Methods for Treating Corneal and Conjunctival Inflammation and Inflammatory Disorders

Abstract

Provided herein are methods for reducing corneal inflammation, reducing inflammatory cell (e.g., dendritic cell) recruitment to the cornea, and treating an corneal inflammatory disorder in a subject that include administering to the subject one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist. Also provided are compositions containing one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist, and kits containing these compositions.

Claims

1. A method of reducing corneal inflammation in a subject, the method comprising administering to a subject having corneal inflammation one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist in an amount sufficient to reduce corneal inflammation in the subject.

2. The method of claim 1, wherein the corneal inflammation is chronic corneal inflammation.

3. The method of claim 1, wherein the corneal inflammation is acute corneal inflammation.

4. The method of claim 1, wherein the corneal inflammation is caused by bacterial infection, fungal infection, parasite infection, viral infection, allergies, dry eye disorder, Fuchs' dystrophy, keratoconus, amyloidosis, lattice dystrophy, Stevens Johnson syndrome, physical corneal injury, Behcet's disease, contact lens wear, corneal graft rejection, dry eye syndrome, or immune keratitis.

5. The method of claim 1, further comprising administering to the subject one or more anti-inflammatory agents.

6. A method of reducing dendritic cell recruitment to the cornea, the method comprising administering to a subject having corneal inflammation one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist in an amount sufficient to reduce dendritic cell recruitment to the cornea.

7. The method of claim 6, wherein the dendritic cell recruitment is recruitment of dendritic cells to the corneal epithelium.

8. The method of claim 6, wherein the dendritic cell recruitment is recruitment of dendritic cells to the anterior or posterior stroma of the cornea.

9. The method of claim 6, further comprising selecting a subject having eye inflammation or a corneal inflammatory disorder.

10. The method of claim 6, further comprising administering to the subject one or more anti-inflammatory agents.

11. A method of treating a corneal inflammatory disorder in a subject, the method comprising administering to a subject having a corneal inflammatory disorder one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist in an amount sufficient to decrease corneal inflammation.

12. The method of claim 11, wherein the corneal inflammatory disorder is keratitis.

13. The method of claim 12, wherein the keratitis is non-infectious keratitis.

14. The method of claim 12, wherein the keratitis is infectious keratitis.

15. The method of claim 11, further comprising selecting a subject having a corneal inflammatory disorder.

16. The method of claim 11, further comprising administering to the subject one or more anti-inflammatory agents.

17. The method of claim 11, wherein the MadCAM-1 antagonist is an antibody or an antigen-binding antibody fragment that binds specifically to MadCAM-1.

18. The method of claim 17, wherein the antibody is a fully human antibody or humanized antibody.

19. The method of claim 17, wherein the antigen-binding antibody fragment is selected from the group of: a Fab fragment, a F(ab)2 fragment, and a scFv fragment.

20. The method of claim 11, wherein the 47 integrin antagonist is an antibody or an antigen-binding antibody fragment that binds specifically to 47 integrin.

21.-43. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG. 1 is a diagram of the eye showing the inflammatory cell populations present in the corneal epithelium and the corneal stroma under normal conditions (normal cornea) and during inflammation (inflamed cornea).

[0059] FIG. 2 is a diagram showing the injection of calcein-labeled dendritic cells into the carotid artery of a mouse and the intravital imaging of the cornea of the mouse. The mice used to perform these experiments can be control mice in which inflammation has not been induced or a mouse eye inflammation model.

[0060] FIG. 3 is a picture of an exemplary suture made in a mouse eye inflammation model.

[0061] FIG. 4A is an in vitro confocal micrograph showing the limbal vessel in a control (untreated) mouse following injection of FITC-dextran.

[0062] FIG. 4B is an in vitro confocal micrograph showing the presence of calcein-labeled dendritic cells present in the limbal vessel of a mouse following induction of inflammation (suture-induced inflammation).

[0063] FIG. 4C is an in vitro confocal micrograph showing the presence of calcein-labeled dendritic cells present in the limbal vessel of a mouse that received an anti-VCAM-1 antibody prior to induction of eye inflammation (suture-induced inflammation).

[0064] FIG. 5 is a graph showing the percentage of rolling calcein-labeled dendritic cells in the limbal vessel compared to the total number of passing calcein-labeled dendritic cells in the limbal vessel in a mouse receiving no treatment or receiving an anti-P-selection antibody (anti-P-Sel), an anti-L-selectin antibody (anti-L-Sel), an anti-E-selectin antibody (anti-E-Sel), or an anti-PSGL-1 antibody (anti-PSGL-1).

[0065] FIG. 6 is a graph showing the percentage of rolling calcein-labeled dendritic cells in the limbal vessel compared to the total number of passing calcein-labeled dendritic cells in the limbal vessel in a mouse eye inflammation model receiving no treatment or receiving an anti-P-selectin antibody (anti-P-Sel), an anti-L-selectin antibody (anti-L-Sel), a combination of an anti-P-selectin antibody and an anti-L-selectin antibody (anti-P+L Sel), or an anti-CD44 antibody prior to induction of eye inflammation (suture-induced inflammation).

[0066] FIG. 7 is a graph showing the percentage of rolling calcein-labeled dendritic cells in the limbal vessel compared to the total number of passing calcein-labeled dendritic cells in the limbal vessel in a mouse receiving no treatment or receiving an anti-VCAM-1 antibody (anti-VCAM-1), an anti-ICAM-1 antibody (anti-ICAM-1), or an anti-MadCAM-1 antibody.

[0067] FIG. 8 is a graph showing the percentage of rolling calcein-labeled dendritic cells in the limbal vessel compared to the total number of passing calcein-labeled dendritic cells in the limbal vessel in a mouse eye inflammation model receiving no treatment or receiving an anti-VCAM-1 antibody (anti VCAM-1), an anti-ICAM-1 antibody (anti ICAM-1), or an anti-MadCAM-1 antibody (anti MadCAM-1) prior to induction of eye inflammation (suture-induced inflammation).

[0068] FIG. 9 is a graph showing the percentage of calcein-labeled dendritic cells that stick to the limbal vessel for greater than 30 seconds compared to the total number of calcein-labeled dendritic cells passing in the limbal vessel in a mouse receiving no treatment or receiving an anti-VCAM-1 antibody (anti-VCAM-1), an anti-ICAM-1 antibody (anti-ICAM-1), an anti-MadCAM-1 antibody (anti-Mad-CAM), or PTX.

[0069] FIG. 10 is a graph showing the percentage of calcein-labeled dendritic cells that stick to the limbal vessel for greater than 30 seconds compared to the total number of calcein-labeled dendritic cells passing in the limbal vessel in a mouse eye inflammation model receiving no treatment or receiving an anti-VCAM-1 antibody (anti-VCAM-1 antibody), an anti-ICAM-1 antibody (anti-ICAM), an anti-MadCAM-1 antibody (anti-MadCAM), or PTX.

[0070] FIG. 11 is a graph showing the number of calcein-labeled dendritic cells present in the corneal epithelium, the corneal anterior stroma, or the corneal posterior stroma in a mouse untreated or treated with an anti-47-antibody (a4b7), or in a mouse eye inflammation model that is untreated or treated with an anti-47-antibody (a4b7) prior to induction of inflammation (suture-induced inflammation).

[0071] FIG. 12 is a graph showing the number of calcein-labeled dendritic cells present in the corneal epithelium, the corneal anterior stroma, or the corneal posterior stroma in a mouse treated with a control rat IgG or an anti-MadCAM-1-antibody (anti-MadCam), or in a mouse eye inflammation model that is treated with a control rat IgG or an anti-MadCam-antibody (anti-MadCAM) prior to induction of inflammation (suture-induced inflammation).

[0072] FIG. 13 is a graph showing the RT-PCR data for the expression of MadCAM-1 in the corneal limbal tissue of control mice (steady state mice) or mice having eye inflammation (suture-induced inflammation), or in Peyer's patches of control mice (steady state mice) (positive control).

DETAILED DESCRIPTION OF THE INVENTION

[0073] The invention is based, at least in part, on the discovery that antibodies that specifically bind to MadCAM-1, L-selectin, E-selectin, or 47 integrin decrease the migration of dendritic cells to the cornea in a mouse model of corneal inflammation. In view of this discovery, provided herein are methods for reducing corneal and/or conjunctival inflammation, reducing inflammatory cell (e.g., dendritic cell) recruitment to the cornea and/or the conjunctiva, and treating a corneal inflammatory disorder and/or a conjunctival inflammatory disorder in a subject that include administering one or more (e.g., two, three, four, or five) of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and a E-selectin antagonist to a subject. Also provided are compositions containing one or more (e.g., two, three, four, or five) of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and a S-selectin antagonist, as well as kits containing these compositions. Additional aspects and exemplary embodiments of these methods, compositions, and kits are described herein.

Corneal Inflammation

[0074] Corneal inflammation is a condition that commonly results in the development of one or more symptoms in a subject. Non-limiting examples of such symptoms of corneal inflammation include: redness of the cornea; irritation, itchiness, burning, and/or dryness of the cornea; pain in the eye; excess tears or other discharge from an eye; difficulty opening an eyelid; blurred vision; sensitivity to light; and swelling around the eye. A subject can be diagnosed or identified as having corneal inflammation based on the observation or detection of one or more (e.g., at least two, three, or four) symptoms or physical characteristics of corneal inflammation selected from the group of: redness of the cornea; irritation, itchiness, burning, and/or dryness of the cornea; pain in the eye; excess tears or other discharge from an eye; difficulty opening an eyelid; blurred vision; sensitivity to light; swelling around the eye; and an elevated number of immunological cells present in the cornea (e.g., an elevated number of T-lymphocytes (e.g., effector T-cells), dendritic cells, stimulated monocytes, macrophages, B-cells, natural killer cells, eosinophils, and/or mast cells present in the cornea of the subject) (e.g., as compared to the same subject prior to the development of corneal inflammation, a control subject that does not have an eye disorder (a healthy subject), or a threshold value). In some embodiments, the detection of an elevated level of the number of immunological cells present in the cornea can be accomplished through the use of in vivo confocal microscopy using methods known in the art (see, e.g., the methods described in Cruzat et al., Semin. Ophthalmol. 25:171-177, 2010).

[0075] In some embodiments, the intensity, frequency, or duration of one or more symptoms of corneal inflammation can vary within the subject at any given time. For example, a subject having corneal inflammation can have one or more symptoms that are more prominent or more severe than other symptoms of corneal inflammation (depending on the subject and depending on the cause of the corneal inflammation).

[0076] In some embodiments, a subject having corneal inflammation (e.g., a low level of corneal inflammation) may only present with one or more symptoms that can only be detected using a microscopic technique (e.g., in vivo confocal microscopy) of the cornea. In some embodiments, a subject can present with both symptoms that can be detected without the use of a microscopic technique and symptoms that can only be detected using a microscopic technique. In some embodiments, a subject can be diagnosed or identified as having corneal inflammation by a medical professional (e.g., a physician, a physician's assistant, a nurse, a nurse's assistant, or a laboratory professional).

[0077] Corneal inflammation can be caused by a variety of factors. Non-limiting examples of causes of corneal inflammation include bacterial infection, fungal infection, parasite infection, viral infection (e.g., herpes simplex or herpes zoster), allergies, dry eye disorder, Fuchs' dystrophy, keratoconus, amyloidosis, lattice dystrophy, Stevens Johnson syndrome, physical corneal injury, Behcet's disease, contact lens wear, corneal graft rejection, dry eye syndrome, or immune keratitis (e.g., peripheral ulcerative keratitis). Additional causes of corneal inflammation are known in the art.

[0078] In some embodiments, the corneal inflammation is acute corneal inflammation. In some embodiments, the corneal inflammation is chronic corneal inflammation. In some embodiments, a subject having corneal inflammation has already been diagnosed as having a corneal inflammatory disorder (e.g., any of the corneal inflammatory disorders described herein). In some embodiments, the subject may already be receiving a treatment for corneal inflammation. In some embodiments, the subject may be resistant or show little responsiveness to a previous treatment for corneal inflammation. In some embodiments, the subject can be an infant, a child, or an adult (e.g., at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old). In some embodiments, the subject is a female. In some embodiments, the subject is a male.

Corneal Inflammatory Disorders

[0079] Corneal inflammatory disorders are a family of disorders of the eye that are characterized by two of more of the following features in a subject: an elevated number of T-lymphocytes (e.g., effector T-cells) in a cornea, an elevated number of dendritic cells in a cornea, an elevated number of macrophages in a cornea, an elevated number of stimulated monocytes in a cornea, an elevated number of B-cells in a cornea, an elevated number of natural killer cells in a cornea, an elevated number of eosinophils in a cornea, an elevated number of mast cells in a cornea, an elevated level of redness in a cornea, pain in an eye, irritation, itchiness, burning, and/or dryness of a cornea, excess tears or other discharge from an eye, difficulty opening an eyelid, blurred vision, sensitivity to light, and swelling around an eye (e.g., as compared to levels in the same subject prior to development of corneal inflammation, a subject that does not have an eye disorder (a healthy subject), or a threshold value). Non-limiting examples of corneal inflammatory disorders include allergy, corneal abrasion, puncture, or trauma (including surgically-induced trauma), keratitis (e.g., both non-infectious and infectious keratitis), corneal autoimmune disease, or corneal allograft or xenograft rejection. In some embodiments, the corneal inflammatory disorder is infectious keratits (e.g., bacterial keratitis, fungal keratitis, viral keratitis, or parasitic keratitis). A non-limiting example of viral keratitis is herpes simplex keratitis or herpes zoster ophthalmicus. Additional examples of corneal inflammatory disorders are known in the art.

[0080] Methods for diagnosing a corneal inflammatory disorder in a subject are known in the art. For example, a subject can be diagnosed or identified as having a corneal inflammatory disorder by the observation or detection of one or more (e.g., at least two, three, or four) symptoms or physical characteristics selected from the group of: redness of the cornea; irritation, itchiness, burning, and/or dryness of the cornea; pain in the eye; excess tears or other discharge from an eye; difficulty opening an eyelid; blurred vision; sensitivity to light; swelling around the eye; and an elevated number of immunological cells present in the cornea (e.g., an elevated number of T-lymphocytes (e.g., effector T-cells, dendritic cells, macrophages, stimulated monocytes, B-cells, natural killer cells, eosinophils, and/or mast cells present in the cornea of the subject) (e.g., as compared to the levels in the same subject prior to the development of a corneal inflammatory disorder, a subject that does not have an eye disorder (a healthy subject), or a threshold value). In some embodiments, the detection of an elevated level of the number of immunological cells present in the cornea can be accomplished through the use of in vivo confocal microscopy using methods known in the art (see, e.g., the methods described in Cruzat et al., Semin. Ophthalmol. 25:171-177, 2010).

[0081] In some embodiments, the intensity, frequency, or duration of one or more symptoms of a corneal inflammatory disorder can vary within the subject at any given time. For example, a subject having a corneal inflammatory disorder can have one or more symptoms that are more prominent or more severe than other symptoms of a corneal inflammatory disorder (depending on the subject and depending on specific corneal inflammatory disorder).

[0082] In some embodiments, a subject having a corneal inflammatory disorder may only present with one or more symptoms that can only be detected using a microscopic technique (e.g., in vivo confocal microscopy) to visualize the cornea. In some embodiments, a subject can present with both symptoms that can be detected without the use of a microscopic technique and symptoms that can only be detected using a microscopic technique. In some embodiments, a subject can be diagnosed or identified as having a corneal inflammatory disorder by a medical professional (e.g., a physician, a physician's assistant, a nurse, a nurse's assistant, or a laboratory professional).

[0083] In some embodiments, a subject has already been diagnosed as having a corneal inflammatory disorder (e.g., any of the corneal inflammatory disorders described herein). In some embodiments, the subject may already be receiving a treatment for corneal inflammation. In some embodiments, the subject may be resistant or show little responsiveness to a previous treatment for corneal inflammation. In some embodiments, the subject can be an infant, a child, or an adult (e.g., at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old). In some embodiments, the subject is a female. In some embodiments, the subject is a male.

Conjunctival Inflammation

[0084] Conjunctival inflammation is a condition that commonly results in the development of one or more symptoms in a subject. Non-limiting examples of such symptoms of conjunctival inflammation include: redness of the white of the eye or eyelid; irritation, itchiness, burning, and/or dryness of the eye; pain in the eye; excess tears or other discharge from an eye; difficulty opening an eyelid; blurred vision; sensitivity to light; and swelling around the eye. A subject can be diagnosed or identified as having conjunctival inflammation based on the observation or detection of one or more (e.g., at least two, three, or four) symptoms or physical characteristics of conjunctival inflammation selected from the group of: redness of the white of the eye or eyelid; irritation, itchiness, burning, and/or dryness of the eye; pain in the eye; excess tears or other discharge from an eye; difficulty opening an eyelid; blurred vision; sensitivity to light; swelling around the eye; and an elevated number of immunological cells present in the conjunctiva (e.g., an elevated number of T-lymphocytes (e.g., effector T-cells), dendritic cells, stimulated monocytes, macrophages, B-cells, natural killer cells, eosinophils, and/or mast cells present in the conjunctiva of the subject) (e.g., as compared to the same subject prior to the development of conjunctival inflammation, a control subject that does not have an eye disorder (a healthy subject), or a threshold value). In some embodiments, the detection of an elevated level of the number of immunological cells present in the conjunctiva can be accomplished through the use of in vivo confocal microscopy using methods known in the art (see, e.g., the methods described in Cruzat et al., Semin. Ophthalmol. 25:171-177, 2010).

[0085] In some embodiments, the intensity, frequency, or duration of one or more symptoms of conjunctival inflammation can vary within the subject at any given time. For example, a subject having conjunctival inflammation can have one or more symptoms that are more prominent or more severe than other symptoms of conjunctival inflammation (depending on the subject and depending on the cause of the conjunctival inflammation).

[0086] In some embodiments, a subject having conjunctival inflammation (e.g., a low level of conjunctival inflammation) may only present with one or more symptoms that can only be detected using a microscopic technique (e.g., in vivo confocal microscopy) of the conjunctiva. In some embodiments, a subject can present with both symptoms that can be detected without the use of a microscopic technique and symptoms that can only be detected using a microscopic technique. In some embodiments, a subject can be diagnosed or identified as having conjunctival inflammation by a medical professional (e.g., a physician, a physician's assistant, a nurse, a nurse's assistant, or a laboratory professional).

[0087] Conjunctival inflammation can be caused by a variety of factors. Non-limiting examples causes of conjunctival inflammation include viruses, bacteria (e.g., gonorrhea or chlamydia), irritants (e.g., shampoos, dirt, smoke, or chlorine), an allergen, or contact lens wear. Additional causes of conjunctival inflammation are known in the art.

[0088] In some embodiments, the conjunctival inflammation is acute conjunctival inflammation. In some embodiments, the conjunctival inflammation is chronic conjunctival inflammation. In some embodiments, a subject having conjunctival inflammation has already been diagnosed as having a conjunctival inflammatory disorder (e.g., any of the conjunctival inflammatory disorders described herein). In some embodiments, the subject may already be receiving a treatment for conjuctival inflammation. In some embodiments, the subject may be resistant or show little responsiveness to a previous treatment for conjunctival inflammation. In some embodiments, the subject can be an infant, a child, or an adult (e.g., at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old). In some embodiments, the subject is a female. In some embodiments, the subject is a male.

Conjunctival Inflammatory Disorders

[0089] Conjunctival inflammatory disorders are a family of disorders of the eye that are characterized by two of more of the following features in a subject: an elevated number of T-lymphocytes (e.g., effector T-cells) in a conjunctiva, an elevated number of dendritic cells in a conjunctiva, an elevated number of macrophages in a conjunctiva, an elevated number of stimulated monocytes in a conjunctiva, an elevated number of B-cells in a conjunctiva, an elevated number of natural killer cells in a conjunctiva, an elevated number of eosinophils in a conjunctiva, an elevated number of mast cells in a conjunctiva, an elevated level of redness in the white of an eye or an eyelid, pain in an eye, irritation, itchiness, burning, and/or dryness of an eye, excess tears or other discharge from an eye, difficulty opening an eyelid, blurred vision, sensitivity to light, and swelling around an eye (e.g., as compared to levels in the same subject prior to development of conjunctival inflammation, a subject that does not have an eye disorder (a healthy subject), or a threshold value). Non-limiting examples of conjunctival inflammatory disorders include viral conjunctivitis, bacterial conjunctivis, fungal conjunctivitis, parasitic conjunctivitis, or allergic conjunctivitis. Additional examples of conjunctival inflammatory disorders are known in the art.

[0090] Methods for diagnosing a conjunctival inflammatory disorder in a subject are known in the art. For example, a subject can be diagnosed or identified as having a conjunctival inflammatory disorder by the observation or detection of one or more (e.g., at least two, three, or four) symptoms or physical characteristics selected from the group of: redness of the cornea; irritation, itchiness, burning, and/or dryness of an eye; pain in the eye; excess tears or other discharge from an eye; difficulty opening an eyelid; blurred vision; sensitivity to light; swelling around the eye; and an elevated number of immunological cells present in the conjunctiva (e.g., an elevated number of T-lymphocytes (e.g., effector T-cells, dendritic cells, macrophages, stimulated monocytes, B-cells, natural killer cells, eosinophils, and/or mast cells present in the conjunctiva of the subject) (e.g., as compared to the levels in the same subject prior to the development of a conjunctival inflammatory disorder, a subject that does not have an eye disorder (a healthy subject), or a threshold value). In some embodiments, the detection of an elevated level of the number of immunological cells present in the conjunctiva can be accomplished through the use of in vivo confocal microscopy using methods known in the art (see, e.g., the methods described in Cruzat et al., Semin. Ophthalmol. 25:171-177, 2010).

[0091] In some embodiments, the intensity, frequency, or duration of one or more symptoms of a conjunctival inflammatory disorder can vary within the subject at any given time. For example, a subject having a conjuctival inflammatory disorder can have one or more symptoms that are more prominent or more severe than other symptoms of a conjunctival inflammatory disorder (depending on the subject and depending on specific conjunctival inflammatory disorder).

[0092] In some embodiments, a subject having a conjunctival inflammatory disorder may only present with one or more symptoms that can only be detected using a microscopic technique (e.g., in vivo confocal microscopy) to visualize the conjunctiva. In some embodiments, a subject can present with both symptoms that can be detected without the use of a microscopic technique and symptoms that can only be detected using a microscopic technique. In some embodiments, a subject can be diagnosed or identified as having a conjunctival inflammatory disorder by a medical professional (e.g., a physician, a physician's assistant, a nurse, a nurse's assistant, or a laboratory professional).

[0093] In some embodiments, a subject has already been diagnosed as having a conjunctival inflammatory disorder (e.g., any of the conjunctival inflammatory disorders described herein). In some embodiments, the subject may already be receiving a treatment for conjunctival inflammation. In some embodiments, the subject may be resistant or show little responsiveness to a previous treatment for conjunctival inflammation. In some embodiments, the subject can be an infant, a child, or an adult (e.g., at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old). In some embodiments, the subject is a female. In some embodiments, the subject is a male.

Antagonists

[0094] Provided herein, in part, are methods for decreasing corneal and/or conjunctival inflammation, decreasing immunological cell (e.g., dendritic cell) migration to the cornea and/or conjunctiva, and treating a corneal inflammatory disorder and/or conjunctival inflammatory disorder that include the administration of one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and a E-selectin antagonist to a subject.

[0095] Mucosal vascular addressin cell adhesion molecule 1 (MadCAM-1), also known as addressin, binds to 47 integrin, L-selectin, and 41 integrin. Mature human MadCAM-1 binds to 47 integrin through its two conserved immunoglobulin superfamily (IgSF) domains (see, e.g., Tan et al., Structure 15:793-801, 1998). Conserved residues in human MadCAM-1 that are important for integrin binding (e.g., 47 and 41 integrin) include the CD loop of the first IgSF domain and amino acid residues Asp42 and Arg70 (see, Tan et al., supra). In contrast, the amino acids that are glycosylated in MadCAM-1 are important for L-selectin binding (see, Tan et al., supra). A precursor form of MadCAM-1 is processed to remove a signal peptide. The mature form of MadCAM-1 contains a short cytoplasmic domain, a transmembrane domain, a mucin-like domain, and the two IgSF domains (see, Leung et al., Immunol. Cell. Biol. 74:490-496, 1996; Shyjan et al., J. Immunol. 156:2851-2857, 1996). Non-limiting examples of human mature, processed forms of MadCAM-1 protein are SEQ ID NOs: 1-7.

[0096] Non-limiting examples of MadCAM-1 antagonists include antibodies and antigen-binding antibody fragments that specifically bind to MadCAM-1. Additional non-limiting examples of MadCAM-1 antagonists include soluble 47 agents, soluble 41 agents, and soluble L-selectin molecules. Exemplary embodiments and aspects of these MadCAM-1 antagonists are described below.

[0097] Alpha-4 beta-7 integrin is a heterodimer of the 4 integrin and the 74 integrin that is constitutively expressed on the surface of leukocytes including lymphocytes, monocytes, eosinophils and basophils (see Hemler et al., J. Biol. Chem. 262:11478-11485, 1987; and Bochner et al., J. Exp. Med. 173:1553-1556, 1991). A non-limiting example of the sequence of human mature, processed 4 integrin is SEQ ID NO: 8, and a non-limiting example of the sequence of human mature, process 7 integrin is SEQ ID NO: 9.

[0098] Non-limiting examples of 47 integrin antagonists include antibodies and antigen-binding antibody fragments that specifically bind to 47 integrin. Additional non-limiting examples of 47 integrin antagonists include soluble MadCAM-1 molecules, small molecule 47 integrin antagonists, and antibodies or antigen-binding fragments that bind to 4 integrin or 7 integrin. Exemplary embodiments and aspects of these 47 antagonists are described below.

[0099] L-selectin, also known as CD62L, is a cell adhesion molecule expressed in the plasma membrane of leukocytes. It contains several domains, including a signal sequence, transmembrane domain, and cytoplasmic domain. L-selectin binds to GlyCAM-1, CD34, MadCAM-1, and PSGL-1. A non-limiting example of the sequence of human mature, processed L-selectin is SEQ ID NO: 19.

[0100] Non-limiting examples of L-selectin antagonists are antibodies or an antigen-binding antibody fragments that specifically bind to L-selectin, or a soluble MadCAM-1 agent, a soluble CD34 molecule, a soluble PSGL-1 molecule, or a soluble GlyCAM-1 agent. Additional examples of L-selectin antagonists are small molecules (e.g., the small molecule L-selectin antagonists described herein or known in the art).

[0101] E-selectin, also known as CD62E, endothelial-leukocyte adhesion molecule 1 (ELAM-1), or leukocyte-endothelial cell adhesion molecule 2 (LECAM2), is a cell adhesion molecule expressed on endothelial cells (e.g., endothelial cells activated by cytokines). It contains several domains, including a signal sequence, transmembrane domain, and cytoplasmic domain. E-selectin binds to sialylated carbohydrates (e.g., carbohydrates of the Lewis X and Lewis A families) present on proteins expressed by leukocytes (e.g., monocytes, granulocytes, and T-lymphocytes).

[0102] Non-limiting examples of E-selectin antagonists are antibodies or an antigen-binding antibody fragments that specifically bind to E-selectin. Additional examples of E-selectin antagonists are small molecules (e.g., the small molecule E-selectin antagonists described herein or known in the art).

Antibodies and Antigen-Binding Antibody Fragments

[0103] Antibodies that specifically bind to MadCAM-1, 47, 4 integrin, 7 integrin, L-selectin, or E-selectin can be, for example, polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific), human antibodies, chimeric antibodies (e.g., human-mouse chimera), single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and IgA.sub.2), or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG.sub.1 antibody or antigen-binding fragment thereof. In other embodiments, the antibody or antigen-binding fragment thereof is an IgG4 antibody or antigen-binding fragment thereof. Immunoglobulins may have both a heavy and light chain.

[0104] Antibodies and antibody fragments as referred to herein include variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments. Examples of antibodies and antigen-binding fragments thereof include, but are not limited to: single-chain Fvs (scFvs), Fab fragments, Fab fragments, F(ab)2, disulfide-linked Fvs (sdFvs), Fvs, and fragments containing either a VL or a VH domain. The term single chain Fv or scFv as used herein refers to a polypeptide comprising at least one VL domain of an antibody linked to at least one VH domain of an antibody.

[0105] An isolated MadCAM-1, 47, 4 integrin, 7 integrin, L-selectin, or E-selectin, or a fragment thereof (e.g., at least 7, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 amino acids of a wild type form of MadCAM-1, 4 integrin, 7 integrin, 47 integrin, L-selectin, or E-selectin) can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivitizing agent, for example, malimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl.sub.2, or R.sup.1NCNR, where R and R.sup.1 are different alkyl groups. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times).

[0106] An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus). An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide (e.g., a fragment of MadCAM-1, such as the soluble MadCAM-1 molecules described herein, a fragment of 47 integrin, such as the soluble 47 agents described herein, a fragment of 4 integrin, a fragment of 7 integrin, a fragment of L-selectin, such as the soluble L-selectin agents described herein, or a fragment of E-selectin). The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.

[0107] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with MadCAM-1, 4 integrin, 7 integrin, 47 integrin, L-selectin, or E-selectin, or an antigenic peptide thereof (e.g., a fragment of MadCAM-1, such as the soluble MadCAM-1 molecules described herein, a fragment of 47 integrin, such as the soluble 47 agents described herein, a fragment of 4 integrin, a fragment of 7 integrin, a fragment of L-selectin, such as the soluble L-selectin molecules described herein, or a fragment of E-selectin) as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized MadCAM-1, 47, 4 integrin, 7 integrin, L-selectin, or E-selectin, or fragment thereof. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or trioma techniques. The technology for producing hybridomas is well known (see, generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y.). Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.

[0108] As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against a polypeptide can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide or a peptide fragment containing the epitope of interest. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP* Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening an antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; WO 92/18619; WO 91/17271; WO 92/2079; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; Fuchs et al., Bio/Technology 9:1370-1372, 1991; Hay et al., Hum. Antibod. Hybridomas 3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989; and Griffiths et al., FHBO J. 12:725-734, 1993.

[0109] In some embodiments of any of the methods described herein, the antibodies or antigen-binding fragments are human antibodies, humanized antibodies, or chimeric antibodies that contain a sequence from a human antibody (e.g., a human immunoglobulin constant domain or human immunoglobulin variable domain framework regions). Humanized antibodies are chimeric antibodies that contain a minimal sequence derived from non-human (e.g., mouse) immunoglobulin. In some embodiments, a humanized antibody is a human antibody that has been engineered to contain at least one complementary determining region (CDR) present in a non-human antibody (e.g., a mouse, rat, rabbit, or goat antibody). In some embodiments, a humanized antibody or fragment thereof can contain all three CDRs of a light chain and/or a heavy chain of a non-human antibody that specifically binds to MadCAM-1 or a fragment thereof, a non-human antibody that specifically binds to 47 or a fragment thereof, a non-human antibody that specifically binds to L-selectin or a fragment thereof, or a non-human antibody that specifically binds to E-selectin or a fragment thereof. In some embodiments, the framework region residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) antibody residues. In some embodiments, the humanized antibodies can contain residues which are not found in the human antibody or in the non-human (e.g., mouse) antibody. Methods for making a humanized antibody from a non-human (e.g., mouse) monoclonal antibody are known in the art. Additional non-limiting examples of making a chimeric (e.g., humanized) antibody are described herein.

[0110] In some embodiments, the antibodies are chimeric antibodies that contain a light chain immunoglobulin that contains the light chain variable domain of a non-human antibody (e.g., a mouse antibody) or at least one CDR of a light chain variable domain of a non-human antibody (e.g., a mouse antibody) and the constant domain of a human immunoglobulin light chain (e.g., human chain constant domain). In some embodiments, the antibodies are chimeric antibodies that contain a heavy chain immunoglobulin that contains the heavy chain variable domain of a non-human (e.g., a mouse antibody) or at least one CDR of a heavy chain variable domain of a non-human (e.g., a mouse antibody) and the constant domain of a human immunoglobulin heavy chain (e.g., a human IgG heavy chain constant domain). In some embodiments, the chimeric antibodies contain a portion of a constant (Fc domain) of a human immunoglobulin.

[0111] In some embodiments, the antibodies or antigen-binding fragments thereof can be multi-specific (e.g., multimeric). For example, the antibodies can take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole immunoglobulin molecules or of F(ab).sub.2 fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent. Individual monomers within an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers. For example, individual antibodies within a multimer may have the same or different binding specificities.

[0112] Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG.sub.1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers. Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acethylthio-acetate) (available, for example, from Pierce Biotechnology, Inc., Rockford, Ill.) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997). Antibody homodimers can be converted to Fab2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0113] In some embodiments, the multi-specific antibody is a bi-specific antibody. Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers (see, for example, WO 96/27011).

[0114] Bi-specific antibodies include cross-linked or heteroconjugate antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a variety of cross-linking techniques.

[0115] Methods for generating bi-specific antibodies from antibody fragments are also known in the art. For example, bi-specific antibodies can be prepared using chemical linkage. Brennan et al. (Science 229:81, 1985) describes a procedure where intact antibodies are proteolytically cleaved to generate F(ab)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab TNB derivatives is then reconverted to the Fab thiol by reduction with mercaptoethylamine, and is mixed with an equimolar amount of another Fab TNB derivative to form the bi-specific antibody.

[0116] Additional methods have been developed to facilitate the direct recovery of Fab-SH fragments from E. coli, which can be chemically coupled to form bi-specific antibodies. Shalaby et al. (J. Exp. Med 175:217-225, 1992) describes the production of a fully-humanized bi-specific antibody F(ab)2 molecule. Each Fab fragment was separately secreted from E. coli and subjected to direct chemical coupling in vitro to form the bi-specific antibody.

[0117] Additional techniques for making and isolating bi-specific antibody fragments directly from recombinant cell culture have also been described. For example, bi-specific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol. 148:1547-1553, 1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.

[0118] The diabody technology described by Hollinger et al. (Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, 1993) is an additional method for making bi-specific antibody fragments. The fragments contain a heavy chain variable domain (V.sub.H) connected to a light chain variable domain (V.sub.L) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V.sub.H and V.sub.L domains of one fragment are forced to pair with the complementary V.sub.L and V.sub.H domains of another fragment, thereby forming two antigen-binding sites. Another method for making bi-specific antibody fragments by the use of single-chain Fv (sFv) dimers has been described in Gruber et al. (J. Immunol. 153:5368, 1994). Alternatively, the bi-specific antibody can be a linear or single-chain antibody produced using the methods described, for example, in Zapata et al. (Protein Eng. 8:1057-1062, 1995). In some embodiments the antibodies have more than two antigen-binding sites. For example, tri-specific antibodies can be prepared as described in Tutt et al. (J. Immunol. 147:60, 1991).

[0119] Alternatively, antibodies can be made to multimerize through recombinant DNA techniques. IgM and IgA naturally form antibody multimers through the interaction with the mature J chain polypeptide. Non-IgA or non-IgM molecules, such as IgG molecules, can be engineered to contain the J chain interaction domain of IgA or IgM, thereby conferring the ability to form higher order multimers on the non-IgA or non-IgM molecules (see, for example, Chintalacharuvu et al., Clin. Immunol. 101:21-31, 2001, and Frigerio et al., Plant Physiol. 123:1483-1494, 2000). IgA dimers are naturally secreted into the lumen of mucosa-lined organs. This secretion is mediated through the interaction of the J chain with the polymeric IgA receptor (pIgR) on epithelial cells. If secretion of an IgA form of an antibody (or of an antibody engineered to contain a J chain interaction domain) is not desired, it can be greatly reduced by expressing the antibody molecule in association with a mutant J chain that does not interact well with pIgR (Johansen et al., J. Immunol., 167:5185-192, 2001). ScFv dimers can also be formed through recombinant techniques known in the art. An example of the construction of scFv dimers is given in Goel et al. (Cancer Res. 60:6964-71, 2000). Antibody multimers may be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography.

[0120] Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin). The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human).

[0121] Any of the antibodies or antigen-binding fragments described herein may be conjugated to a label (e.g., a radioisotope, fluorophore, chromophore, or the like) or a therapeutic agent (e.g., a proteinaceous or small molecule therapeutic agent).

[0122] In some embodiments of any of the methods described herein, the antibody or antigen-binding fragment thereof binds to an epitope on MadCAM-1, 47, 4 integrin, or 7 integrin with an KD equal to or less than 110.sup.7 M, a KD equal to or less than 110.sup.8 M, a KD equal to or less than 510.sup.8 M, a KD equal to or less than 510.sup.9M, or a KD equal to or less than 110.sup.9M under physiological conditions (e.g., in phosphate buffered saline).

[0123] Methods for detecting the ability of an antibody or antigen-binding antibody fragment to bind to MadCAM-1, 47, 4 integrin, 7 integrin, L-selectin, or E-selectin can be performed using methods known in the art, e.g., surface plasmon resonance (SPR).

[0124] Additional exemplary antibodies that specifically bind to MadCAM-1 are known in the art (e.g., U.S. Patent Application Publication No. 20090214558; herein incorporated by reference; and MadCAM-1 antibodies commercially available from Abbiotec, Santa Cruz Technology, and Hycult Biotech). Additional exemplary antibodies that specifically bind to 47 are known in the art (e.g., natalizumab, vedolizumab, and the humanized antibodies described in U.S. Pat. Nos. 5,840,299; 6,602,503; 7,482,003; and 7,618,630; and U.S. Patent Applications Nos. 2010/0203042 and 2009/0202527 (each listed patent and patent application is incorporated herein by reference). In some embodiments, an 47 antagonist is an antibody or an antigen-binding fragment thereof that can bind to 4 and/or 7. An exemplary humanized antibody that binds to human 4 integrin is natalizumab. Additional examples of humanized antibodies that bind to human 4 integrin are described in U.S. Patent Application Publication Nos. 2010/0081793. An exemplary humanized antibody that binds to human 7 integrin is rhuMAb Beta7.

[0125] Additional exemplary antibodies that specifically bind to L-selectin are known in the art (e.g., aselizumab, HuDREG-55, HuEP5C7, and DREG-200). Additional exemplary antibodies that specifically bind to E-selectin are known in the art (e.g., HuEP5C7, CDP850, and CDP-850).

[0126] In some embodiments, the antibodies or antigen-binding fragments described herein are isolated or purified (e.g., at least 70%, 75%, 80%, 85%, 90%, 96%, 97%, 98%, or 99% pure by dry weight).

[0127] Soluble L-Selectin Molecules

[0128] The mature form of L-selectin contains the following domains: a lectin domain, an epiderminal growth factor domain, two SCR domains, a transmembrane domain and a cytoplasmic domain. A soluble L-selectin molecule contains an amino acid sequence that is at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, or 200 amino acids) in a wild type form of L-selectin (e.g., a precursor or processed L-selectin, e.g., any one of SEQ ID NOS: 10 and 19) that is soluble at a physiological pH and has the ability to specifically bind to MadCAM-1. In some embodiments, the soluble L-selectin lacks one or more (e.g., two, three, or four of its domains) of its domains. In some embodiments, the soluble L-selectin molecule lacks its signal sequence, its transmembrane domain, and its cytoplasmic domain. In some embodiments, the soluble L-selectin molecule contains an additional amino acid sequence (e.g., a sequence that stabilizes the protein or increases the protein's half-life in vivo, e.g., an Fc domain or serum albumin).

[0129] In some embodiments, the soluble L-selectin molecule contains a sequence that is at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, or 200 amino acids) present between amino acids 171 and amino acid 385 in SEQ ID NO: 10 shown below. The mRNA encoding precursor L-selectin is also shown below. Soluble L-selectin molecules can be generated using molecular biology skills known in the art. The ability of a soluble L-selectin molecule to bind to MadCAM-1, can be performed using cell binding assays (e.g., cells expressing one or more of MadCAM-1), competitive binding assays with antibodies that specifically bind to MadCAM-1, or surface plasmon resonance (SPR).

Precursor human L-selectin polypeptide (SEQ ID NO: 10) with the signal peptide underlined and the transmembrane domain shown in bold.

TABLE-US-00001 (SEQIDNO:10) MGCRRTREGPSKAMIFPWKCQSTQRDLWNIFKLWGWTMLC CDFLAHHGTDCWTYHYSEKPMNWQRARRFCRDNYTDLVAI QNKAEIEYLEKTLPFSRSYYWIGIRKIGGIWTWVGTNKSL TEEAENWGDGEPNNKKNKEDCVEIYIKRNKDAGKWNDDAC HKLKAALCYTASCQPWSCSGHGECVEIINNYTCNCDVGYY GPQCQFVIQCEPLEAPELGTMDCTHPLGNFSFSSQCAFSC SEGINLIGIEETTCGPFGNWSSPEPTCQVIQCEPLSAPDL GIMNCSHPLASFSFTSACTFICSEGTELIGKKKTICESSG IWSNPSPICQKLDKSFSMIKEGDYNPLFIPVAVMVTAFSG LAFIIWLARRLKKGKKSKRSMNDPY
An exemplary cDNA encoding precursor human L-selectin is SEQ ID NO: 15.

[0130] Soluble PSGL-1 Agents

[0131] The precursor form of PSGL-1 contains a signal sequence that is cleaved to form the mature protein. A soluble PSGL-1 molecule contains an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids) in a wild type form of PSGL-1 (e.g., a precursor or processed PSGL-1, e.g., SEQ ID NO: 21) that is soluble at a physiological pH and has the ability to specifically bind to L-selectin. In some embodiments, the soluble PSGL-1 lacks one or more (e.g., two, three, or four of its domains) of its domains. In some embodiments, the soluble PSGL-1 molecule lacks its signal sequence, its transmembrane domain, and its cytoplasmic domain. In some embodiments, the soluble PSGL-1 molecule contains an additional amino acid sequence (e.g., a sequence that stabilizes the protein or increases the protein's half-life in vivo, e.g., an Fc domain or serum albumin).

[0132] In some embodiments, the soluble PSGL-1 molecule contains a sequence that is at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids) present between amino acids 18 and amino acid 320 in SEQ ID NO: 21 shown below. The mRNA encoding precursor PSGL-1 is also shown below. Soluble PSGL-1 molecules can be generated using molecular biology skills known in the art. The ability of a soluble PSGL-1 molecule to specifically bind to L-selectin, can be performed using cell binding assays (e.g., cells expressing L-selectin), competitive binding assays with antibodies that specifically bind to L-selectin, or surface plasmon resonance (SPR).

Precursor human PSGL-1 polypeptide (SEQ ID NO: 21) with the signal peptide underlined and the transmembrane domain shown in bold.

TABLE-US-00002 (SEQIDNO:21) 1 MPLQLLLLLILLGPGNSLQLWDTWADEAEKALGPLLARDRRQATEYEYLDYDFLPETEPP 61 EMLRNSTDTTPLTGPGTPESTTVEPAARRSTGLDAGGAVTELTTELANMGNLSTDSAAME 121 IQTTQPAATEAQTTQPVPTEAQTTPLAATEAQTTRLTATEAQTTPLAATEAQTTPPAATE 181 AQTTQPTGLEAQTTAPAAMEAQTTAPAAMEAQTTPPAAMEAQTTQTTAMEAQTTAPEATE 241 AQTTQPTATEAQTTPLAAMEALSTEPSATEALSMEPTTKRGLFIPFSVSSVTHKGIPMAA 301 SNLSVNYPVGAPDHISVKQCLLAILILALVATIFFVCTVVLAVRLSRKGHMYPVRNYSPT 361 EMVCISSLLPDGGEGPSATANGGLSKAKSPGLTPEPREDREGDDLTLHSFLP
An exemplary cDNA encoding precursor human PSGL-1 is SEQ ID NO: 22.

[0133] Soluble CD34 Molecules

[0134] The precursor form of CD34 contains a signal sequence that is cleaved to form the mature protein. A soluble CD34 molecule contains an amino acid sequence that is at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, or 250 amino acids) in a wild type form of CD34 (e.g., a precursor or processed CD34, e.g., SEQ ID NO: 23) that is soluble at a physiological pH and has the ability to specifically bind to L-selectin. In some embodiments, the soluble CD34 lacks one or more (e.g., two, three, or four of its domains) of its domains. In some embodiments, the soluble CD34 molecule lacks its signal sequence, its transmembrane domain, and its cytoplasmic domain. In some embodiments, the soluble CD34 molecule contains an additional amino acid sequence (e.g., a sequence that stabilizes the protein or increases the protein's half-life in vivo, e.g., an Fc domain or serum albumin).

[0135] In some embodiments, the soluble CD34 molecule contains a sequence that is at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, or 250 amino acids) present between amino acids 32 and amino acid 290 in SEQ ID NO: 23 shown below. The mRNA encoding precursor CD34 is also shown below. Soluble CD34 molecules can be generated using molecular biology skills known in the art. The ability of a soluble CD34 molecule to bind to L-selectin, can be performed using cell binding assays (e.g., cells expressing L-selectin), competitive binding assays with antibodies that specifically bind to L-selectin, or surface plasmon resonance (SPR).

Precursor human CD34 polypeptide (SEQ ID NO: 23) with the signal peptide underlined and the transmembrane domain shown in bold.

TABLE-US-00003 (SEQIDNO:23) 1 MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQGTFSNVSTNVSYQE 61 TTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTV 121 FTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGI 181 REVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRP 241 QCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLIALVTSGAL 301 LAVLGITGYFLMNRRSWSPTGERLGEDPYYTENGGGQGYSSGPGTSPEAQGKASVNRGAQ 361 ENGTGQATSRNGHSARQHVVADTEL
An exemplary cDNA encoding precursor human CD34 is SEQ ID NO: 24.

Soluble GlyCAM-1 Molecules

[0136] The precursor form of GlyCAM-1 contains a signal sequence that is cleaved to form the mature protein. A soluble GlyCAM-1 molecule contains an amino acid sequence that is at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 10 or 20 amino acids) in a wild type form of GlyCAM-1 (e.g., a precursor or processed GlyCAM-1, e.g., SEQ ID NO: 25) that is soluble at a physiological pH and has the ability to specifically bind to L-selectin. In some embodiments, the soluble GlyCAM-1 molecule lacks its signal sequence. In some embodiments, the soluble GlyCAM-1 molecule contains an additional amino acid sequence (e.g., a sequence that stabilizes the protein or increases the protein's half-life in vivo, e.g., an Fc domain or serum albumin).

[0137] In some embodiments, the soluble GlyCAM-1 molecule contains a sequence that is at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 10 or 20 amino acids) present between amino acids 19 and amino acid 47 in SEQ ID NO: 25 shown below. The mRNA encoding precursor GlyCAM-1 is also shown below. Soluble GlyCAM-1 molecules can be generated using molecular biology skills known in the art. The ability of a soluble GlyCAM-1 molecule to bind to L-selectin, can be performed using cell binding assays (e.g., cells expressing L-selectin), competitive binding assays with antibodies that specifically bind to L-selectin, or surface plasmon resonance (SPR).

Precursor human GlyCAM-1 polypeptide (SEQ ID NO: 25) with the signal peptide underlined.

TABLE-US-00004 (SEQIDNO:25) 1 MKFFMVLLPASLASTSLAILDVESGLLPQLSVLLSNRLRGKTCQTGP
An exemplary cDNA encoding precursor human GlyCAM-1 is SEQ ID NO: 26.

[0138] Soluble 47 Agents

[0139] Alpha-4, beta-7 integrin is a heterodimer that is composed of 4 integrin and 7 integrin. Both the 4 integrin and the 4 integrin contain a number of domains including a signal sequence, a transmembrane domain, and a cytoplasmic domain. A soluble 47 agent is a composition that contains a protein (e.g., a single polypeptide or heterodimeric protein) containing an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 850 amino acids) in a wild type form of 4 integrin (e.g., precursor or processed 4 integrin, e.g., any one of SEQ ID NOS: 8 and 12) and an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 amino acids) in a wild type form of 7 integrin (e.g., precursor or processed 7 integrin, e.g., any one of SEQ ID NOS: 9 and 13) that is soluble at physiological pH and has the ability to specifically bind to MadCAM-1. In some embodiments, the amino acid sequence that is at least 95% identical to a contiguous sequence in 4 integrin lacks the signal sequence, the transmembrane domain, and cytoplasmic domain of 4 integrin, and/or the amino acid sequence that is at least 95% identical to a contiguous sequence in 7 integrin lacks the signal sequence, the transmembrane domain, and cytoplasmic domain of 7 integrin. In some embodiments, the soluble 47 agent contains a polypeptide that contains an additional amino acid sequence (e.g., a sequence that stabilizes the polypeptide or increases the polypeptide's half-life in vivo, e.g., an Fc domain or bovine serum albumin).

[0140] In some embodiments, the amino acid sequence that is at least 95% identical to a contiguous sequence in 4 integrin contains a sequence that is at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 850 amino acids) present between amino acids 34 and amino acid 977 in SEQ ID NO: 12. In some embodiments, the amino acid sequence that is at least 95% identical to a contiguous sequence in 7 integrin contains a sequence that is at least 95% identical (e.g., at least 96%, 97%, 98%, 99%, or 100% identical) to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 amino acids) present between amino acids 20 and amino acid 723 in SEQ ID NO: 13. The mRNA encoding 4 and the mRNA encoding 7 are also shown below. Soluble 47 agents can be generated using molecular biology skills known in the art. The ability of a soluble 47 agent to bind to MadCAM-1, can be performed using cell binding assays (e.g., cells expressing one or more of MadCAM-1), competitive binding assays with antibodies that specifically bind to MadCAM-1, or surface plasmon resonance (SPR).

Precursor human 4 integrin polypeptide (SEQ ID NO: 12) with the signal sequence underlined and the transmembrane domain shown in bold.

TABLE-US-00005 (SEQIDNO:12) 1 MAWEARREPGPRRAAVRETVMLLLCLGVPTGRPYNVDTESALLYQGPHNTLFGYSVVLHS 61 HGANRWLLVGAPTANWLANASVINPGAIYRCRIGKNPGQTCEQLQLGSPNGEPCGKTCLE 121 ERDNQWLGVTLSRQPGENGSIVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRI 181 APCYQDYVKKFGENFASCQAGISSFYTKDLIVMGAPGSSYWTGSLFVYNITTNKYKAFLD 241 KQNQVKFGSYLGYSVGAGHFRSQHTTEVVGGAPQHEQIGKAYIFSIDEKELNILHEMKGK 301 KLGSYFGASVCAVDLNADGFSDLLVGAPMQSTIREEGRVFVYINSGSGAVMNAMETNLVG 361 SDKYAARFGESIVNLGDIDNDGFEDVAIGAPQEDDLQGAIYIYNGRADGISSTFSQRIEG 421 LQISKSLSMFGQSISGQIDADNNGYVDVAVGAFRSDSAVLLRTRPVVIVDASLSHPESVN 481 RTKFDCVENGWPSVCIDLTLCFSYKGKEVPGYIVLFYNMSLDVNRKAESPPRFYFSSNGT 541 SDVITGSIQVSSREANCRTHQAFMRKDVRDILTPIQIEAAYHLGPHVISKRSTEEFPPLQ 601 PILQQKKEKDIMKKTINFARFCAHENCSADLQVSAKIGFLKPHENKTYLAVGSMKTLMLN 661 VSLFNAGDDAYETTLHVKLPVGLYFIKILELEEKQINCEVTDNSGVVQLDCSIGYIYVDH 721 LSRIDISFLLDVSSLSRAEEDLSITVHATCENEEEMDNLKHSRVTVAIPLKYEVKLTVHG 781 FVNPTSFVYGSNDENEPETCMVEKMNLTFHVINTGNSMAPNVSVEIMVPNSFSPQTDKLF 841 NILDVQTTTGECHFENYQRVCALEQQKSAMQTLKGIVRFLSKTDKRLLYCIKADPHCLNF 901 LCNFGKMESGKEASVHIQLEGRPSILEMDETSALKFEIRATGFPEPNPRVIELNKDENVA 961 HVLLEGLHHQRPKRYFTIVIISSSLLLGLIVLLLISYVMWKAGFFKRQYKSILQEENRRD 1021 SWSYINSKSNDD
Precursor human 7 integrin polypeptide (SEQ ID NO: 13) with the signal sequence underlined and the transmembrane region shown in bold.

TABLE-US-00006 (SEQIDNO:13) 1 MVALPMVLVLLLVLSRGESELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPS 61 CAWCKQLNFTASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGA 121 TQLAPQRVRVTLRPGEPQQLQVRFLRAEGYPVDLYYLMDLSYSMKDDLERVRQLGHALLV 181 RLQEVTHSVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTRLERCQSPFSFHHVLSLTGDAQ 241 AFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRNVSRLLVFTSDDTFHTAGDGKL 301 GGIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAVTSAALPVYQELS 361 KLIPKSAVGELSEDSSNVVQLIMDAYNSLSSTVTLEHSSLPPGVHISYESQCEGPEKREG 421 KAEDRGQCNHVRINQTVTFWVSLQATHCLPEPHLLRLRALGFSEELIVELHTLCDCNCSD 481 TQPQAPHCSDGQGHLQCGVCSCAPGRLGRLCECSVAELSSPDLESGCRAPNGTGPLCSGK 541 GHCQCGRCSCSGQSSGHLCECDDASCERHEGILCGGFGRCQCGVCHCHANRTGRACECSG 601 DMDSCISPEGGLCSGHGRCKCNRCQCLDGYYGALCDQCPGCKTPCERHRDCAECGAFRTG 661 PLATNCSTACAHTNVTLALAPILDDGWCKERTLDNQLFFFLVEDDARGTVVLRVRPQEKG 721 ADHTQAIVLGCVGGIVAVGLGLVLAYRLSVEIYDRREYSRFEKEQQQLNWKQDSNPLYKS 781 AITTTINPRFQEADSPTL
An exemplary cDNA encoding human 4 integrin is SEQ ID NO: 27. An exemplary cDNA encoding human 7 integrin is SEQ ID NO: 16.

Soluble 41 Agents

[0141] Alpha-4, beta-1 integrin is a heterodimer that is composed of 4 integrin and 1 integrin. Both the 4 integrin and the 1 integrin contain a number of domains including a signal sequence, a transmembrane domain, and a cytoplasmic domain. A non-limiting example of a human mature, processed 1 integrin protein is SEQ ID NO: 20. A soluble 41 agent is a composition that contains a protein (e.g., a single polypeptide or a heterodimeric protein) that contains an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, 99% or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 850 amino acids) in a wild type form of 4 integrin (e.g., precursor or processed 4 integrin, e.g., any one of SEQ ID NOS: 8 and 12) and an amino acid sequence that is at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 amino acids) in a wild type form of 1 integrin (e.g., precursor or processed 1 integrin, e.g., any one of SEQ ID NOS: 17 and 20) that is soluble at physiological pH and has the ability to specifically bind to MadCAM-1. In some embodiments, the amino acid sequence that is at least 95% identical to a contiguous sequence in 4 integrin lacks the signal sequence, the transmembrane domain, and cytoplasmic domain of 4 integrin, and/or the amino acid sequence that is at least 95% identical to a contiguous sequence in 1 integrin lacks the signal sequence, the transmembrane domain, and cytoplasmic domain of 1 integrin. In some embodiments, the soluble 41 agent contains a polypeptide that contains an additional amino acid sequence (e.g., a sequence that stabilizes the polypeptide or increases the polypeptide's half-life in vivo, e.g., an Fc domain or serum albumin).

[0142] In some embodiments, the amino acid sequence that is at least 95% identical to a contiguous sequence in 4 integrin contains a sequence that is at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 850 amino acids) present between amino acids 34 and amino acid 977 in SEQ ID NO: 12. In some embodiments, the amino acid sequence that is at least 95% identical to a contiguous sequence in 1 integrin contains a sequence that is at least 95% identical (e.g., at least 96%, 97%, 98%, 99%, or 100% identical) to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 amino acids) present between amino acid 21 and amino acid 728 in SEQ ID NO: 17. The mRNA encoding 4 is shown above and the mRNA encoding 1 is shown below. Soluble 41 agents can be generated using molecular biology skills known in the art. The ability of a soluble 41 agent to bind to MadCAM-1, can be performed using cell binding assays (e.g., cells expressing one or more of MadCAM-1), competitive binding assays with antibodies that specifically bind to MadCAM-1, or surface plasmon resonance (SPR).

Precursor human 1 integrin polypeptide (SEQ ID NO: 17) with the signal sequence underlined and the transmembrane domain shown in bold.

TABLE-US-00007 1 MNLQPIFWIGLISSVCCVFAQTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEGMPT 61 SARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQIQPQQLV 121 LRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDF 181 RIGFGSFVEKTVMPYISTTPAKLRNPCTSEQNCTSPFSYKNVLSLTNKGEVFNELVGKQR 241 ISGNLDSPEGGFDAIMQVAVCGSLIGWRNVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQ 301 CHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVTEEFQPVYKELKNLIPKSAVGTL 361 SANSSNVIQLIIDAYNSLSSEVILENGKLSEGVTISYKSYCKNGVNGTGENGRKCSNISI 421 GDEVQFEISITSNKCPKKDSDSFKIRPLGFTEEVEVILQYICECECQSEGIPESPKCHEG 481 NGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVCGQCVCRK 541 RDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCE 601 ASNGQICNGRGICECGVCKCTDPKFQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDT 661 CTQECSYFNITKVESRDKLPQPVQPDPVSHCKEKDVDDCWFYFTYSVNGNNEVMVHVVEN 721 PECPTGPDIIPIVAGVVAGIVLIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTGEN 781 PIYKSAVTTVVNPKYEGK
An exemplary cDNA encoding human 1 integrin is SEQ ID NO: 18.

[0143] Soluble MadCAM-1 Molecules

[0144] The precursor form of MadCAM-1 contains a short cytoplasmic domain, a transmembrane domain, a mucin-like domain, two IgSF domains, and a signal sequence (see, Leung et al., Immunol. Cell. Biol. 74:490-496, 1996; Shyjan et al., J. Immunol. 156:2851-2857, 1996). A soluble MadCAM-1 molecule contains an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, 99%, or 100%) identical to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, 225, 250, or 275 amino acids) in a wild type form of MadCAM-1 (e.g., precursor or processed MadCAM-1, e.g., any one of SEQ ID NOS: 1-7 and 11) that is soluble at a physiological pH and has the ability to specifically bind to 47 integrin, L-selectin, or 41 integrin. In some embodiments, the soluble MadCAM-1 lacks one or more (e.g., two, three, or four of its domains) of its domains. In some embodiments, the soluble MadCAM-1 molecule lacks its signal sequence, its transmembrane domain, and its cytoplasmic domain. In some embodiments, the soluble MadCAM-1 molecule contains an additional amino acid sequence (e.g., a sequence that stabilizes the protein or increases the protein's half-life in vivo, e.g., a Fc domain or serum albumin).

[0145] In some embodiments, the soluble MadCAM-1 molecule contains a sequence that is at least 95% identical (e.g., at least 96%, 97%, 98%, 99%, or 100%) to a contiguous sequence (e.g., at least 50, 75, 100, 125, 150, 175, 200, 225, 250, or 275 amino acids) present between amino acid 19 and amino acid 318 in SEQ ID NO: 11 shown below. The mRNA encoding precursor MadCAM-1 is also shown below. Soluble MadCAM-1 molecules can be generated using molecular biology skills known in the art. The ability of a soluble MadCAM-1 molecule to bind to 41, 47, or L-selectin can be performed using cell binding assays (e.g., cells expressing one or more of 41, 47, or L-selectin), competitive binding assays with antibodies that specifically bind to one or more of 41, 47, or L-selectin, or surface plasmon resonance (SPR).

Precursor human MadCAM-1 polypeptide (SEQ ID NO: 11) with the signal sequence underlined and the transmembrane domain shown in bold.

TABLE-US-00008 (SEQIDNO:11) 1 MDFGLALLLAGLLGLLLGQSLQVKPLQVEPPEPVVAVALGASRQLTCRLACADRGASVQW 61 RGLDTSLGAVQSDTGRSVLTVRNASLSAAGTRVCVGSCGGRTFQHTVQLLVYAFPDQLTV 121 SPAALVPGDPEVACTAHKVTPVDPNALSFSLLVGGQELEGAQALGPEVQEEEEEPQGDED 181 VLFRVTERWRLPPLGTPVPPALYCQATMRLPGLELSHRQAIPVLHSPTSPEPPDTTSPES 241 PDTTSPESPDTTSQEPPDTTSPEPPDKTSPEPAPQQGSTHTPRSPGSTRTRRPEISQAGP 301 TQGEVIPTGSSKPAGDQLPAALWTSSAVLGLLLLALPTYHLWKRCRHLAEDDTHPPASLR 361 LLPQVSAWAGLRGTGQVGISPS
An exemplary cDNA encoding human MadCAM-1 is SEQ ID NO: 14.

Small Molecule 47 Integrin Antagonists

[0146] Non-limiting examples of 47 integrin antagonists include a number of small molecules. Non-limiting examples of these small molecule 47 integrin antagonists include: CT301, thiocarbamates (e.g., U.S. Pat. Nos. 7,015,323 and 7,166,600; each of which is incorporated by reference), sulfasalazines (e.g., U.S. Patent Application Publication No. 2009/0264478; herein incorporated by reference), barbituric acid derivatives (see, e.g., Harriman et al., Bioorg. Med. Chem. Lett. 18:2509-2512, 2008), sulfonamides (e.g., WO 98/53818), TR14035 (see, e.g., Cortijo et al., Br. J. Pharmacol. 147:661-670, 2006), cyclic hexapeptides (e.g., P10 cyclo (Leu-Asp-Thr-Ala-D-Pro-Ala), the peptides described Baer et al., J. Med. Chem. 44:2586-2592, 2001, CWLDVC (TBC 772), and the peptides described in J. Immunol. 158:4180-4186, 1996), tripeptides (see, e.g., the peptides described in Shroff et al., Bioorg. Med. Chem. Lett. 13:1601-1606, 1998), and other small peptides (see, e.g., Schroff et al., Bioorg. Med. Chem. Lett. 6:2495-2500, 1996). Additional examples of small molecule 47 integrin antagonists include (2S)-3-(2,5-dichlorobiphenyl-4-yl)-2-({[1-(2-methoxybenzoyl)piperidin-3-yl]carbonyl}amino) propanoic acid, BIO5192, AMD 15057, BIO-1211, N-acyl phenylalanine analogues (see, e.g., Chen et al., Bioorg. Med. Chem. Lett. 10:725-727, 2000), and the small molecules described in Mackenzie et al., (Exp. Cell. Res. 276:90-100, 2002), Lobb et al. (Expert. Opin. Invest. Drugs 8:935-945, 1999), Lee et al. (Bioorg. Med. Chem. 17:977-980, 2009), U.S. Patent Application Publication No. 2002/0091142 (incorporated by reference herein), and WO 98/004247. Non-limiting examples of small molecule 47 integrin antagonists include the cyclic peptides described in Boer et al. (J. Med. Chem. 44:2586-2592, 2001) (e.g., cyclic hexapeptide P10 cyclo (Leu-Asp-Thr-Ala-D-Pro-Ala), P25 cyclo(Leu-Asp-Thr-Ala-D-Pro-Phe), P28 cyclo(Leu-Asp-Thr-Asp-D-Pro-Phe), P29 cyclo(Leu-Asp-Thr-Asp-D-Pro-His), and P30 cyclo(Leu-Asp-Thr-Asp-D-Pro-Tyr). Additional examples of small molecule 47 integrin antagonists are known in the art.

Small Molecule E-Selectin and L-Selectin Antagonists

[0147] In some embodiments, the E-selectin and/or L-selectin antagonist can be a small molecule. Non-limiting examples of small molecules that can act as both an E-selectin antagonist and an L-selectin antagonist include TCB-1269, efomycines (see, e.g., those described in Wienrich et al., J. Invest. Dermatol. 126:882-889, 2006), trihydroxybenzene molecules (see, e.g., those described in Kranich et al., J. Med. Chem. 50:1101-1115, 2007), bimosiamose, and GMI-1070. Additional examples of small molecule L-selectin antagonists and E-selectin antagonists are known in the art.

Methods of Reducing Corneal Inflammation and/or Conjunctival Inflammation

[0148] Provided herein are methods of reducing corneal inflammation and/or conjunctival inflammation in a subject that include administering to a subject one or more (e.g., two, three, four, or five) of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist.

[0149] In some embodiments, the corneal inflammation is chronic corneal inflammation. In some embodiments, the corneal inflammation is acute corneal inflammation. In some embodiments, the corneal inflammation can be caused by bacterial infection, fungal infection, parasite infection, viral infection (e.g., herpes simplex or herpes zoster), allergy, dry eye disorder, Fuchs' dystrophy, keratoconus, amyloidosis, lattice dystrophy, Stevens Johnson syndrome, physical corneal injury (e.g., abrasion, puncture or trauma, e.g., surgical trauma), Behcet's disease, contact lens wear, corneal graft rejection, dry eye syndrome, or immune keratitis (e.g., peripheral ulcerative keratitis). Additional causes of corneal inflammation are known in the art.

[0150] In some embodiments, the conjunctival inflammation is chronic conjunctival inflammation. In some embodiments, the conjunctival inflammation is acute conjunctival inflammation. In some embodiments, the conjunctival inflammation can be caused by viruses, bacteria (e.g., gonorrhea or chlamydia), irritants (e.g., shampoos, dirt, smoke, or chlorine), an allergen, or contact lens wear. Additional causes of conjunctival inflammation are known in the art.

[0151] A reduction in corneal inflammation can be detected by the observance or detection a decrease in one or more (e.g., at least two, three, or four) of the following: the number of T-lymphocytes (e.g., effector T-cells) in a cornea, the number of dendritic cells in a cornea, the number of macrophages in a cornea, the number of stimulated monocytes in a cornea, the number of B-cell in a cornea, the number of natural killer cells present in a cornea, the number of eosinophils in a cornea, the number of mast cells in a cornea, the level of redness in a cornea, pain in an eye, irritation, itchiness, burning, and/or dryness of the cornea, swelling around the eye, sensitivity to light, the amount of discharge from an eye, difficulty opening an eyelid, and blurred vision (e.g., as compared to the same subject prior to receiving a treatment (e.g., any of the treatments described herein) or compared to threshold value). In some embodiments, a reduction in corneal inflammation can be assessed by physical examination of the subject (e.g., through the use of microscopic techniques (e.g., in vivo confocal microscopy) or through examination techniques that do not require the use of a microscope). In some embodiments, a reduction in corneal inflammation can be assessed by a medical professional (e.g., a physician, a physician's assistant, a nurse, a nurse's assistant, or a technician).

[0152] A reduction in conjunctival inflammation can be detected by the observance or detection a decrease in one or more (e.g., at least two, three, or four) of the following: the number of T-lymphocytes (e.g., effector T-cells) in a conjunctiva, the number of dendritic cells in a conjuctiva, the number of macrophages in a conjunctiva, the number of stimulated monocytes in a conjunctiva, the number of B-cells in a conjunctiva, the number of natural killer cells present in a conjunctiva, the number of eosinophils in a conjunctiva, the number of mast cells in a conjunctiva, the level of redness in the white of an eye or in an eyelid, pain in an eye, irritation, itchiness, burning, and/or dryness of an eye, swelling around the eye, sensitivity to light, the amount of discharge from an eye, difficulty opening an eyelid, and blurred vision (e.g., as compared to the same subject prior to receiving a treatment (e.g., any of the treatments described herein) or compared to threshold value). In some embodiments, a reduction in conjunctival inflammation can be assessed by physical examination of the subject (e.g., through the use of microscopic techniques (e.g., in vivo confocal microscopy) or through examination techniques that do not require the use of a microscope). In some embodiments, a reduction in conjunctival inflammation can be assessed by a medical professional (e.g., a physician, a physician's assistant, a nurse, a nurse's assistant, or a technician).

[0153] Some embodiments further include selecting a subject having corneal inflammation and/or conjunctival inflammation (e.g., any type of corneal inflammation and/or conjunctival inflammation described herein or corneal inflammation and/or conjunctival inflammation caused by any of the factors described herein) or a corneal inflammatory disorder and/or a conjunctival inflammatory disorder (e.g., any of the corneal inflammatory disorders and/or conjunctival inflammatory disorders described herein).

[0154] In some embodiments, the MadCAM-1 antagonist can be an antibody (e.g., any of the antibodies described herein) or an antigen-binding antibody fragment (e.g., any of the antibody fragments described herein) that specifically binds to MadCAM-1. In some embodiments, the MadCAM-1 antagonist can be a soluble L-selectin molecule (e.g., any of the soluble L-selectin molecules described herein), a soluble 47 agent (e.g., any of the soluble 47 agents described herein), or a soluble 41 agent (e.g., any of the soluble 41 agents described herein).

[0155] In some embodiments, the 47 integrin antagonist can be an antibody (e.g., any of the antibodies described herein) or an antigen-binding antibody fragment (e.g., any of the antibody fragments described herein) that specifically binds to 47 integrin, 4 integrin, or integrin. In some embodiments, the 47 antagonist is a soluble MadCAM-1 molecule (e.g., any of the soluble MadCAM-1 molecules described herein). In some embodiments, the 47 integrin antagonist is a small molecule (a small molecule 47 integrin antagonist) (e.g., any of the small molecule 47 integrin antagonists described herein or known in the art).

[0156] In some embodiments, the L-selectin antagonist can be an antibody (e.g., any of the antibodies described herein) or an antigen-binding antibody fragment (e.g., any of the antibody fragments described herein) that specifically binds to L-selectin. In some embodiments, the L-selectin antagonist is a soluble MadCAM-1 molecule (e.g., any of the soluble MadCAM-1 molecules described herein), a soluble CD34 molecule (e.g., any of the soluble CD34 molecules described herein), a soluble PSGL-1 molecule (e.g., any of the soluble PSGL-1 molecules described herein), or a soluble GlyCAM-1 molecule (e.g., any of the soluble GlyCAM-1 molecules described herein). In some embodiments, the L-selectin antagonist is a small molecule (a small molecule L-selectin antagonist) (e.g., any of the small molecule L-selectin antagonists described herein or known in the art).

[0157] In some embodiments, the E-selectin antagonist can be an antibody (e.g., any of the antibodies described herein) or an antigen-binding antibody fragment (e.g., any of the antibody fragments described herein) that specifically binds to E-selectin. In some embodiments, the E-selectin antagonist is a small molecule (a small molecule E-selectin antagonist) (e.g., any of the small molecule E-selectin antagonists described herein or known in the art).

[0158] In some embodiments, when two or more agents (e.g., MadCAM-1 antagonist(s), 47 integrin antagonist(s), L-selectin antagonist(s), and/or E-selectin antagonist(s)) are administered to the subject, the two or more agents can be any combination of the agents described herein (e.g., a combination of at least one antibody or antigen-binding antibody fragment and at least one soluble selectin-L molecule, soluble MadCAM-1 molecule, soluble 47 agent, soluble 41 agent, soluble CD34 molecule, soluble PSGL-1 molecule, and/or soluble GlyCAM-1 molecule; a combination of two or more antibodies or antigen-binding antibody fragments; a combination of two or more of a soluble selectin-L molecule, a soluble MadCAM-1 molecule, a soluble 47 agent, a soluble 41 agent, a soluble CD34 molecule, a soluble PSGL-1 molecule, and a soluble GlyCAM-1 molecule; a combination of one or more antibodies or antigen-binding antibody fragments and one or more small molecule 47 integrin antagonists, small molecule L-selectin antagonists, and small molecule E-selectin antagonists; or a combination of two or more small molecule 47 integrin antagonists, small molecule L-selectin antagonists, and small molecule E-selectin antagonists). In some embodiments where two or more agents (selected from a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist) are administered, the two or more agents can be formulated in a single composition (e.g., any of the compositions described herein). In some embodiments, one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, an L-selectin antagonist, and a E-selectin antagonist are formulated for ocular administration (e.g., an eye drop formulation). In some embodiments, one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, an L-selectin antagonist, and a E-selectin antagonist are formulated for systemic administration (e.g., oral, intramuscular, intravenous, intaarterial, subcutaneous, or intraperitoneal injection).

[0159] In some embodiments, the subject is administered one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and a E-selectin antagonist by intravenous, intaarterial, intramuscular, ocular, nasal, subcutaneous, or intraperitoneal administration. The amount of one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist can be the amount (e.g., the amount of one or more agents) that results in a observable or detectable decrease in one or more physical characteristics of corneal inflammation and/or conjunctival inflammation (e.g., any of the physical characteristics of corneal inflammation and/or conjunctival inflammation described herein). A medical professional can determine the appropriate dosage of one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist to administer to the subject based on a number of factors (e.g., the subject's age, general health, sex, and body weight). Exemplary dosages of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist to be administered to the subject are described herein. In some embodiments, the one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist are formulated in a physiologically acceptable excipient or buffer. In some embodiments where two or more agents (agents from the group of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist) are administered to the subject, the agents can, e.g., be administered in separate compositions (e.g., by the same (e.g., ocular administration) or different routes of administration (e.g., any combination of the various routes of administration described herein, e.g., one composition administered by ocular administration and one composition administered by subcutaneous or oral administration)).

[0160] In some embodiments, one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist are administered to the subject at least once every two months (e.g., at least once every month, at least once every two weeks, at least once a week, or at least once, twice, or three times a day). In some embodiments, the subject can be periodically administered one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist over a period of at least one week (e.g., at least one month, two months, six months, one year, and two years).

[0161] In some embodiments, a subject is administered at least one or both of a MadCAM-1 antagonist (e.g., any of the MadCAM-1 antagonists described herein) and an 47 integrin antagonist (e.g., any of the 47 integrin antagonists described herein). In some embodiments, a MadCAM-1 antagonist and an 47 integrin antagonist are formulated in the same composition (e.g., a composition for oral or ocular administration). In some embodiments, a MadCAM-1 antagonist and an 47 integrin antagonist are formulated in separate compositions. In some embodiments, a MadCAM-1 antagonist and an 47 integrin antagonist are administered to the subject at least once every two months (e.g., once every month, once every two week, or at least once a day (e.g., twice a day, three times a day, four times a day, or five times a day)).

[0162] Some embodiments further include administering to the subject one or more additional agents (e.g., an antibiotic, anti-parasitic agent, an anti-viral agent, an anti-fungal agent, and an anti-inflammatory agent). In some embodiments, the one or more additional agents are present in the same formulation with one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist.

[0163] In some embodiments, the subject has previously been diagnosed or identified as having corneal inflammation and/or conjunctival inflammation, or as having a corneal inflammatory disorder and/or a conjunctival inflammatory disorder. In some embodiments, the subject does not present with one or more symptoms of corneal inflammation and/or conjunctival inflammation that can be detected without the use of a microscope. In some embodiments, the subject may be resistant or respond poorly to other forms of treatment for corneal inflammation and/or conjunctival inflammation. In some embodiments, the subject was previously administered another treatment for corneal inflammation and/or conjunctival inflammation, and the subject ceases taking the previously administered treatment prior to being administered one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist. In some embodiments, the subject was previously administered another treatment for corneal inflammation and/or conjunctival inflammation, and the subject is administered the previous treatment in addition to one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist.

[0164] In some embodiments, the subject is a child, teenager, or an adult (e.g., at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old). In some embodiments, the subject is a female. In some embodiments, the subject is a male.

Methods of Reducing Inflammatory Cell Recruitment to the Cornea and/or Conjunctiva

[0165] Provided herein are methods of decreasing inflammatory cell (e.g., any of the inflammatory cells described herein, e.g., dendritic cell) recruitment to the cornea and/or conjunctiva in a subject that include administering to a subject one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist. In some embodiments, the decreased inflammatory cell (e.g., dendritic cell) recruitment is inflammatory cell (e.g., dendritic cell) recruitment to the corneal epithelium. In some embodiments, the decreased inflammatory cell (e.g., dendritic cell) recruitment is inflammatory cell (e.g., dendritic cell) recruitment to the anterior or posterior stroma of the cornea. In some embodiments, the decrease inflammatory cell (e.g., dendritic cell) recruitment is inflammatory cell (e.g., dendritic cell) recruitment to the conjunctiva.

[0166] A decrease in inflammatory cell (e.g., dendritic cell) recruitment to the cornea in a subject can be indirectly detected by the observance or detection of a decrease in one or more (e.g., at least two, three, or four) of the following: the number of other immunological cells present in the cornea (e.g., one or more of the number of T-lymphocytes (e.g., T-cell effector cells), macrophages, stimulated monocytes, B-cells, natural killer cells, eosinophils, and mast cells), the level of redness in a cornea, pain in an eye, irritation, itchiness, burning, and/or dryness of the cornea, swelling around the eye, sensitivity to light, the amount of discharge from an eye, difficulty opening eyelid, and blurred vision (e.g., as compared to the same subject prior to receiving a treatment (e.g., any of the treatments described herein), compared to a subject that has corneal inflammation or a corneal inflammatory disease, or compared to threshold value). In some embodiments, a decrease in inflammatory cell (e.g., dendritic cell) recruitment to the cornea in a subject can be directly detected by observance or detection of a decrease in the number of one or more types of inflammatory cells (e.g., dendritic cells) present in the cornea (e.g., the corneal epithelium or the anterior or posterior stroma of the cornea) (e.g., as compared to the same subject prior to receiving a treatment (e.g., any of the treatments described herein) or compared to threshold value). The detection of the presence of different immunological cell types in the cornea of the subject can be determined using a microscopic technique (e.g., in vivo confocal microscopy as described herein and other techniques known in the art). In some embodiments, a decrease in the migration of inflammatory cells (e.g., dendritic cells) to the cornea can be indirectly assessed through examination techniques that do not require the use of a microscope). In some embodiments, a decrease in the migration of inflammatory cells (e.g., dendritic cells) (using any of the methods described herein or known in the art) can be assessed by a medical professional (e.g., a physician, a physician's assistant, a nurse, a nurse's assistant, or a technician).

[0167] A decrease in inflammatory cell (e.g., dendritic cell) recruitment to the conjunctiva in a subject can be indirectly detected by the observance or detection of a decrease in one or more (e.g., at least two, three, or four) of the following: the number of other immunological cells present in the conjunctiva (e.g., one or more of the number of T-lymphocytes (e.g., T-cell effector cells), macrophages, stimulated monocytes, B-cells, natural killer cells, eosinophils, and mast cells), the level of redness in the white of an eye or an eyelid, pain in an eye, irritation, itchiness, burning, and/or dryness of an eye, swelling around the eye, sensitivity to light, the amount of discharge from an eye, difficulty opening eyelid, and blurred vision (e.g., as compared to the same subject prior to receiving a treatment (e.g., any of the treatments described herein), compared to a subject that has conjunctival inflammation or a conjunctival inflammatory disease, or compared to threshold value). In some embodiments, a decrease in inflammatory cell (e.g., dendritic cell) recruitment to the conjunctiva in a subject can be directly detected by observance or detection of a decrease in the number of one or more types of inflammatory cells (e.g., dendritic cells) present in the conjunctiva (e.g., as compared to the same subject prior to receiving a treatment (e.g., any of the treatments described herein) or compared to threshold value). The detection of the presence of different immunological cell types in the conjunctiva of the subject can be determined using a microscopic technique (e.g., in vivo confocal microscopy as described herein and other techniques known in the art). In some embodiments, a decrease in the migration of inflammatory cells (e.g., dendritic cells) to the conjunctiva can be indirectly assessed through examination techniques that do not require the use of a microscope). In some embodiments, a decrease in the migration of inflammatory cells (e.g., dendritic cells) (using any of the methods described herein or known in the art) can be assessed by a medical professional (e.g., a physician, a physician's assistant, a nurse, a nurse's assistant, or a technician).

[0168] Some embodiments further include selecting a subject having corneal inflammation and/or conjunctival inflammation (e.g., any type of corneal inflammation and/or conjunctival inflammation described herein, or corneal inflammation and/or conjunctival inflammation caused by any of the factors described herein) or a corneal inflammatory disorder and/or a conjunctival inflammatory disorder (e.g., any of the corneal inflammatory disorders and/or conjunctival inflammatory disorders described herein). Some embodiments include selecting a subject suspected of having or presenting with one or more symptoms of corneal inflammation and/or conjunctival inflammation, or with one or more symptoms of a corneal inflammatory disorder and/or a conjunctival inflammatory disorder (e.g., any of the symptoms described herein). Some embodiments include selecting a subject diagnosed as having corneal inflammation and/or conjunctival inflammation, or diagnosed as having a corneal inflammatory disorder and/or a conjunctival inflammatory disorder. Some embodiments include selecting a subject at increased risk of developing corneal and/or conjunctival inflammation, or at increased risk of developing a corneal inflammatory disorder and/or a conjunctival inflammatory disorder.

[0169] In some embodiments, the MadCAM-1 antagonist can be an antibody (e.g., any of the antibodies described herein) or an antigen-binding antibody fragment (e.g., any of the antibody fragments described herein) that specifically binds to MadCAM-1. In some embodiments, the MadCAM-1 antagonist can be a soluble L-selectin molecule (e.g., any of the soluble L-selectin molecules described herein), a soluble 47 agent (e.g., any of the soluble 47 agents described herein), or a soluble 41 agent (e.g., any of the soluble 41 agents described herein).

[0170] In some embodiments, the 47 integrin antagonist can be an antibody (e.g., any of the antibodies described herein) or an antigen-binding antibody fragment (e.g., any of the antibody fragments described herein) that specifically binds to 47 integrin. In some embodiments, the 47 antagonist is a soluble MadCAM-1 molecule (e.g., any of the soluble MadCAM-1 molecules described herein). In some embodiments, the 47 integrin antagonist is a small molecule (a small molecule 47 integrin antagonist) (e.g., any of the small molecule 47 integrin antagonists described herein or known in the art).

[0171] In some embodiments, the L-selectin antagonist can be an antibody (e.g., any of the antibodies described herein) or an antigen-binding antibody fragment (e.g., any of the antibody fragments described herein) that specifically binds to L-selectin. In some embodiments, the L-selectin antagonist is a soluble MadCAM-1 molecule (e.g., any of the soluble MadCAM-1 molecules described herein), a soluble CD34 molecule (e.g., any of the soluble CD34 molecules described herein), a soluble PSGL-1 molecule (e.g., any of the soluble PSGL-1 molecules described herein), or a soluble GlyCAM-1 molecule (e.g., any of the soluble GlyCAM-1 molecules described herein). In some embodiments, the L-selectin antagonist is a small molecule (a small molecule L-selectin antagonist) (e.g., any of the small molecule L-selectin antagonists described herein or known in the art).

[0172] In some embodiments, the E-selectin antagonist can be an antibody (e.g., any of the antibodies described herein) or an antigen-binding antibody fragment (e.g., any of the antibody fragments described herein) that specifically binds to E-selectin. In some embodiments, the E-selectin antagonist is a small molecule (a small molecule E-selectin antagonist) (e.g., any of the small molecule E-selectin antagonists described herein or known in the art).

[0173] In some embodiments, when two or more agents (e.g., MadCAM-1 antagonist(s), 47 integrin antagonist(s), L-selectin antagonist(s), and/or E-selectin antagonist(s)) are administered to the subject, the two or more agents can be any combination of the agents described herein (e.g., a combination of at least one antibody or antigen-binding antibody fragment and at least one soluble selectin-L molecule, soluble MadCAM-1 molecule, soluble 47 agent, soluble 41 agent, soluble CD34 molecule, soluble PSGL-1 molecule, and/or soluble GlyCAM-1 molecule; a combination of two or more antibodies or antigen-binding antibody fragments; a combination of two or more of a soluble selectin-L molecule, a soluble MadCAM-1 molecule, a soluble 47 agent, a soluble 41 agent, a soluble CD34 molecule, a soluble PSGL-1 molecule, and a soluble GlyCAM-1 molecule; a combination of one or more antibodies or antigen-binding antibody fragments and one or more small molecule 47 integrin antagonists, small molecule L-selectin antagonists, and small molecule E-selectin antagonists; or a combination of two or more small molecule 47 integrin antagonists, small molecule L-selectin antagonists, and small molecule E-selectin antagonists). In some embodiments where two or more agents (selected from a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist) are administered, the two or more agents can be formulated in a single composition (e.g., any of the compositions described herein). In some embodiments, one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, an L-selectin antagonist, and a E-selectin antagonist are formulated for ocular administration (e.g., an eye drop formulation). In some embodiments, one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, an L-selectin antagonist, and a E-selectin antagonist are formulated for systemic administration (e.g., oral, intramuscular, intravenous, intaarterial, subcutaneous, or intraperitoneal injection).

[0174] In some embodiments, the subject is administered one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and a E-selectin antagonist by intravenous, intaarterial, intramuscular, ocular, nasal, subcutaneous, or intraperitoneal administration. The amount of one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist can be the amount (e.g., the amount of one or more agents) that results in a observable or detectable decrease in inflammatory cell (e.g., dendritic cell) recruitment to the cornea and/or conjunctiva, and/or decreases one or more physical characteristics of corneal inflammation and/or conjunctival inflammation (e.g., any of the physical characteristics of corneal inflammation and/or conjunctival inflammation described herein). A medical professional can determine the appropriate dosage of one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist to administer to the subject based on a number of factors (e.g., the subject's age, general health, sex, and body weight). Exemplary dosages of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist to be administered to the subject are described herein. In some embodiments, the one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist are formulated in a physiologically acceptable excipient or buffer. In some embodiments where two or more agents (agents from the group of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist) are administered to the subject, the agents can, e.g., be administered in separate compositions (e.g., by the same (e.g., ocular administration) or different routes of administration (e.g., any combination of the various routes of administration described herein, e.g., one composition administered by ocular administration and one composition administered by subcutaneous or oral administration)).

[0175] In some embodiments, one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist are administered to the subject at least once every two months (e.g., at least once every month, at least once every two weeks, at least once a week, or at least once, twice, or three times a day). In some embodiments, the subject can be periodically administered one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist over a period of at least one week (e.g., at least one month, two months, six months, one year, and two years).

[0176] In some embodiments, a subject is administered at least one or both of a MadCAM-1 antagonist (e.g., any of the MadCAM-1 antagonists described herein) and an 47 integrin antagonist (e.g., any of the 47 integrin antagonists described herein). In some embodiments, a MadCAM-1 antagonist and an 47 integrin antagonist are formulated in the same composition (e.g., a composition for oral or ocular administration). In some embodiments, a MadCAM-1 antagonist and an 47 integrin antagonist are formulated in separate compositions. In some embodiments, a MadCAM-1 antagonist and an 47 integrin antagonist are administered to the subject at least once every two months (e.g., once every month, once every two week, or at least once a day (e.g., twice a day, three times a day, four times a day, or five times a day)).

[0177] Some embodiments further include administering to the subject one or more additional agents (e.g., an antibiotic, anti-parasitic agent, an anti-viral agent, an anti-fungal agent, and an anti-inflammatory agent). In some embodiments, the one or more additional agents are present in the same formulation with one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist. In some embodiments, the subject has previously been diagnosed or identified as having corneal inflammation and/or conjunctival inflammation, or as having a corneal inflammatory disorder and/or a conjunctival inflammatory disorder. In some embodiments, the subject is at increased risk of developing corneal and/or conjunctival inflammation, or is at increased risk of developing a corneal inflammatory disorder and/or a conjunctival inflammatory disorder. In some embodiments, the subject is suspected of having or presents with one or more symptoms of corneal inflammation and/or conjunctival inflammation, or is suspected of having or presents with one or more symptoms of a corneal inflammatory disorder and/or a conjunctival inflammatory disorder. In some embodiments, the subject does not present with one or more symptoms of corneal inflammation and/or conjunctival inflammation that can be detected without the use of a microscope. In some embodiments, the subject may be resistant or respond poorly to other forms of treatment for corneal inflammation and/or conjunctival inflammation. In some embodiments, the subject was previously administered another treatment for corneal inflammation and/or conjunctival inflammation, and the subject ceases taking the previously administered treatment prior to being administered one or more of a MadCAM-lantagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist. In some embodiments, the subject was previously administered another treatment for corneal inflammation and/or conjunctival inflammation, and the subject is administered the previous treatment in addition to one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist.

[0178] In some embodiments, the subject is a child, teenager, or an adult (e.g., at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old). In some embodiments, the subject is a female. In some embodiments, the subject is a male.

Methods of Treating Corneal Inflammatory Disorders and/or Conjunctival Inflammatory Disorders

[0179] Provided herein are methods of treating a corneal inflammatory disorder (e.g., any of the corneal inflammatory disorders described herein or known in the art) and/or a conjunctival inflammatory disorder (e.g., any of the conjuctival inflammatory disorders described herein or known in the art) in a subject that include administering to a subject one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, and L-selectin antagonist, and an E-selectin antagonist.

[0180] Successful treatment of a corneal inflammatory disorder in a subject can be detected by the observance or detection of a decrease in one or more (e.g., at least two, three, or four) of the following: the number of T-lymphocytes (e.g., effector T-cells) in a cornea, the number of dendritic cells in a cornea, the number of macrophages in a cornea, the number of stimulated monocytes in a cornea, the number of B-cells in a cornea, the number of natural killer cells in a cornea, the number of eosinophils in a cornea, the number of mast cells in a cornea, the level of redness in a cornea, pain in an eye, irritation, itchiness, burning, and/or dryness of the cornea, swelling around the eye, sensitivity to light, the amount of discharge from an eye, difficulty opening an eyelid, and blurred vision (e.g., as compared to the same subject prior to receiving a treatment (e.g., any of the treatments described herein), compared to a subject having corneal inflammation or a corneal inflammatory disease, or compared to threshold value). In some embodiments, a treatment of a corneal inflammatory disorder can be assessed by physical examination of the subject (e.g., through the use of microscopic techniques (e.g., in vivo confocal microscopy) or through examination techniques that do not require the use of a microscope). In some embodiments, successful treatment of a corneal inflammatory disorder can be assessed by a medical professional (e.g., a physician, a physician's assistant, a nurse, a nurse's assistant, or a technician).

[0181] Successful treatment of a conjuctival inflammatory disorder in a subject can be detected by the observance or detection of a decrease in one or more (e.g., at least two, three, or four) of the following: the number of T-lymphocytes (e.g., effector T-cells) in a conjunctiva, the number of dendritic cells in a conjunctiva, the number of macrophages in a conjunctiva, the number of stimulated monocytes in a conjunctiva, the number of B-cells in a conjunctiva, the number of natural killer cells in a conjunctiva, the number of eosinophils in a conjunctiva, the number of mast cells in a conjunctiva, the level of redness in a white of an eye or an eyelid, pain in an eye, irritation, itchiness, burning, and/or dryness of an eye, swelling around the eye, sensitivity to light, the amount of discharge from an eye, difficulty opening an eyelid, and blurred vision (e.g., as compared to the same subject prior to receiving a treatment (e.g., any of the treatments described herein), compared to a subject having conjunctival inflammation or a conjunctival inflammatory disease, or compared to threshold value). In some embodiments, a treatment of a conjunctival inflammatory disorder can be assessed by physical examination of the subject (e.g., through the use of microscopic techniques (e.g., in vivo confocal microscopy) or through examination techniques that do not require the use of a microscope). In some embodiments, successful treatment of a conjunctival inflammatory disorder can be assessed by a medical professional (e.g., a physician, a physician's assistant, a nurse, a nurse's assistant, or a technician).

[0182] Some embodiments further include selecting a subject having corneal inflammation and/or conjunctival inflammation (e.g., any type of corneal inflammation and/or conjunctival inflammation described herein, or corneal inflammation and/or conjunctival inflammation caused by any of the factors described herein) or a corneal inflammatory disorder and/or a conjunctival inflammatory disorder (e.g., any of the corneal inflammatory disorders and/or conjunctival inflammatory disorders described herein). Some embodiments include selecting a subject suspected of having or presenting with one or more symptoms of corneal inflammation and/or conjunctival inflammation, or with one or more symptoms of a corneal inflammatory disorder and/or a conjunctival inflammatory disorder (e.g., any of the symptoms described herein). Some embodiments include selecting a subject diagnosed as having corneal inflammation and/or conjunctival inflammation, or diagnosed as having a corneal inflammatory disorder and/or a conjunctival inflammatory disorder. Some embodiments include selecting a subject at increased risk of developing corneal and/or conjunctival inflammation, or at increased risk of developing a corneal inflammatory disorder and/or a conjunctival inflammatory disorder.

[0183] In some embodiments, the MadCAM-1 antagonist can be an antibody (e.g., any of the antibodies described herein) or an antigen-binding fragment (e.g., any of the antigen-binding antibody fragments described herein) that specifically binds to MadCAM-1. In some embodiments, the MadCAM-1 antagonist can be a soluble L-selectin molecule (e.g., any of the soluble L-selectin molecules described herein), a soluble 47 agent (e.g., any of the soluble 47 agents described herein), or a soluble 41 agent (e.g., any of the soluble 41 agents described herein).

[0184] In some embodiments, the 47 integrin antagonist can be an antibody (e.g., any of the antibodies described herein) or an antigen-binding antibody fragment (e.g., any of the antibody fragments described herein) that specifically binds to 47 integrin. In some embodiments, the 47 antagonist is a soluble MadCAM-1 molecule (e.g., any of the soluble MadCAM-1 molecules described herein). In some embodiments, the 47 integrin antagonist is a small molecule (a small molecule 47 integrin antagonist) (e.g., any of the small molecule 47 integrin antagonists described herein or known in the art). In some embodiments, the L-selectin antagonist can be an antibody (e.g., any of the antibodies described herein) or an antigen-binding antibody fragment (e.g., any of the antibody fragments described herein) that specifically binds to L-selectin. In some embodiments, the L-selectin antagonist is a soluble MadCAM-1 molecule (e.g., any of the soluble MadCAM-1 molecules described herein), a soluble CD34 molecule (e.g., any of the soluble CD34 molecules described herein), a soluble PSGL-1 molecule (e.g., any of the soluble PSGL-1 molecules described herein), or a soluble GlyCAM-1 molecule (e.g., any of the soluble GlyCAM-1 molecules described herein). In some embodiments, the L-selectin antagonist is a small molecule (a small molecule L-selectin antagonist) (e.g., any of the small molecule L-selectin antagonists described herein or known in the art).

[0185] In some embodiments, the E-selectin antagonist can be an antibody (e.g., any of the antibodies described herein) or an antigen-binding antibody fragment (e.g., any of the antibody fragments described herein) that specifically binds to E-selectin. In some embodiments, the E-selectin antagonist is a small molecule (a small molecule E-selectin antagonist) (e.g., any of the small molecule E-selectin antagonists described herein or known in the art).

[0186] In some embodiments, when two or more agents (e.g., MadCAM-1 antagonist(s), 47 integrin antagonist(s), L-selectin antagonist(s), and/or E-selectin antagonist(s)) are administered to the subject, the two or more agents can be any combination of the agents described herein (e.g., a combination of at least one antibody or antigen-binding antibody fragment and at least one soluble selectin-L molecule, soluble MadCAM-1 molecule, soluble 47 agent, soluble 41 agent, soluble CD34 molecule, soluble PSGL-1 molecule, and/or soluble GlyCAM-1 molecule; a combination of two or more antibodies or antigen-binding antibody fragments; a combination of two or more of a soluble selectin-L molecule, a soluble MadCAM-1 molecule, a soluble 47 agent, a soluble 41 agent, a soluble CD34 molecule, a soluble PSGL-1 molecule, and a soluble GlyCAM-1 molecule; a combination of one or more antibodies or antigen-binding antibody fragments and one or more small molecule 47 integrin antagonists, small molecule L-selectin antagonists, and small molecule E-selectin antagonists; or a combination of two or more small molecule 47 integrin antagonists, small molecule L-selectin antagonists, and small molecule E-selectin antagonists). In some embodiments where two or more agents (selected from a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist) are administered, the two or more agents can be formulated in a single composition (e.g., any of the compositions described herein). In some embodiments, one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, an L-selectin antagonist, and a E-selectin antagonist are formulated for ocular administration (e.g., an eye drop formulation). In some embodiments, one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, an L-selectin antagonist, and a E-selectin antagonist are formulated for systemic administration (e.g., oral, intramuscular, intravenous, intaarterial, subcutaneous, or intraperitoneal injection).

[0187] In some embodiments, the subject is administered one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and a E-selectin antagonist by intravenous, intaarterial, intramuscular, ocular, nasal, subcutaneous, or intraperitoneal administration. The amount of one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist can be the amount (e.g., the amount of one or more agents) that results in a observable or detectable decrease in one or more physical characteristics of corneal inflammation and/or conjunctival inflammation (e.g., any of the physical characteristics of corneal inflammation and/or conjunctival inflammation described herein). A medical professional can determine the appropriate dosage of one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist to administer to the subject based on a number of factors (e.g., the subject's age, general health, sex, and body weight). Exemplary dosages of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist to be administered to the subject are described herein. In some embodiments, the one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist are formulated in a physiologically acceptable excipient or buffer. In some embodiments where two or more agents (agents from the group of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist) are administered to the subject, the agents can, e.g., be administered in separate compositions (e.g., by the same (e.g., ocular administration) or different routes of administration (e.g., any combination of the various routes of administration described herein, e.g., one composition administered by ocular administration and one composition administered by subcutaneous or oral administration)).

[0188] In some embodiments, one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist are administered to the subject at least once every two months (e.g., at least once every month, at least once every two weeks, at least once a week, or at least once, twice, or three times a day). In some embodiments, the subject can be periodically administered one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist over a period of at least one week (e.g., at least one month, two months, six months, one year, and two years).

[0189] In some embodiments, a subject is administered at least one or both of a MadCAM-1 antagonist (e.g., any of the MadCAM-1 antagonists described herein) and an 47 integrin antagonist (e.g., any of the 47 integrin antagonists described herein). In some embodiments, a MadCAM-1 antagonist and an 47 integrin antagonist are formulated in the same composition (e.g., a composition for oral or ocular administration). In some embodiments, a MadCAM-1 antagonist and an 47 integrin antagonist are formulated in separate compositions. In some embodiments, a MadCAM-1 antagonist and an 47 integrin antagonist are administered to the subject at least once every two months (e.g., once every month, once every two week, or at least once a day (e.g., twice a day, three times a day, four times a day, or five times a day)).

[0190] Some embodiments further include administering to the subject one or more additional agents (e.g., an antibiotic, anti-parasitic agent, an anti-viral agent, an anti-fungal agent, and an anti-inflammatory agent). In some embodiments, the one or more additional agents are present in the same formulation with one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist.

[0191] In some embodiments, the subject has previously been diagnosed or identified as having corneal inflammation and/or conjunctival inflammation, or as having a corneal inflammatory disorder and/or a conjunctival inflammatory disorder. In some embodiments, the subject is at increased risk of developing corneal and/or conjunctival inflammation, or is at increased risk of developing a corneal inflammatory disorder and/or a conjunctival inflammatory disorder. In some embodiments, the subject is suspected of having or presents with one or more symptoms of corneal inflammation and/or conjunctival inflammation, or is suspected of having or presents with one or more symptoms of a corneal inflammatory disorder and/or a conjunctival inflammatory disorder. In some embodiments, the subject does not present with one or more symptoms of corneal inflammation and/or conjunctival inflammation that can be detected without the use of a microscope. In some embodiments, the subject may be resistant or respond poorly to other forms of treatment for corneal inflammation and/or conjunctival inflammation. In some embodiments, the subject was previously administered another treatment for corneal inflammation and/or conjunctival inflammation, and the subject ceases taking the previously administered treatment prior to being administered one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist. In some embodiments, the subject was previously administered another treatment for corneal inflammation and/or conjunctival inflammation, and the subject is administered the previous treatment in addition to one or more of a MadCAM-1 antagonist, an 47 integrin antagonist, a L-selectin antagonist, and an E-selectin antagonist.

[0192] In some embodiments, the subject is a child, teenager, or an adult (e.g., at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old). In some embodiments, the subject is a female. In some embodiments, the subject is a male.

Compositions

[0193] Also provided are compositions containing one or more (e.g., at least two, three, four, or five) MadCAM-1 antagonist(s) (e.g., any of the MadCAM-1 antagonists described herein or known in the art), 47 integrin antagonist(s) (e.g., any of the 47 integrin antagonists described herein or known in the art), L-selectin antagonist(s) (e.g., any of the L-selectin antagonists described herein or known in the art), and E-selectin antagonist(s) (e.g., any of the E-selectin antagonists described herein or known in the art). In some embodiments, the compositions contain one or more pharmaceutically acceptable excipients or buffers. In some embodiments, the compositions are formulated as a liquid. In some embodiments, the compositions are formulated for ocular administration (e.g., eye drop formulations). In some embodiments, the compositions are formulated for systemic administration (e.g., for oral, intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous administration). The methods described herein can include administering these compositions.

[0194] Also provided are compositions containing one or more (e.g., at least two, three, four, or five) MadCAM-1 antagonist(s) (e.g., any of the MadCAM-1 antagonists described herein or known in the art), and/or one or more (e.g., at least two, three, or four) 47 integrin antagonist(s) (e.g., any of the 47 integrin antagonists described here in or known in the art). In some embodiments, the compositions contain one or more pharmaceutically acceptable excipients or buffers. In some embodiments, the compositions are formulated as a liquid. In some embodiments, the compositions are formulated for ocular administration (e.g., eye drop formulations). In some embodiments, the compositions are formulated as a solid (e.g., a pill or capsule). In some embodiments, the compositions are formulated as a slow-release formulation.

[0195] Pharmaceutical compositions are formulated to be compatible with their intended route of administration, whether ocular or parenteral (e.g., intravenous, intradermal, subcutaneous, transmucosal (e.g., nasal sprays are formulated for inhalation), or transdermal (e.g., topical ointments, salves, gels, patches or creams as generally known in the art). The compositions can include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents; antibacterial or antifungal agents such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and isotonic agents such as sugars (e.g., dextrose), polyalcohols (e.g., manitol or sorbitol), or salts (e.g., sodium chloride). Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811). Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes or multiple dose vials. Where required (as in, for example, injectable formulations), proper fluidity can be maintained by, for example, the use of a coating such as lecithin, or a surfactant. Absorption of the active ingredient can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0196] Where oral administration is intended, the agent can be included in pills, capsules, troches and the like and 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 corn starch; a lubricant such as magnesium stearate; 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.

[0197] The compositions described herein can be formulated for ocular or parenteral administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage). Toxicity and therapeutic efficacy of compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. One can, for example, determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population), the therapeutic index being the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to target that agent to the site of the affected tissue (the aim being to minimize potential damage to unaffected cells and, thereby, reduce side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0198] In some embodiments, the amount of a MadCAM-1 antagonist present (or each MadCAM-1 antagonist when more than one MadCAM-1 antagonist is present) in a single dose of the composition is between 1 mg and 50 mg, 5 mg and 100 mg, 10 mg to 100 mg, 20 mg to 50 mg, 50 mg to 100 mg, 75 mg to 150 mg, 100 mg to 200 mg, 150 mg to 250 mg. In some embodiments, the amount of a 47 integrin antagonist present (or each 47 integrin antagonist when more than one 47 integrin antagonist is present) in a single dose of the composition is between 1 mg and 50 mg, 5 mg and 100 mg, 10 mg to 100 mg, 20 mg to 50 mg, 50 mg to 100 mg, 75 mg to 150 mg, 100 mg to 200 mg, 150 mg to 250 mg. In some embodiments, the amount of a L-selectin antagonist present (or each L-selectin antagonist when more than one L-selectin antagonist is present) in a single dose of the composition is between 1 mg and 50 mg, 5 mg and 100 mg, 10 mg to 100 mg, 20 mg to 50 mg, 50 mg to 100 mg, 75 mg to 150 mg, 100 mg to 200 mg, 150 mg to 250 mg. In some embodiments, the amount of an E-selectin antagonist present (or each E-selectin antagonist when more than one E-selectin antagonist is present) in a single dose of the composition is between 1 mg and 50 mg, 5 mg and 100 mg, 10 mg to 100 mg, 20 mg to 50 mg, 50 mg to 100 mg, 75 mg to 150 mg, 100 mg to 200 mg, 150 mg to 250 mg.

[0199] Some embodiments of the compositions further include one or more additional agents (e.g., one or more (e.g., two, three, four, or five), e.g., agents selected from the group of antibiotics, anti-parasitic agents, an anti-viral agents, an anti-fungal agents, and an anti-inflammatory agents.

Kits

[0200] Also provided herein are kits that contain any of the compositions described herein. In some embodiments, the kits can further include an item for use in administering a composition (e.g., any of the compositions described herein) to the subject (e.g., a syringe, e.g., a pre-filled syringe). In some embodiments, the kits contain one or more doses (e.g., at least two, three, four, five, or six doses) of any of the compositions described herein. In some embodiments, the kit further contains instructions for administering the composition (or a dose of the composition) to a subject having corneal inflammation and/or conjunctival inflammation, or having a corneal inflammatory disorder and/or conjunctival disorder. In some embodiments, the kit further contains instructions for performing any of the methods described herein (e.g., any of the methods of decreasing corneal inflammation and/or conjunctival inflammation, any of the methods of decreasing inflammatory cell (e.g., dendritic cell) migration to the cornea and/or conjunctiva, and any of the methods of treating a corneal inflammatory disorder and/or conjunctival inflammatory disorder described herein). The invention is further described in the following example, which does not limit the scope of the invention described in the claims.

EXAMPLE

Example 1. In Vivo Studies of Dendritic Cell Recruitment to the Cornea

[0201] A set of experiments was performed in a mouse eye inflammation model to study the role of various adhesion molecules in the rolling and adhesion behavior of dendritic cells at the limbal vessel at the cornea.

[0202] Materials and Methods

[0203] In these experiments, corneal bone marrow-derived dendritic cells (cDC) were purified from Flt-3 melanoma treated mice using L-selectin columns (Miltenyi, Boston, Mass.) and labeled with calcein. Balb/c mice were either left untreated (steady state) or corneal inflammation was induced by suturing one of the eyes of the mouse (suture-induced corneal inflammation) (see, e.g., FIG. 3). One week later calcein-labeled dDC cells (210.sup.6 cells) were injected retrograde through the carotid artery (via catheter) in small boluses of 20-50 L into the mice (with or without one or more the antibodies described below), and visualized in the blood stream of the contralateral corneal and limbal vasculature using epifluorescent intravital microscopy (EF-IVM; IVM, 500 Mikron Instruments) (n=5 per group) (FIG. 2). (EF-IVM is described in Mempel et al., Curr. Opin. Immunol. 39:2925-2935, 2009.) Specifically, a low-lag silicon-intensified target camera (VE1000SIT; Dage MTI) for ulta-low light real-time video recordings was used with a video-triggered stroboscope system (Chadwick Helmuth), and a time base generator (For-A, Co., Ltd.). Video analysis was carried out at a reduced speed. Rolling (cells that interacted visibly with the endothelium and traveled at a slower velocity than the main blood stream) and non-interacting cells were counted in each microvessel section and averaged. The rolling fraction (percentage of rolling calcein-labeled dendritic cells in the limbal vessel compared to the total number of passing calcein-labeled dendritic cells in the limbal vessel) and sticking efficiency (percentage of calcein-labeled dendritic cells that stick to the limbal vessel wall compared to the total number of passing calcein-labeled dendritic cells) were measured.

[0204] The effect of different adhesion molecules on dendritic cell recruitment was determined by intravenous injection with 100 microliters of antibodies that specifically bind to P-selectin, E-selectin, L-selectin, ICAM-1, VCAM-1, or MadCAM-1 at the time the calcein-labelled cells were injected (thirty minutes before intravital microscopy recordings were taken). In the case of L-selectin, the labeled dendritic cells were treated with 10 l of 0.5 mg/ml anti-selectin antibody for 30 minutes at 4 degrees, and then excess antibody was washed away with PBS prior to injection through the carotid artery. Pertussis toxin (PTX) treatment of the labeled dendritic cells was performed by incubating 0.8 ng of PTX with 20 million labeled dendritic cells for 2 hours at 37 C. Corneal inflammation by suturing was initiated 7 days prior to imaging and or treatment. Homing (recruitment) of the calcein-labeled dendritic cells to normal or inflamed corneas was also studied by ex vivo confocal microscopy (Olympus Fluoview 1000), twenty-four hours after intravenous injection of fluorescently-labeled dendritic cells (with or without antibodies that specifically bind to P-selectin, E-selectin, L-selectin, ICAM-1, VCAM-1, or MadCAM-1).

[0205] Results

[0206] An initial set of experiments show that the injected calcein-labeled dendritic cells are recruited to the limbal vessel in mice that are untreated and mice with suture-induced eye inflammation (both before and after treatment with an anti-VCAM-1 antibody) (see, FIGS. 4A-C).

[0207] An additional set of experiments was performed to determine the role of different adhesion molecules in dendritic cell rolling in the limbal vessel of the cornea in both untreated (steady state) mice and in mice with suture-induced inflammation. In these experiments, the mice were left untreated or corneal inflammation was induced (suture-induced inflammation), a week later the mice were administered calcein-labelled dendritic cells with or without anti-P-selectin antibodies, anti-L-selectin antibodies, anti-E-selectin antibodies, an anti-PSGL-1 antibodies, a combination of anti-P-selectin and anti-L-selectin antibodies, anti-CD44 antibodies, anti-VCAM-1 antibodies, or anti-MadCAM-1 antibodies, and intravital images were obtained thirty minutes later. In the case of anti-L-selectin antibody treatment, the labeled dendritic cells were treated with the antibody for 30 minutes at 4 C., and washed with phosphate buffered saline prior to injection. Antibody treatment was performed 30 minutes prior to imaging and seven days after suture induced inflammation) in the in vitro confocal microscopy experiments, and 24 hours in advance of the homing experiments.

[0208] The resulting data show that anti-P-selectin antibodies, anti-PSGL-1 antibodies, and anti-VCAM-1 antibodies significantly decrease the rolling fraction of dendritic cells in the limbal vessel in untreated (steady state) mice (FIGS. 5 and 7). Additional data show that anti-P-selectin antibodies, anti-E-selectin antibodies, a combination of anti-P-selectin and anti-L-selectin antibodies, anti-VCAM-1 antibodies, and anti-MadCAM-1 antibodies significantly decrease the rolling fraction of dendritic cells in the limbal vessel in mice following stimulation of eye inflammation (suture-induced eye inflammation) (FIGS. 6 and 8).

[0209] An additional set of experiments was performed to determine the role of different adhesion molecules in dendritic cell sticking in the limbal vessel of the cornea in both untreated (steady state) mice and in mice with suture-induced eye inflammation. In these experiments, the mice were administered the calcein-labelled cells with or without anti-VCAM-1 antibodies, anti-ICAM-1 antibodies, or anti-MadCAM-1 antibodies, thirty minutes before intravital images were obtained.

[0210] The resulting data show that anti-VCAM-1 antibodies and anti-MadCAM-1 antibodies significantly decrease the sticking efficiency of dendritic cells in the limbal vessel in untreated (steady state) mice, as well as in mice following induction of eye inflammation (suture-induced eye inflammation) (FIGS. 9 and 10, respectively).

[0211] Additional experiments were performed to determine the number of labeled dendritic cells that localized to the corneal epithelium, the corneal anterior stroma, and the corneal posterior stroma in normal (steady state) mice or in mice with suture-induced eye inflammation (inflamed). In these experiments, the two groups of mice were administered calcein-labelled cells treated with a control rat IgG, anti-47 antibodies, or L-selectin antibodies prior to injection, or the mice were administered anti-MadCAM-1 antibodies. In case of anti-47 antibodies the dendritic cells were treated with 10 L of 0.5 mg/ml antibody for 30 minutes at 4 C., and washed with phosphate buffered saline prior to tail vein injection of the cells to the steady state or inflamed mice. The data show that administration of anti-47 antibodies or administration of anti-MadCAM-1 antibodies significantly decreases dendritic cell migration to the corneal epithelium, the corneal anterior stroma, and the corneal posterior stroma in both untreated (steady state) mice and mice with suture-induced eye inflammation (FIGS. 11 and 12, respectively). These data, for example, indicate that agents that antagonize either 47 integrin or MadCAM-1 can be used to reduce dendritic cell migration to the corneal epithelium, the corneal anterior stroma, and the corneal posterior stroma in a subject, can be used to reduce corneal inflammation, and can be used to treat a corneal inflammatory disorder (via their ability to decrease dendritic cell recruitment to the cornea).

[0212] RT-PCR was also performed to confirm the expression of MadCAM-1 in the limbal/cornea tissue. The results show that the expression of MadCAM-1 is increased in the limbal/corneal tissue in an inflamed eye, but shows low expression in the limbal/tissue in a normal (steady state) eye.

OTHER EMBODIMENTS

[0213] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.