities, as incubation of lymphocytes with Hermes-1 or with HA did not have any effect on lymphocyte attachment to rat lymph node HEV.

Immunohistochemistry Studies.

Analysis of sequential, 6 μm thick, frozen sections of skin biopsy from patients with acGVHD was performed. The location of adhering cells correlated with the pattern of 10286-012W01 / BWH 899

immunohistochemical staining for CD34, indicating that both human PBMC and rat TDL adhered to endothelial structures in the papillary/upper reticular dermis (Sackstein et al., Blood, 101(2):771-8, 2003). Comparison of the sites of adhering lymphocytes with endothelial structures and with the pattern of histochemical staining for HA revealed concordance. Sites with intense HA staining corresponded with vascular endothelium, and were the locations of the prominent lymphocyte adherence. Interestingly, no lymphocyte binding was observed on endothelial stractures which did not display HA deposition. Moreover, staining with anti-CD31 and anti-CD34 mAb showed no evidence of HA deposition within lymphatic endothelial structures (CD34-, CD31+ vessels) and there was no binding to lymphatic vessels. Skin sections treated with hyaluronidase prior to the incubation with bPG revealed no staining, showing specificity of bPG binding to HA deposits (data not shown).

Lymphocytes do not bind to HA-coated plates.

Based on the finding that normal PBMC and TDL bound HA in skin sections of acGVHD (under shear stress conditions that mimic blood flow), adherence assays on HA-coated plates were performed in order to investigate the binding of lymphocytes to the immobilized HA. These assays were performed under both static and shear conditions using rotatory and tilt platforms.

Under either static or shear conditions, there was no lymphocyte binding to HA immobilized on plates, even using input HA concentration in excess of 10 mg/mL. On the other hand, KGla cells adhered readily to HA-coated plates. KGla adherence was CD44- and HA- specific, as evidenced by abrogation following treatment of cells with Hermes-1 mAb and treatment of HA-coated plates with hyaluronidase prior to the addition of KGla cells. The finding that CD44 expressed on lymphocytes will bind to HA naturally expressed on endothelium of skin in acute GVHD whereas the same lymphocytes do not engage HA immobilized on plates provides evidence that the HA deposited on inflammatory endothelium is specialized to promote CD44-mediated lymphocyte adhesion. These data suggest that the structure and/or the microarchitecture of the HA produced in situ is different than HA available from routine tissue sources. At present, the only available technique to identify this difference is the shear-based Stamper- Woodruff assay we have employed here. It should be noted that all present histochemisfry techniques would not be capable of detecting this molecular difference, as 10286-012W01 / BWH 899

staining intensity is a function of HA concentration. Nonetheless, our results demonstrating the concordance of lymphocyte adherence with discrete HA deposits on endothelium serve as a new method to diagnose inflammation-specific HA deposition. For acute GVHD, this combination of functional assay and histochemical detection of HA leads to a marked improvement in our capacity to diagnose this condition. Indeed, despite extensive analysis of the histopathology of GVHD, there are no pafhognomonic features of this condition. The characteristic perivascular dermal lymphocytic infiltrates in acGVHD are the earliest feature of this entity (Woodruff et al., Transplant Proc, 8:675-684, 1976), but are not specific for GVHD alone and can occur in a variety of other cutaneous diseases and reactions. Moreover, many skin eraptions occur post-transplant in response to the preparative regimen (LeBoit, J Am Acad Dermatol, 20:236-241, 1989), hypersensitivity to drags, infections and even the recovery of leukocytes (Horn, JCutan Pathol., 21:385-392, 1994; Glass et al., J Am Acad Dermatol, 24:455-459, 1996). Our data indicate that the Stamper- Woodruff assay is both sensitive and specific for the diagnosis of this condition.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

10286-012W01 / BWH 899WHAT IS CLAIMED IS:

1. A method for inhibiting interaction of CD44 with hyaluronan, the method comprising:

5 contacting a hyaluronan-expressing cell or hyaluronan with an agent that decreases synthesis or expression of hyaluronan, thereby decreasing interaction of CD44 with hyaluronan.

2. The method of claim 1, wherein the agent degrades hyaluronan. o

3. The method of claim 2, wherein the agent is a glycosidase.

4. The method of claim 3, wherein the glycosidase is hyaluronidase.

5 5. The method of claim 1, wherein the agent is a sugar analog.

6. The method of claim 5, wherein the sugar analog is fluorinated.

7. The method of claim 6, wherein the sugar analog is 2-acetamido-2-deoxy- 0 l,4,6-tri-O-acetyl-3-deoxy-3-fluoro-D-glucopyranose (3-F-GlcNAc) or 2-acetamido-2- deoxy-l,3,6-tri-O-acetyl-4-deoxy-4-fluoro-D-glucopyranose (4-F-GlcNAc).

8. The method of claim 1, wherein the agent inhibits a hyaluronan synthase.

5 9. The method of claim 8, wherein the agent is a small inhibitory RNA (siRNA) of a hyaluronan synthase.

10. The method of claim 1 , wherein the contacting is performed in vivo.

0 11. The method of claim 1 , wherein the hyaluronan-expressing cell or hyaluronan is contacted in the presence of CD44 or a CD44-expressing cell. 10286-012W01 / BWH 899

12. The method of claim 11 , wherein the hyaluronan-expressing cell or hyaluronan is contacted in the presence of a CD44-expressing cell, and the CD44-expressing cell is a leukocyte.

13. The method of claim 12, wherein the leukocyte is a mature leukocyte.

14. The method of claim 13, wherein the leukocyte is a lymphocyte.

15. The method of claim 14, wherein the lymphocyte is a T cell.

16. A method for inhibiting interaction of CD44 with hyaluronan, the method comprising: contacting a hyaluronan-expressing cell or hyaluronan with an agent that decreases binding of hyaluronan to CD44, thereby decreasing interaction of CD44 with hyaluronan.

17. The method of claim 16, wherein the agent is a hyaluronan antagonist.

18. The method of claim 17, wherem the hyaluronan antagonist is is a small molecule, a peptide, a carbohydrate, a peptide-carbohydrate complex, or a nucleic acid.

19. The method of claim 18, wherein the hyaluronan antagonist is a carbohydrate selected from the group consisting of: hyaluronan hexasaccharides, chondiOitin, or high molecular weight hyaluronan.

20. The method of claim 19, wherein the agent inhibits a sialidase.

21. A method for treating a disorder characterized by CD44-hyaluronan interactions, the method comprising: 10286-012W01 / BWH 899

administering to a subject an agent that decreases the expression or synthesis of hyaluronan, to thereby treat the disorder.

22. The method of claim 21, wherein the method further comprises identifying a 5 subject having or at risk for having a CD44-hyaluronan mediated disorder.

23. The method of claim 21, wherein the method includes administering the agent at a dose sufficient to reduce the expression or synthesis of hyaluronan, and/or inhibit interaction of CD44 with hyaluronan, but does not inhibit interaction of CD44 with another

0 ligand.

24. The method of claim 23, wherein the agent is a sugar analog.

25: The method of claim 24, wherein the sugar analog is 3-F-GlcNAc or 4-F- 5 GlcNAc.

26. The method of claim 23, wherein the agent inhibits a hyaluronan synthase.

27. The method of claim 23, wherein the agent is administered topically, !0 subcutaneously, intradermally, intravenously, orally, transmucosally, or rectally.

28. The method of claim 23, wherein the disorder is a cancer.

29. The method of claim 28, wherein the cancer is a leukemia. !5

30. The method of claim 23, wherein the disorder is a hematopoietic disorder.

31. The method of claim 23, wherein the disorder is an inflammatory disorder.

iθ 32. The method of claim 31 , wherein the disorder is a cutaneous inflammatory disorder. 10286-012W01 / BWH 899

33. The method of claim 32, wherein the disorder is psoriasis, contact dermatitis, or graf versus host disease.

5 34. The method of claim 31, wherein the disorder is acute rheumatoid arthritis, multiple sclerosis, Guillain-Barre syndrome, Crohn's disease, ulcerative colitis, inflammatory bowel disease, lupus erythematosus, insulin-dependent diabetes mellitus, psoriatic arthritis, sarcoidosis, hypersensivity pneumonitis, ankylosing spondylitis and related spoldyloartliropathies, Reiter's syndrome and systemic sclerosis, Hashimoto's thyroiditis and

0 Grave's disease, graft rejection, atopic conditions, asthma, allergy, and allergic rhinitis.

35. The method of claim 33, further comprising the step of detennining a change in CD44-hyaluronan interaction in the subject.

5 36. A method for treating a disorder characterized by CD44-hyaluronan interactions, the method comprising: identifying a subject suffering from or at risk for a CD44-hyaluronan mediated disorder; administering to a subject an agent that decreases binding of hyaluronan to Ό CD44, thereby treating the disorder.

37. A kit for treating a disorder characterized by CD44-hyaluronan interactions, the kit comprising: an agent that decreases the expression or synthesis of hyaluronan.; and 5 instructions for administering the agent to a subject suffering from or at risk for a disorder characterized by CD44-hyaluronan interactions.

38. The kit of claim 37, wherein the agent degrades hyaluronan.

0 39. The kit of claim 37, wherein the agent is a glycosidase. 10286-012W01 / BWH 899

40. The kit of claim 39, wherein the glycosidase is hyaluronidase.

41. The kit of claim 37, wherein the agent is a sugar analog.

42. The kit of claim 41 , wherein the sugar analog is fluorinated.

43. The kit of claim 42, wherein the sugar analog is 3-F-GlcNAc or 4-F-GlcNAc.

44. The kit of claim 37, wherein the agent inhibits a hyaluronan synthase.

45. The kit of claim 44, wherein the agent is a small inhibitory RNA (siRNA).

46. A kit for treating a disorder characterized by CD44-hyaluronan interactions, the kit comprising: an agent that decreases binding of hyaluronan to CD44; and instructions for administering the agent to a subject suffering from or at risk for a disorder characterized by CD44-hyaluronan interactions.

47. The method of claim 46, wherein the agent is a small molecule, a peptide, a carbohydrate, a peptide-carbohydrate complex, or a nucleic acid.

48. The method of claim 47, wherein the agent increases sialylation of CD44.

49. The method of claim 48, wherein the agent inhibits a sialidase.

5

50. The method of claim 47, wherein the protein is a peptide or an antibody or antigen-binding fragment thereof.

51. A method for identifying an agent useful in the treatment of a CD44- ) hyaluronan mediated disorder, the method comprising: contacting a biological sample with a test compound; and 10286-012W01 / BWH 899

evaluating the ability of the test compound to reduce a CD44-hyaluronan interaction, wherein a reduction in a CD44-hyaluronan interaction is an indication that the test compound is an agent useful in the treatment of a CD44-hyaluronan mediated disorder.

52. The method of claim 51, wherein the agent is a small molecule, a peptide, a carbohydrate, a peptide-carbohydrate complex, or a nucleic acid.

53. The method of claim 51 , wherein the biological sample comprises cells.

i 54. The method of claim 51 , wherein the biological sample comprises CD44- expressing cells.

55. The method of claim 54 wherein the CD44-expressing cells are leukocytes.

.

56. The method of claim 51 , wherein the evaluating comprises performing a binding assay.

57. The method of claim 56, wherein the biological sample further comprises a tissue section.

3

58. The method of claim 51 , wherein the disorder is a cutaneous disorder.

59. The method of claim 58, wherein the cutaneous disorder is acute cutaneous graft versus host disease.

5

60. A method for identifying a compound useful in the treatment of a CD44- hyaluronan mediated disorder, the method comprising: providing a compound that decreases synthesis or expression of hyaluronan; and o evaluating the ability of the compound to decrease a CD44-hyaluronan interaction.