WO2018132639A1

WO2018132639A1 - Methods and kits for the diagnosis and/or prognosis of ocular cicatricial pemphigoid

Info

Publication number: WO2018132639A1
Application number: PCT/US2018/013453
Authority: WO
Inventors: Charles Stephen FOSTER; Razzaque AHMED
Original assignee: Foster Charles Stephen; Ahmed Razzaque
Priority date: 2017-01-13
Filing date: 2018-01-12
Publication date: 2018-07-19
Also published as: WO2018132639A8

Abstract

The invention provides methods of diagnosing ocular cicatricial pemphigoid (OCP) in a subject, by determining the level or concentration of autoantibodies that bind β4 integrin in a biological sample obtained from the subject. In embodiments, the concentration of anti-p4 integrin autoantibodies is determined using an ELISA. Methods of monitoring the course of OCP, methods of treating OCP, and kits for diagnosing OCP are also provided.

Description

METHODS AND KITS FOR THE DIAGNOSIS AND/OR PROGNOSIS OF OCULA

CICATRICIAL PEMPHIGOID

RELATED APPLICATIONS

[001] This application claims priority to U.S. Provisional Application No. 62/446,080, filed on January 13, 2017, the entire contents of which are expressly incorporated herein by reference.

SEQUENCE LISTING

[002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 10, 2018, is named SEQ_LIST_126558_00320_2018_01_10 and is 37.8 KB in size.

BACKGROUND

[003] Mucous membrane pemphigoid (MMP) is a systemic autoimmune disease characterized by recurrent blistering of mucous membranes and skin, resulting in excessive and often irreversible scar tissue formation. MMP lesions commonly affect the mouth and gums, but can also affect mucous membranes and squamous epithelia in other parts of the body, such as the genitals, nose, oropharynx, esophagus and larynx. Involvement of the conjunctiva, known as ocular cicatricial pemphigoid (OCP), occurs in up to 70% of MMP cases.

[004] OCP is characterized clinically by scarring of the conjunctiva. OCP progresses slowly over the course of years from chronic conjunctivitis to subepithelial fibrosis, fornix foreshortening, symblepharon, and ankylobelepharon formation with ocular keratinization. In some cases, corneal scarring can result in blindness without proper treatment.

[005] OCP is a rare disease, affecting between 1 in 12,000 to 1 in 60,000 people. Consequently, clinical expertise is rare, and practitioners often misdiagnose or fail to diagnose the disorder. Symptoms are nonspecific, particularly early in disease, and include chronic irritation, dryness and a grittiness sensation in the eye. Chronic conjunctivitis, a symptom of OCP, can often be confused with infective aetiologies. Currently, the mean duration of symptoms prior to diagnosis is 2.8 years. Delay in diagnosis enables the disease to progress to later stages in many patients, and delays receipt of appropriate therapy. As a result, patients often require harsh therapeutic regimens to treat late- stage disease, which could have been avoided if a diagnosis was made earlier. Generally, standard diagnosis of OCP requires obtaining a biopsy sample from eye tissue, and identifying a homogeneous linear deposition of immunoreactions, including IgG, IgA, IgM, and complement 3 component (C3), along the basement membrane zone (BMZ) of the conjunctiva. This procedure is invasive, leads to further inflammation of the eye, and does not easily exclude OCP if negative. Thus, a diagnostic test that can quickly, accurately and noninvasively detect OCP is needed.

SUMMARY OF THE INVENTION

[006] The present invention is based, at least in part, on the discovery that an elevated serum or plasma concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin can indicate that a subject has or is at risk for developing ocular cicatricial pemphigoid (OCP). These autoantibodies are present at an elevated concentration in the serum or plasma of OCP subjects relative to subjects who do not have OCP. The methods and assays described herein can be used to diagnose and select a therapy for OCP in subjects having or suspected of having OCP.

[007] In one aspect, the invention provides a method of detecting the level or concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin in a serum or plasma sample derived from a subject known or suspected of having OCP. In one embodiment, the level or concentration of the autoantibodies is detected using an enzyme-linked immunosorbent assay (ELISA). In an exemplary embodiment, the level or concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin is detected by contacting the serum or plasma sample with a β4 integrin polypeptide, or a fragment thereof, and detecting the formation of a complex between the β4 integrin polypeptide, or fragment thereof, and autoantibodies that bind thereto. In one embodiment, the β4 integrin polypeptide can comprise SEQ ID NO: l, or a fragment thereof, e.g., a fragment containing 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 75 or more, or 100 or more amino acids of SEQ ID NO: 1. In one embodiment, the autoantibodies are detected by contacting the serum or plasma sample with a β4 integrin fragment comprising the cytoplasmic domain of β4 integrin, or a portion thereof, for example, a portion comprising amino acid residues 1489- 1822, amino acid residues 1489- 1572, or amino acid residues 1489 - 1510 of the full-length human β4 integrin sequence, e.g. , the human β4 integrin sequence provided as SEQ ID NO: l.

[008] In another aspect, the invention provides a method of diagnosing Ocular

Cicatricial Pemphigoid (OCP) in a subject, by determining the concentration of

autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin in a serum or plasma sample from the subject, wherein an increase in the concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin relative to that in a normal subject is indicative of OCP in the subject. In one embodiment, the concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin is determined using an ELISA assay.

[009] In some embodiments, an increase of about 1.5-fold or more (e.g., an increase of at least about 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11- fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000- fold or more) in the level or concentration of serum autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin relative to the serum level or concentration of said antibodies in a normal subject, is indicative of OCP. In an exemplary embodiment, the increase is an increase of about 2-fold or more. In another embodiment, the increase is an increase of about 3-fold or more. In another embodiment, the increase is an increase of about 10-fold or more.

[010] In some embodiments, an increase of about 50% or more (e.g., an increase of at least about 50%, 75%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more) in the level or concentration of serum autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin relative to the level or concentration in a normal subject is indicative of OCP.

[011] In other embodiments, a serum concentration of autoantibodies that specifically bind to the cytoplasmic domain of β4 integrin of about 100 ng/mL or more (e.g., about 100 ng/mL, 150 ng/mL, 200 ng/mL, 250 ng/mL, 300 ng/mL, 350 ng/mL, 400 ng/mL, 450 ng/mL, 500 ng/mL, 550 ng/mL, 600 ng/mL, 650 ng/mL, 700 ng/mL, 750 ng/mL, 800 ng/mL, 850 ng/mL, 900 ng/mL, 950 ng/mL, 1000 ng/mL, 1100 ng/mL, 1200 ng/mL, 1300 ng/mL, 1400 ng/mL, 1500 ng/mL, 1600 ng/mL, 1700 ng/mL, 1800 ng/mL, 1900 ng/mL, 2000 ng/mL, 2500 ng/mL, 3000 ng/mL, 4000 ng/mL, 5000 ng/mL, 6000 ng/mL, 7000 ng/mL, 8000 ng/mL, 9000 ng/mL, 10,000 ng/mL or more) is indicative of OCP in the subject.

[012] In some embodiments, the autoantibodies are detected using a β4 integrin polypeptide or a β4 integrin polypeptide fragment. The fragment can include all or a portion of the β4 integrin cytoplasmic domain. For example, the β4 integrin fragment can contain at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In another embodiment, the fragment can include a portion of β4 integrin comprising at least amino acid residues 1489- 1822, amino acid residues 1489- 1572, amino acid residues 1489 - 1510, or amino acid residues 1689- 1702 of the full-length human β4 integrin sequence, e.g., the human β4 integrin sequence provided as SEQ ID NO: l . In some embodiments, the autoantibodies are IgG, IgM, or IgA antibodies, or combinations thereof. In an exemplary embodiment, the autoantibodies are IgG antibodies. In another exemplary embodiment, the autoantibodies are IgM antibodies.

[013] In some embodiments, the method further involves administering a

therapeutically effective amount of an agent for treating OCP to the subject. The agent can be, for example, a chemo therapeutic agent or an immune suppressive agent, or a combination thereof. In some embodiments, the agent is mitomycin C, methotrexate, mycophenolate mofetil, cyclophosphamide, rituximab, intravenous immunoglobulin (IVIg), azathioprine, a TNF-a inhibitor, cyclosporins, prednisone, diaminodiphenylsulfone, CellCept, or any combination thereof. In some embodiments, one or more of the foregoing agents is administered to a subject determined to have a serum or plasma concentration of

autoantibodies that specifically bind to the cytoplasmic domain of β4 integrin of greater than or equal to about 100 ng/mL (e.g., about 100 ng/mL, 150 ng/mL, 200 ng/mL, 250 ng/mL, 300 ng/mL, 350 ng/mL, 400 ng/mL, 450 ng/mL, 500 ng/mL, 550 ng/mL, 600 ng/mL, 650 ng/mL, 700 ng/mL, 750 ng/mL, 800 ng/mL, 850 ng/mL, 900 ng/mL, 950 ng/mL, or about 1000 ng/mL, 1100 ng/mL, 1200 ng/mL, 1300 ng/mL, 1400 ng/mL, 1500 ng/mL, 1600 ng/mL, 1700 ng/mL, 1800 ng/mL, 1900 ng/mL, 2000 ng/mL, 2500 ng/mL, 3000 ng/mL, 4000 ng/mL, 5000 ng/mL, 6000 ng/mL, 7000 ng/mL, 8000 ng/mL, 9000 ng/mL, 10,000 ng/mL or more). In other embodiments, one or more of the foregoing agents is administered to a subject determined to have a 1.5-fold or greater increase (e.g., an increase of at least about 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more) in the serum or plasma concentration of autoantibodies that specifically bind to the cytoplasmic domain of β4 integrin, relative to the serum or plasma concentration of the autoantibodies in a normal subject. In other embodiments, one or more of the foregoing agents is administered to a subject determined to have an increase of about 50% or more (e.g., an increase of at least about 50%, 75%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more) in the level or concentration of serum or plasma autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin relative to the serum or plasma level or concentration in a normal subject.

[014] In one aspect, the invention provides a method of diagnosing Ocular Cicatricial Pemphigoid (OCP) in a subject, comprising contacting a plasma or serum sample from the subject with a β4 integrin peptide bound to a solid support; incubating the plasma or serum sample with the solid support for a period of time sufficient to allow antibodies in the plasma or serum sample to specifically bind the β4 integrin peptide; washing the solid support to remove components of the plasma or serum sample that do not specifically bind the β4 integrin peptide; contacting the solid support with a detection antibody, or antigen-binding fragment thereof, that specifically binds the antibodies in the plasma or serum sample bound to the β4 integrin peptide, wherein the detection antibody is coupled to a detectable label; determining the level of the antibodies in the plasma or serum sample that specifically bind the β4 integrin peptide; and comparing the level of the antibodies in the plasma or serum sample that specifically bind the β4 integrin peptide to a suitable control, wherein the suitable control is indicative of the level of antibodies that specifically bind the β4 integrin peptide in a plasma or serum sample from a normal subject; wherein an increase in the level of antibodies that specifically bind the β4 integrin peptide in the plasma or serum sample relative to the suitable control indicates that the subject has OCP. In one embodiment, the sample is a serum sample. In another embodiment, the sample is a plasma sample.

[015] In one embodiment, the β4 integrin peptide comprises amino acid residues 1489- 1510 of human β4 integrin (SEQ ID NO:4). In another embodiment, the β4 integrin peptide comprises amino acid residues 1489- 1572 of human β4 integrin (SEQ ID NO:5). In one embodiment, the β4 integrin peptide comprises amino acid residues 1489- 1822 of human β4 integrin (SEQ ID NO:6). In another embodiment, the β4 integrin peptide comprises amino acid residues 1689- 1702 of human β4 integrin (SEQ ID NO:7). In one embodiment, the β4 integrin peptide comprises SEQ ID NO: l . In some embodiments, β4 integrin peptides that contain a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater identity with the foregoing peptides can be used in the assays and methods described herein.

[016] In one embodiment, the detection antibody specifically binds the constant region of one or more immunoglobulins selected from the group consisting of IgG, IgM, IgA, IgD, and IgE. For example, the detection antibody can specifically bind the constant region of an IgG immunoglobulin and/or the constant region of an IgM immunoglobulin.

[017] In one embodiment, the solid support can be a microtiter plate. Other solid supports known in the art can also be employed, for example, solid supports useful for column chromatography, e.g. , agarose, sepharose, etc.

[018] The detectable label can be, for example, an enzymatic label, a fluorophore label, a chromophore label, or a radio label. In exemplary embodiments, the detectable label provides a quantitative output reading that can be correlated with the level of antibody bound to the solid support.

[019] In one embodiment, the methods described herein further comprises

administering a therapeutically effective amount of an agent for treating OCP to the subject. In one embodiment, the agent is a chemo therapeutic agent or an immune suppressive agent, or a combination thereof. For example, the agent can include mitomycin C, methotrexate, mycophenolate mofeti!, cyclophosphamide, rituximab, intravenous immunoglobulin (IVIg), azathioprine, a TNF-a inhibitor, cyclosporine, prednisone, diaminodiphenyisulfone, or any combination thereof.

[020] In one aspect, the invention provides a method of treating Ocular Cicatricial Pemphigoid (OCP) in a subject, comprising selecting a subject having an increased serum concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin relative to that in a normal subject, and administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an OCP therapeutic, thereby treating OCP in the subject. The OCP therapeutic can include, in some embodiments, a chemo therapeutic agent or an immune suppressive agent, or a combination thereof. For example, the OCP therapeutic can be mitomycin C, methotrexate, mycophenolate mofetil, cyclophosphamide, rituximab, intravenous immunoglobulin (IVIg), azathioprine, a TNF-a inhibitor, cyclosporine, prednisone, diaminodiphenyisulfone, CellCept, or a combination thereof. [021] In some embodiments, the serum concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin is determined using an ELISA assay.

[022] In one embodiment, a subject having a serum concentration of autoantibodies that specifically bind to the cytoplasmic domain of β4 integrin of about 100 ng/mL or more (e.g., about 100 ng/mL, 150 ng/mL, 200 ng/mL, 250 ng/mL, 300 ng/mL, 350 ng/mL, 400 ng/mL, 450 ng/mL, 500 ng/mL, 550 ng/mL, 600 ng/mL, 650 ng/mL, 700 ng/mL, 750 ng/mL, 800 ng/mL, 850 ng/mL, 900 ng/mL, 950 ng/mL, or about 1000 ng/mL, 1100 ng/mL, 1200 ng/mL, 1300 ng/mL, 1400 ng/mL, 1500 ng/mL, 1600 ng/mL, 1700 ng/mL, 1800 ng/mL, 1900 ng/mL, 2000 ng/mL, 2500 ng/mL, 3000 ng/mL, 4000 ng/mL, 5000 ng/mL, 6000 ng/mL, 7000 ng/mL, 8000 ng/mL, 9000 ng/mL, 10,000 ng/mL or more) is selected for treatment with the OCP therapeutic.

[023] In another embodiment, a subject having an increase of about 1.5-fold or more (e.g., an increase of at least about 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 50-fold, 100-fold, 200- fold, 500-fold, 1000-fold or more) in the serum level or concentration of autoantibodies that specifically bind to the cytoplasmic domain of β4 integrin, relative to the serum level or concentration of said autoantibodies in a normal subject, is selected for treatment with the OCP therapeutic.

[024] In another embodiment, a subject having an increase of about 50% or more (e.g., an increase of at least about 50%, 75%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more) in the serum level or concentration of autoantibodies that specifically bind to the cytoplasmic domain of β4 integrin, relative to the serum level or concentration of said autoantibodies in a normal subject, is selected for treatment with the OCP therapeutic.

[025] In one embodiment, the autoantibodies specifically bind a β4 integrin

polypeptide or a β4 integrin polypeptide fragment. The β4 integrin polypeptide can include all or a portion of the β4 integrin cytoplasmic domain. For example, the β4 integrin polypeptide can contain at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In another embodiment, the fragment can include a portion of β4 integrin comprising at least amino acid residues 1489- 1822, amino acid residues 1489- 1572, or amino acid residues 1489 - 1510 of the full-length human β4 integrin sequence, e.g., the human β4 integrin sequence provided as SEQ ID NO: l . In some embodiments, the autoantibodies are IgG, IgM, or IgA antibodies, or combinations thereof. In an exemplary embodiment, the autoantibodies are IgG antibodies. In another exemplary embodiment, the autoantibodies are IgM antibodies.

[026] In one aspect, the invention provides a method of monitoring the course of Ocular Cicatricial Pemphigoid (OCP) in a subject, comprising determining a concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin in a first serum or plasma sample obtained from the subject, and determining a concentration of

autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin in a second serum or plasma sample obtained from the subject during or after treatment with an OCP therapeutic, wherein a decrease in the concentration of autoantibodies that specifically bind the cytoplasmic domain of β4 integrin in the second serum or plasma sample relative to the first serum or plasma sample indicates that the subject is responding to treatment with the OCP therapeutic. No change or an increase in the level of anti-p4 integrin autoantibodies can indicate that the subject is not responding to the treatment. In one embodiment, the first serum or plasma sample is obtained from the subject prior to treatment with an OCP therapeutic. In another embodiment, the first serum or plasma sample is obtained from the subject during the course of treatment with an OCP therapeutic, at a time prior to obtaining the second serum sample.

[027] In some embodiments, a decrease in the concentration of anti-p4 integrin autoantibodies of at least about 2-fold (e.g. , a decrease of at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20- fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more) indicates that a subject is responding to treatment. In some embodiments, a concentration of less than 100 ng/mL (e.g. , less than about 90 ng/mL, 50 ng/mL, 25 ng/mL, 10 ng/mL, 5 ng/mL, 1 ng/mL, 0.1 ng/mL, or 0.01 ng/mL) in the second serum sample obtained during or after treatment with the OCP therapeutic indicates that the subject has entered remission from OCP. In some

embodiments, the concentration of autoantibodies that specifically bind the cytoplasmic domain of β4 integrin is determined using an ELISA assay.

[028] In another aspect, the invention provides a method of diagnosing Ocular

Cicatricial Pemphigoid (OCP) in a subject, comprising performing an ELISA assay to determine the concentration of autoantibodies that bind to the cytoplasmic domain of β4 integrin in a serum sample from the subject, wherein a concentration of about 100 ng/mL or more (e.g. , about 100 ng/mL, 150 ng/mL, 200 ng/mL, 250 ng/mL, 300 ng/mL, 350 ng/mL, 400 ng/mL, 450 ng/mL, 500 ng/mL, 550 ng/mL, 600 ng/mL, 650 ng/mL, 700 ng/mL, 750 ng/mL, 800 ng/mL, 850 ng/mL, 900 ng/mL, 950 ng/mL, or about 1000 ng/mL, 1100 ng/mL, 1200 ng/mL, 1300 ng/mL, 1400 ng/mL, 1500 ng/mL, 1600 ng/mL, 1700 ng/mL, 1800 ng/mL, 1900 ng/mL, 2000 ng/mL, 2500 ng/mL, 3000 ng/mL, 4000 ng/mL, 5000 ng/mL, 6000 ng/mL, 7000 ng/mL, 8000 ng/mL, 9000 ng/mL, 10,000 ng/mL or more) or more is indicative of OCP in the subject. In some embodiments, the autoantibodies are detected using a β4 integrin polypeptide or a β4 integrin polypeptide fragment. The fragment can include all or a portion of the β4 integrin cytoplasmic domain. For example, the β4 integrin fragment can contain at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In another embodiment, the fragment can include a portion of β4 integrin comprising at least amino acid residues 1489- 1822, amino acid residues 1489- 1572, or amino acid residues 1489- 1510 of the full-length human β4 integrin sequence, e.g. , the human β4 integrin sequence provided as SEQ ID NO: l . In some embodiments, the autoantibodies are IgG, IgM, or IgA antibodies, or combinations thereof. In an exemplary embodiment, the autoantibodies are IgG antibodies. In another exemplary embodiment, the autoantibodies are IgM antibodies.

BRIEF DESCRIPTION OF THE FIGURES

[029] FIG. 1A-FIG. 10 depict standard curves used to develop an ELISA for detecting the presence and the concentration of autoantibodies that bind to β4 integrin. Protein concentration levels were assessed using a spectrophotometer at OD450. The concentration of Antigen A used to produce the standard curves ranged from 0 to 2,000 ng/ml, and included concentrations of 2,000 ng/mL, 1,000 ng/mL, 500 ng/mL, 250 ng/mL, 125 ng/mL, 100 ng/mL, 8 ng/mL, and 1 ng/mL. The primary antibody (anti-beta 4 antibody) concentrations used to produce the standard curves ranged from 0 to 1,000 ng/mL, and included 1,000 ng/mL, 500 ng/mL, 250 ng/mL, 125 ng/mL, 100 ng/mL, and 1 ng/mL. FIG. 10 provides a combined regression analysis for the standard curves.

DETAILED DESCRIPTION

[030] The present invention provides novel methods for diagnosing Ocular Cicatricial Pemphigoid (OCP) in a subject. In some embodiments, the methods entail detecting autoantibodies which bind β4 integrin in a suitable sample. Methods of monitoring a subject with OCP, methods of treating OCP, and kits for diagnosing OCP are also provided.

[031] 1. Definitions

[032] In order that the present invention may be more readily understood, certain terms are first defined.

[033] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to co ver both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising, "having," "including," and

"containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value recited or falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited.

[034] The term "about" or "approximately" generally means within 5%. In one embodiment, the term about refers to a number(s) which is within 1% of a given value or range.

[035] The term "antibody", as used herein, has its art-recognized meaning, and encompasses immunoglobulin molecules comprised of four polypeptide chains (two heavy (H) chains and two light (L) chains) inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions of the heavy and light chains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is generally composed of three CDRs and four FRs.

[036] The term "autoantibody," as used herein, refers to an antibody produced by a subject, which reacts with an antigen endogenous to the subject. For example, a subject may generate autoantibodies which recognize a protein expressed by the subject, e.g. , β4 integrin. Such autoantibodies may be deleterious to the subject, and can cause, e.g. , an inflammatory response and/or an autoimmune response in the subject.

[037] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion"), as used herein, refers to a portion of a full-length antibody, generally the target binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')₂ and Fv fragments. The phrase "functional fragment" of an antibody is a compound having qualitative biological activity in common with a full-length antibody. As used herein, "functional fragment" with respect to antibodies, refers to Fv, scFv, F(ab) and F(ab')₂ fragments. An "Fv" fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. An scFv contains one heavy and one light chain variable domain connected by a linker peptide of a size that permits the VH and VL domains to interact to form the target binding site. Collectively, the six CDRs confer target binding specificity to the antibody or antibody fragment. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) can have the ability to recognize and bind target, although at a lower affinity than the entire binding site.

[038] The terms "β4 integrin", "β4 subunit", "integrin beta 4", "β4", or "ITGB4" refer to an integrin β4 polypeptide, also known as CD 104. Integrins are heterodimers of alpha and beta subunits that associate to form transmembrane receptors for extracellular matrix components, e.g., basement membrane proteins. The human β4 integrin subunit associates preferentially with an a6 integrin subunit, and serves as a receptor for laminin. The wild-type amino acid sequence of human β4 integrin is provided in SEQ ID NO: 1. Polymorphisms and alternatively- spliced variants of the nucleotide and amino acid sequences of β4 integrin are known in the art and can be found, for example, in publically available databases such as the NCBI GenBank. For example, polymorphisms of the human β4 integrin gene can be found under GenBank Accession Nos. X51841, X52186, X53587, BC118916, BC126411, and AF011375. Exemplary naturally occurring variants of human β4 integrin have one or more of the following substitutions with respect to the wild-type β4 integrin sequence shown in SEQ ID NO: 1 : R844L, G931D, H1216Q, R1225H, R1281W, T1764S, L1779P. Unless otherwise specified, a "β4 integrin" is a naturally occurring β4 integrin, including naturally occurring variants thereof, and orthologs/paralogs thereof in other species. A "nucleic acid encoding β4 integrin" or a "gene encoding β4 integrin" refers to a nucleic acid molecule (e.g. , DNA, RNA) that encodes an integrin β4 polypeptide, as described herein. An exemplary nucleic acid molecule encoding wild-type β4 integrin is provided in SEQ ID NO: 2. In an embodiment, a β4 integrin or a nucleic acid encoding a β4 integrin is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a β4 integrin sequence, as represented by SEQ ID NO: l or SEQ ID NO: 2. Sequence identity can be determined, e.g. , by comparing sequences using NCBI BLAST (e.g., Megablast with default parameters).

[039] As used herein, the term "biopsy" or "biopsy sample" refers to a sample of tissue (e.g., conjunctiva) that is removed from a subject for the purpose of diagnosing OCP. The biopsy tissue is then examined for the presence or absence of OCP.

[040] The term "capture reagent" refers to a reagent capable of binding and capturing a target molecule in a sample such that under suitable conditions, the capture reagent-target molecule complex can be identified, detected, measured, and/or separated from the rest of the sample. In some embodiments, the capture reagent is immobilized or can be immobilized. In some embodiments, the capture reagent may be an antibody or a mixture of different antibodies that specifically recognize a target protein. In other embodiments, the capture reagent may be an antigen that is specifically bound by a target molecule, e.g. , an

autoantibody.

[041] A "concentration" of an analyte (e.g. , a protein or a nucleic acid) refers to the quantity of an analyte in a unit volume of a test sample.

[042] As used herein, the term "cytoplasmic domain" or "intracellular domain" refers to the portion of a protein that resides in or is located in the cytoplasm of a cell. The β4 integrin protein has an N-terminal extracellular domain, a transmembrane domain, and a C- terminal cytoplasmic or intracellular domain. The cytoplasmic domain of wild-type human β4 integrin is provided as SEQ ID NO: 3, and includes amino acid residues 734 to 1822 of SEQ ID NO: 1.

[043] The term "detection" includes any means of detecting a target molecule, including direct and indirect detection of the molecule. Detecting includes both qualitative and quantitative measurements of a target molecule. In one aspect, a detection method is used to identify the presence of autoantibodies that bind β4 integrin in a patient sample. In another aspect, a detection method is used to quantify the amount of autoantibodies that bind to β4 integrin present in a patient sample. In some embodiments, the relative quantities of autoantibodies that bind to β4 integrin are compared between different subject samples, e.g., between samples collected from subjects suspected of having OCP and control samples collected from subjects without OCP.

[044] The term "dose," as used herein, refers to an amount of an agent, (e.g., a chemotherapeutic agent), that is given to a subject. The dose may be a unit dose,

representing the amount of the agent administered to a subject at one time. The dose may also be expressed as the amount of the agent a subject receives in a specified time period, e.g. , a daily dose, a weekly dose, a monthly dose, etc. The term "dosing", as used herein, refers to the administration of a substance (e.g. , a chemotherapeutic agent) to achieve a therapeutic objective (e.g. , the treatment of an autoimmune disease, including, such as OCP).

[045] A kit is any manufacture (e.g., a package or container) comprising at least one reagent (e.g. a reagent for specifically detecting autoantibodies indicative of OCP in a subject), the manufacture being promoted, distributed, or sold as a unit for performing the methods described herein.

[046] The terms "sample," or "biological sample" as used herein, refers to a composition that is obtained or derived from a subject that contains a molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics. A sample derived from a subject is one that originates from the subject. In one embodiment, a biological sample is a cellular sample. In another embodiment, the sample is an acellular sample. Exemplary samples include, but are not limited to, primary cells, cell lysates, blood, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood- derived cells, urine, cerebrospinal fluid, alveolar fluid, lung lavage, gastric fluid, gastric lavage, peritoneal fluid, saliva, sputum, tears, perspiration or sweat, mucus, wound fluid, nasal discharge, bone marrow sample, tumor lysates, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof.

[047] The term "subject," as used herein, refers to either a human or non-human animal subject. In one embodiment, the subject is a human subject. In another embodiment, the subject is a non-human subject. In some embodiments, the subject is a mammalian subject, e.g. , a primate, mouse, rat, rabbit, sheep, dog, cat, horse, or cow. A "normal subject" or a "control subject" refers to a human or non-human animal with a wild-type phenotype, e.g., a human or non-human animal who is not suffering from a disease or disorder (e.g., ocular cicatricial pemphigoid).

[048] A "therapeutically effective amount" of a therapeutic agent, or combinations thereof, is an amount sufficient to treat or reduce symptoms of a disease in a subject. For example, a therapeutically effective amount of an agent for treating ocular cicatricial pemphigoid can be an amount of an agent that provides an observable therapeutic benefit compared to baseline clinically observable signs and symptoms of OCP, e.g., by reducing dry, irritated, or blistering eyes and/or scarring of the conjunctiva. In one embodiment, a therapeutically effective amount of an agent for treating OCP is an amount that reduces the concentration of autoantibodies that bind to β4 integrin in the serum or plasma of a patient.

[049] The term "treat" is used herein to mean to relieve, reduce or alleviate at least one symptom of a disease in a subject. For example, in relation to ocular cicatricial pemphigoid, the term "treat" includes relieving, reducing, or alleviating dry, irritated, and/or blistering eyes, and/or scarring of the conjunctiva. Accordingly, the term "treat" also encompasses delaying or preventing onset prior to clinical manifestation of a disease or symptom of a disease and/or reducing the risk of de veloping or worsening of a symptom of a disease. The progression of OCP is slowed if at least one symptom of OCP is or is expected to be alleviated, slowed, delayed, or prevented.

[050] 2. Ocular Cicatricial Pemphigoid (OCP)

[051] Autoimmunity is a disease condition that occurs when a subject's immune system produces autoantibodies that specifically recognize the body's own components, i.e. self-antigens. Ocular Cicatricial Pemphigoid (OCP) is an autoimmune vesieulobulkms disease that affects the conjunctiva, and sometimes other squamous epithelia. OCP patients often exhibit diverse and nonspecific clinical symptoms, in serious cases, the disease progresses to irreversibly scar the conjunctiva, causing blindness if left undiagnosed or if treated impro perl .

[052] Patients with subepithelial blistering diseases, such as ocular cicatricial pemphigoid, produce autoantibodies that bind to distinct antigens of the basement membrane zone (BMZ) at the epithelial- sub-epithelial junction of mucosa and skin. Tyagi et al. (1996) PNAS 93: 14714-19. OCP patients have autoantibodies that bind to the β4 subunit of the integrin heterodimer α6β4 at the BMZ of the conjunctiva. Generally, integrins are heterodimeric transmembrane receptor complexes composed of a and β subunits that are involved in cell adhesion to extracellular matrices, signal transduction, and hemidesmosomal assembly. Integrins are involved in the adherence of basal epithelial cells to underlying basement membranes. Evidence suggests that the binding of autoantibodies to β4 integrin in the conjunctiva of OCP patients leads to BMZ separation in conjunctival mucosa and blistering of the eye. For example, human conjunctiva cultured with OCP sera induced BMZ separation in a human conjunctiva organ culture model.

[053] The autoantibodies of some OCP patients bind to an epitope within the cytoplasmic domain of the β4 subunit of the α6β4 integrin heterodimer. Rashid et al. (2013) Invest. Opthalmol. Vis. Sci. 54:7707-16. This epitope is located within amino acid residues 734 and 1822 of the full-length human β4 integrin, as provided in SEQ ID NO: 1. In some patients, the predominant epitope bound by β4 autoantibodies is localized within specific regions of the cytoplasmic domain. For example, the predominant epitope can be localized within amino acid residues 1489- 1822 of full-length human β4 integrin. In other

embodiments, the predominant epitope is located within amino acid residues 1489 - 1572 of full-length human β4 integrin. In some embodiments, the predominant epitope is located within amino acid residues 1489 - 1510 of full-length human β4 integrin. In some patients, autoantibodies recognize multiple epitopes within β4 integrin. Kumari et al. (2001) IOVS. 42(21:379-384.

[054] Current methods of identifying subjects having or at risk for Ocular Cicatricial Pemphigoid (OCP) rely on a combination of factors, including patient medical history and the presence of clinical symptoms suggestive of OCP. Since OCP symptoms are often nonspecific, the disease is usually not diagnosed until later points in disease progression. OCP is often brought to clinical attention when symblepharon becomes evident, e.g., in a slit lamp exam. Immuno-detection of autoantibodies in conjunctival biopsy samples taken from patients is currently considered the best method of diagnosis. However, biopsy samples are difficult to obtain, can result in patient discomfort and inflammation, and sometimes provide inadequate amounts of tissue for definitive diagnosis. Furthermore, the autoantibodies can be difficult to detect and measure because the titer of autoantibodies present in OCP patients is low. [055] 3. Methods of Diagnosing Ocular Cicatricial Pemphigoid (OCP)

[056] In light of the difficulties associated with diagnosing OCP using traditional clinical methods, i.e. , clinical exam and/or conjunctival biopsy, there is a need for a more accurate and less invasive method to diagnose and monitor subjects with OCP.

[057] The present invention provides methods for diagnosing OCP in a subject by determining the level or concentration of circulating autoantibodies that specifically bind to β4 integrin in a sample obtained from a subject. In preferred embodiments, the sample is obtained from the subject non-invasively, e.g. , a blood sample, a serum sample, a plasma sample, etc. As described herein, particular levels of autoantibodies that bind specifically to β4 integrin have been identified which indicate that the subject has or is at risk for developing ocular cicatricial pemphigoid. These autoantibodies are present at elevated levels in the serum, plasma, or blood of OCP patients relative to subjects who do not have OCP (i.e., normal or control subjects). The methods described herein improve the speed, accuracy and cost of diagnosing OCP, and can also decrease the overall cost of treatment by eliminating ineffective therapies.

[058] OCP patients produce autoantibodies that bind to the basal membrane zone of human conjunctiva, where they interact with the cytoplasmic domain of β4 integrin and contribute to basal separation. Surprisingly, these autoantibodies are also elevated at detectable levels outside of the eye in subjects with OCP. For example, elevated levels of autoantibodies that bind to β4 integrin have been found to circulate in the blood, plasma, and serum of OCP patients. Elevated levels of circulating autoantibodies that specifically recognize β4 integrin in blood-derived samples, e.g. , serum or plasma, are predictive of whether the subject has or may develop OCP.

[059] In some embodiments, the invention provides methods of detecting the presence, level, or concentration of autoantibodies that specifically bind β4 integrin in a subject known or suspected to have OCP. In some embodiments, the method of detecting autoantibodies that specifically bind β4 integrin in a subject known or suspected of having OCP involves detecting whether autoantibodies that specifically bind β4 integrin are present in a serum or plasma sample derived from the subject by contacting the serum or plasma sample with a β4 integrin polypeptide, or fragment thereof, e.g., a fragment that contains the β4 integrin cytoplasmic domain, or a portion thereof, and detecting binding between the autoantibodies and the β4 integrin polypeptide, or fragment thereof. Detecting binding between the autoantibodies and the β4 integrin polypeptide can be achieved using any suitable method capable of detecting the formation of an antibody/antigen complex, including those methods described herein. In a preferred embodiment, an ELISA is used to detect formation of a complex between β4 integrin polypeptide, and serum or plasma autoantibodies that specifically bind thereto. In some embodiments, the level and/or concentration of autoantibodies that specifically bind β4 integrin are detected in the sample. In some embodiments, the foregoing method further comprises providing and/or obtaining a biological sample (e.g. , a blood-derived sample, such as serum or plasma) from the subject. Subjects known or suspected of having OCP may be identified by a clinical examination based on the presence of one or more symptoms of OCP, e.g., chronic eye irritation, dryness, grittiness, chronic conjunctivitis, etc.

[060] In some embodiments, the invention provides a method of determining the level or concentration of autoantibodies that bind specifically to β4 integrin in a biological sample obtained from the subject, wherein an increase in the level or concentration of autoantibodies that bind specifically to β4 integrin relative to that in a normal subject is indicative of OCP in the subject.

[061] Accordingly, in one aspect, the invention provides a method of facilitating diagnosis of Ocular Cicatricial Pemphigoid (OCP) in a subject. The method can include determining the level or concentration of autoantibodies that specifically bind to β4 integrin in a biological sample obtained from the subject. An increase in the level or concentration of autoantibodies that specifically bind to β4 integrin, relative to the level or concentration of autoantibodies that bind to β4 integrin in a normal subject as determined relative to a suitable control, is indicative of OCP in the subject.

[062] In some embodiments, the method involves (i) determining the level or concentration of autoantibodies that specifically bind to β4 integrin in a biological sample obtained from the subject, (ii) comparing the level or concentration of autoantibodies to the level or concentration of autoantibodies that specifically bind to β4 integrin in a control sample, and (iii) determining whether the level or concentration of the autoantibodies is increased relative to the level or concentration in a normal subject, as determined relative to the control sample. If the control sample is obtained from or representative of a normal subject, an increase in the level or concentration of the autoantibodies relative to the control sample indicates that the subject has OCP. If the control sample is obtained from or representative of a subject having OCP, a level or concentration of the autoantibodies equivalent to or higher than that present in the control sample indicates that the subject has OCP.

[063] An "increased concentration" or "elevated concentration" of autoantibodies refers to a concentration in a test sample that is greater than the concentration present in an equivalent sample obtained from a normal subject. In a preferred embodiment, the increase is a statistically significant increase. For example, the increased concentration can be increased by at least 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000% or more relative to the concentration in the normal subject. In some embodiments, the increased concentration is increased by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 200- fold, 500-fold, 1000-fold or more relative to the concentration in the normal subject. In some embodiments, the increased concentration is increased at least 1.5-fold to 10-fold, 5- fold to 15-fold, 10-fold to 20-fold, 15-fold to 25-fold, 30-fold to 40-fold, 35-fold to 45-fold, 40-fold to 50-fold, or 50-fold to 100-fold more relative to the concentration in the normal subject. In some embodiments, the increased concentration is at least 2.5-fold to 15-fold more relative to the concentration in the normal subject. In some embodiments, the increased concentration is 3-fold to 5-fold more relative to the concentration in the normal subject. In some embodiments, the increased concentration is at least 8-fold to 15-fold more relative to the concentration in the normal subject. In some embodiments, the increased concentration is at least 3-fold to 4-fold more relative to the concentration in the normal subject. In some embodiments, the increased concentration is at least 12-fold to 13-fold more relative to the concentration in the normal subject.

[064] An "increased level", or an "elevated level", of autoantibodies refers to a level in a test sample that is greater than the level present in an equivalent sample obtained from a normal subject. In a preferred embodiment, the increase is a statistically significant increase. For example, the increased level can be increased by at least 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000% or more relative to the level in the normal subject. In some embodiments, the increased level is increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more relative to the level in the normal subject. In some embodiments, the increased level is increased by 1.5-fold to 10-fold, 5-fold to 15-fold, 10-fold to 20-fold, 15-fold to 25-fold, 30-fold to 40-fold, 35-fold to 45-fold, 40-fold to 50-fold, or 50-fold to 100-fold more relative to the level in the normal subject. In some embodiments, the increased level is increased by 2.5-fold to 15-fold more relative to the level in the normal subject. In some embodiments, the increased level is increased by 2.5-fold to 15-fold more relative to the level in the normal subject.

[065] In some embodiments, the method involves (i) determining the concentration of autoantibodies that specifically bind to β4 integrin in a biological sample obtained from the subject, and (ii) determining whether the concentration of the autoantibodies is above or below a threshold concentration indicative of OCP. A concentration of the autoantibodies in the biological sample at or above the threshold concentration is indicative that the subject has OCP. In some embodiments, the threshold concentration of autoantibodies that specifically bind to β4 integrin in a biological sample that is indicative of OCP is between 2 ng/mL and 5,000 ng/mL. In some embodiments, the threshold concentration of autoantibodies that specifically bind to β4 integrin in a serum or plasma sample that is indicative of OCP is greater than or equal to about 100 ng/mL (e.g. , about 100 ng/mL, 150 ng/mL, 200 ng/mL, 250 ng/mL, 300 ng/mL, 350 ng/mL, 400 ng/mL, 450 ng/mL, 500 ng/mL, 550 ng/mL, 600 ng/mL, 650 ng/mL, 700 ng/mL, 750 ng/mL, 800 ng/mL, 850 ng/mL, 900 ng/mL, 950 ng/mL, 1000 ng/mL, 1100 ng/mL, 1200 ng/mL, 1300 ng/mL, 1400 ng/mL, 1500 ng/mL, 1600 ng/mL, 1700 ng/mL, 1800 ng/mL, 1900 ng/mL, 2000 ng/mL, 2500 ng/mL, 3000 ng/mL, 4000 ng/mL, 5000 ng/mL, 6000 ng/mL, 7000 ng/mL, 8000 ng/mL, 9000 ng/mL, or about 10,000 ng/mL or more). In some embodiments, the threshold concentration of autoantibodies that specifically bind to β4 integrin in a biological sample that is indicative of OCP is between 100 to 200 ng/mL, 200 to 300 ng/mL, 300 to 400 ng/mL, 400 to 500 ng/mL, 500 to 600 ng/mL, 600 to 700 ng/mL, 700 to 800 ng/mL, 800 to 900 ng/mL, 900 to 1000 ng/mL, 1000 to 1500 ng/mL, 1500 to 2000 ng/mL, 2000 to 2500 ng/mL, 3000 to 3500 ng/mL, 3500 to 4000 ng/mL, 4000 to 4500 ng/mL, or 4500 to 5000 ng/mL. A concentration of the autoantibodies in the biological sample below the threshold concentration is indicative that the subject does not have OCP, or is in remission from OCP.

[066] Autoantibodies detected using the methods described herein may be of any known isotype, e.g. , IgG, IgA, IgM, IgE, IgD, or any combination thereof. In one

embodiment, the autoantibodies can include complement 3 component (C3). In one embodiment, the autoantibodies are IgG, IgM, and/or IgA. In an exemplary embodiment, the autoantibodies are IgG. In another exemplary embodiment, the autoantibodies are IgM.

[067] Autoantibodies indicative of OCP specifically bind to β4 integrin. In some embodiments, autoantibodies indicative of OCP specifically bind to epitopes located within the cytoplasmic domain of β4 integrin. Accordingly, in some embodiments, the methods described herein can be performed by determining the level or concentration of

autoantibodies that specifically bind the cytoplasmic domain of β4 integrin. The cytoplasmic domain of β4 integrin contains amino acid residues 734 to 1822 of the full-length human β4 integrin polypeptide, as provided in SEQ ID NO: 1. In some embodiments, the methods described herein can be performed by determining the level or concentration of

autoantibodies that bind to specific epitopes or regions within the cytoplasmic domain of β4 integrin. For example, autoantibodies can be detected that bind to an epitope located within amino acid residues 1489- 1822, amino acid residues 1489- 1572 , and/or amino acid residues 1489- 1510 of full-length human β4 integrin.

[068] In some embodiments of the methods described herein, the level or concentration of autoantibodies that bind specifically to β4 integrin can be determined by contacting the sample with a β4 integrin polypeptide, or a fragment thereof. In some embodiments, methods described herein further comprise detecting a complex formed between the autoantibody and the β4 integrin polypeptide, or fragment thereof. The β4 integrin polypeptide can be a full- length β4 integrin polypeptide. In some embodiments, the polypeptide comprises SEQ ID NO: l . Alternatively, the β4 integrin polypeptide can be a fragment of SEQ ID NO: l, e.g., a fragment of a sufficient length to bind β4 integrin autoantibodies. In some embodiments, the fragment comprises between 5- 1500 contiguous amino acids of SEQ ID NO: l, e.g. , 5- 10 amino acids, 5-20 amino acids, 5-50 amino acids, 5- 100 amino acids, 10-20 amino acids, 10- 50 amino acids, 10- 100 amino acids, 10-200 amino acids, 20-50 amino acids, 20- 100 amino acids, 20-200 amino acids, 20-500, amino acids, etc.

[069] In one embodiment, the autoantibodies are detected using a β4 integrin fragment comprising at least 5 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the autoantibodies are detected using a β4 integrin fragment comprising at least 6 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one

embodiment, the autoantibodies are detected using a β4 integrin fragment comprising at least 7 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the autoantibodies are detected using a β4 integrin fragment comprising at least 8 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the autoantibodies are detected using a β4 integrin fragment comprising at least 9 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 15 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 20 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 25 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 30 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 35 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 40 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 45 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 50 contiguous amino acids of the sequence set forth in SEQ ID NO: 1.

[070] In one embodiment, the autoantibodies are detected using a β4 integrin fragment comprising the cytoplasmic domain of β4 integrin, or a portion thereof. The cytoplasmic domain of β4 integrin contains amino acid residues corresponding to positions 734 to 1822 of SEQ ID NO: 1. In one embodiment, the autoantibodies are detected using a polypeptide comprising SEQ ID NO: 3, or a fragment thereof. In one embodiment, the fragment comprises or consists of amino acid residues 756 to 839 of SEQ ID NO: 3. In another embodiment, the fragment comprises or consists of a fragment of amino acid residues 756 to 839 of SEQ ID NO: 3. In one embodiment, the fragment comprises or consists of amino acid residues 756 to 777 of SEQ ID NO: 3. In another embodiment, the fragment comprises or consists of a fragment of amino acid residues 756 to 777 of SEQ ID NO: 3. In one embodiment, the autoantibodies are detected using a β4 integrin fragment comprising at least 5 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the autoantibodies are detected using a β4 integrin fragment comprising at least 6 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 7 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 8 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 9 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 15 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 20 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 25 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 30 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 35 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 40 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 45 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the

autoantibodies are detected using a β4 integrin fragment comprising at least 50 contiguous amino acids of the sequence set forth in SEQ ID NO: 3.

[071] In one embodiment, the β4 integrin polypeptide or β4 integrin polypeptide fragment is at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: l, or a fragment thereof. In one embodiment, the β4 integrin polypeptide or β4 integrin polypeptide fragment is at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:3, or a fragment thereof.

[072] In some embodiments of the methods described herein, the level or concentration of autoantibodies that bind specifically to β4 integrin can be determined using an enzyme- linked immunosorbent assay (ELISA). For example, an ELISA can be used to quantitatively determine the concentration of anti-p4 integrin autoantibodies in a patient sample.

Accordingly, in some embodiments, the methods described herein comprise performing an ELISA to determine the level or concentration of autoantibodies that bind to β4 integrin in a biological sample from the subject.

[073] Generally, an ELISA uses a capture reagent capable of specifically binding the molecule of interest. For example, the capture reagent may be capable of specifically binding anti-p4 integrin autoantibodies. Exemplary capture reagents include, for example, β4 integrin polypeptides, or fragments thereof, as described herein, e.g., fragments that contain the β4 integrin cytoplasmic domain, or a portion thereof. Other exemplary capture reagents include aptamers or anti-idiotypic antibodies that specifically bind autoantibodies recognizing β4 integrin by mimicking a portion of the target antigen. For example, anti-idiotypic antibodies can contain a variable region structure that mimics a region of the β4 integrin polypeptide. The capture reagent can be immobilized on a solid support, such as a multi-well plate.

[074] Captured anti^4 integrin autoantibodies can then be detected and/or quantified with the use of a detection reagent. In one embodiment, the detection reagent is capable of binding to the complex formed between the capture reagent and the anti^4 integrin autoantibody. For example, the detection reagent can be an antibody (e.g. , a polyclonal or monoclonal antibody, or an antigen-binding fragment thereof) that binds to the anti^4 integrin autoantibody, e.g. , at a region of the autoantibody that is not bound by the capture reagent. In one embodiment, the detection reagent is an antibody or fragment thereof that binds the constant region of autoantibodies from the species of interest. For example, if a human sample is used in the assay, the detection reagent may be an antibody or portion thereof that recognizes human immunoglobulins. In exemplary embodiments, the detection reagent is an anti-human IgG antibody, an anti-human IgM antibody, an anti-human IgA antibody, an anti-human IgE antibody, or an antigen binding fragment thereof. The detection reagent typically contains, e.g., is covalently or non-covalently linked to, a detectable moiety. ELISAs can be based on, for example, colorimetry, chemiluminescence, or fluorometry detection means. In exemplary embodiments, the detectable moiety is a radio-label, a chromophore-label, a fluorophore-label, or an enzyme-label. The detection reagent can be directly labeled by coupling (i.e. , physically linking) a detectable substance to the detection reagent. The detection reagent can alternatively be indirectly labeled by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently- labeled or enzymatic ally- labeled secondary antibody, or labeling of a detection reagent with biotin such that it can be detected with fluorescently labeled streptavidin. In one embodiment, the detectable antibody is a primary antibody that binds specifically to β4 integrin-binding autoantibodies. In one embodiment, the detectable antibody is a secondary antibody that binds specifically to a primary antibody that binds to β4 integrin-binding autoantibodies.

[075] As a non- limiting example, an ELISA can be performed using a biological sample, e.g., a serum or plasma sample, in which the sample is contacted with a capture reagent that comprises a β4 integrin polypeptide, or a fragment thereof. The sample is incubated with the capture reagent, and washed to remove unbound components of the biological sample. Complexes containing the capture reagent bound to anti-p4 integrin antibodies present in the biological sample are detected using a detection reagent comprising an antibody that binds the constant region of the bound anti-p4 integrin antibodies. The detection reagent is coupled to an enzymatic label which converts a colorless substrate to a colored product, in proportion to the amount of detection reagent present. After the detection reagent has been allowed to bind to the captured anti-p4 integrin antibodies, unbound detection reagent is removed by washing, and the colorometric substrate is added. The intensity of the colored product can be measured by standard means, e.g. , using a

spectrophotometer or a plate reader. The intensity can be compared against a standard curve to quantify the concentration of anti-p4 integrin autoantibodies present in the biological sample.

[076] In another embodiment, the ELISA capture reagent used for detecting anti-p4 integrin autoantibodies is an antibody, or fragment thereof, that binds to the constant region of autoantibodies in the biological sample. In the embodiment, the detection reagent can comprise a β4 integrin polypeptide, or a fragment thereof, directly or indirectly coupled to a detectable label.

[077] The foregoing methods can be adapted to detect the level or concentration of anti-p4 integrin autoantibodies using different ELISA formats known in the art, for example, direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, etc.

[078] In another embodiment, the level or concentration of autoantibodies that bind specifically to β4 integrin is determined using mass spectrometry. For example, matrix- associated laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) or surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) can be used to quantitatively determine the level or concentration of autoantibodies that bind to β4 integrin in a biological sample. Autoantibodies that bind to β4 integrin can be isolated from the sample prior to mass spectrometry by binding to a capture reagent, immobilized, for example, on a protein-binding chip (Wright, G.L., Jr., et al. (2002) Expert Rev Mol Diagn 2:549; Li, J., et al. (2002) Clin Chem 48: 1296; Laronga, C, et al. (2003) Dis Markers 19:229; Petricoin, E.F., et al. (2002) 359:572; Adam, B.L., et al. (2002) Cancer Res 62:3609; Tolson, J., et al. (2004) Lab Invest 84:845; Xiao, Z., et al. (2001) Cancer Res 61:6029) . Methods for performing mass spectrometry analysis are described in, for example, U.S. Patent Nos. 5,622,824, 5,605,798 and 5,547,835, the entire contents of each of which are incorporated herein by reference.

[079] Other means useful for determining the level of autoantibodies that bind specifically to β4 integrin in a sample include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods and assays such as fluid or gel precipitation reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassay (RIA), immunofluorescent assays, and Western blotting.

[080] The level or concentration of anti-p4 integrin autoantibodies can be determined in a biological sample derived from a subject known or suspected of having OCP. A sample derived from a subject is one that originates from a subject. Such a sample can be further processed after it is obtained from the subject. Biological samples suitable for performing the methods described herein may be obtained from any source. Preferably, the biological sample is obtained non-invasively. For example, the sample can be a non-biopsy sample. In exemplary embodiments, the sample is a sample that contains circulating antibodies. In one embodiment, the biological sample is a bodily fluid, e.g., a fluid containing circulating antibodies. In one embodiment, the sample is a blood sample or a blood-derived sample. Exemplary blood-derived samples include a serum sample or a plasma sample. Other bodily fluids include, for example, urine, saliva, lymph, cerebrospinal fluid, tears, sweat, semen, etc. In some embodiments, the methods described herein further comprise obtaining a biological sample from the subject. The subject can be a subject known or suspected of having OCP. In some embodiments, the subject can be a subject at risk of developing OCP. In some embodiments, the subject is undergoing treatment for OCP. In one embodiment, more than one type of sample is collected from the subject.

[081] Whether or not the level or concentration of autoantibodies in a biological sample from a test subject is increased relative to the concentration of autoantibodies in a normal subject may be ascertained by comparing the level or concentration of autoantibodies in the test subject sample to the level or concentration of autoantibodies in a suitable control. The skilled person can select an appropriate control for the assay in question. For example, a control may be a biological sample derived from a known subject, e.g. , a subject known to be a normal subject, or a subject known to have OCP. If the control is obtained from a normal subject, a statistically significant increase in the level or concentration of anti-p4 integrin autoantibodies in a sample obtained from a test subject relative to the control is indicative that the subject has OCP. If the control is obtained from a subject known to have OCP, levels comparable to or higher than such a control are indicative of OCP, reflective of a difference with respect to the level or concentration of anti-p4 integrin autoantibodies present in a comparable sample from a normal subject.

[082] Exemplary control samples useful for determining the level or concentration of anti-p4 integrin autoantibodies in a normal subject include a biological sample (e.g. , a serum sample, a plasma sample, a blood sample, etc.) obtained from a healthy subject not afflicted with OCP, pooled samples from healthy subjects, a value representative of a level in a healthy subject, etc. In some embodiments, a suitable control can be a known standard value or range of values representative of a "normal" subject, i.e. , a subject not afflicted with OCP.

[083] The term "known standard level" or "control level" refers to an accepted or predetermined level of autoantibodies that bind β4 integrin in a normal subject, which can be used for comparison with the le vel of autoantibodies in a test subject. In one embodiment, the control expression level of autoantibodies that bind the cytoplasmic domain of β4 integrin is based on the level of autoantibodies in a sample(s) from a subject(s) having slow disease progression. In another embodiment, the control level of autoantibodies that bind the cytoplasmic domain of β4 integrin is based on the expression level in a sample from a subject or subjects having rapid disease progression. In another embodiment, the control level of autoantibodies that bind the cytoplasmic domain of β4 integrin is based on the level of autoantibodies in a sample(s) from an unaffected, i.e., non-disease, subject(s), i.e., a subject who does not have OCP. In yet another embodiment, the control level of autoantibodies that bind the cytoplasmic domain of β4 integrin is based on the level of autoantibodies in a sample from a subject(s) prior to the administration of a therapy for OCP.

[084] In one embodiment, a control may also be a reference standard. A reference standard serves as a reference level for comparison, such that test samples can be compared to the reference standard in order to infer the disease status of a subject. A reference standard may be representative of the level of one or more autoantibodies in a known subject, e.g. , a subject known to be a normal subject, or a subject known to have OCP. Likewise, a reference standard may be representative of the level of one or more autoantibodies in a population of known subjects, e.g. , a population of subjects known to be normal subjects, or a population of subjects known to have OCP. The reference standard may be obtained, for example, by pooling samples from a plurality of individuals and determining the level of one or more autoantibodies in the pooled samples, to thereby produce a standard over an averaged population. Such a reference standard represents an average level of one or more

autoantibodies among a population of individuals. A reference standard may also be obtained, for example, by averaging the level of one or more autoantibodies determined to be present in individual samples obtained from a plurality of individuals. Such a standard is also representative of an average level of one or more autoantibodies among a population of individuals. A reference standard may also be a collection of values each representing the level of one or more autoantibodies in a known subject in a population of individuals. In certain embodiments, test samples may be compared against such a collection of values in order to infer the disease status of a subject. In certain embodiments, the reference standard is an absolute value. In such embodiments, test samples may be compared against the absolute value in order to infer whether a subject has or is at risk for OCP. In a one embodiment, a comparison between the level of one or more autoantibodies in a sample relative to a control is made by executing a software classification algorithm. The skilled person can readily envision additional controls that may be appropriate depending on the assay in question. The aforementioned controls are exemplary, and are not intended to be limiting.

[085] 4. Monitoring OCP Progression

[086] The level or concentration of autoantibodies that bind specifically to β4 integrin can be used to monitor the status of Ocular Cicatricial Pemphigoid (OCP) in a subject. In general, an increase in the concentration of autoantibodies is associated with disease progression, while a decrease is associated with improvement.

[087] The level or concentration of autoantibodies that bind to β4 integrin can be determined in accordance with the methods described herein in two or more samples collected from the same subject over a period of time, so as to monitor the course of disease in the subject. In this manner, it is possible to determine whether the level or concentration of anti-p4 integrin autoantibodies has increased, decreased, or remained the same at different points in time. An increase in the level or concentration of anti-p4 integrin autoantibodies over time indicates that OCP is progressing in the subject, while a decrease in the level or concentration of anti-p4 integrin autoantibodies over time indicates that OCP is improving in the subject.

[088] In another embodiment, the concentration of autoantibodies that bind specifically to β4 integrin can be used to monitor the effectiveness of a therapy for OCP. In general, an increase in the concentration of autoantibodies during the course of treatment with an OCP therapeutic is associated with disease progression and signals poor therapeutic effectiveness in the subject. Accordingly, an increase in the concentration of autoantibodies may signal a need to discontinue or alter the treatment given to the subject. A decrease in the

concentration of autoantibodies during the course of treatment with an OCP therapeutic is associated with disease improvement and signals responsiveness to the therapeutic. Levels that decrease to a level at or below those representative of a normal may indicate that OCP has entered remission.

[089] Preferably, comparable samples (e.g. samples obtained from the source (serum, plasma, blood, etc.)) are used at the different time points. Samples can be obtained from the subject for testing at multiple intervals. In one embodiment, the samples are obtained before and after treatment. For example, the samples can be collected from the subject before and after the subject has received treatment with an OCP therapeutic. In another embodiment, the samples are obtained at separate times during the course of treatment. In exemplary embodiments, the samples are obtained at intervals of 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 6 months, 1 year, 18 months, 2 years or 3 years or more.

[090] In one embodiment, the invention provides a method of monitoring the course of Ocular Cicatricial Pemphigoid (OCP) in a subject, by determining a concentration of autoantibodies that bind specifically to β4 integrin in a first biological sample obtained from the subject prior to treatment with an OCP therapeutic; and determining a concentration of autoantibodies that bind specifically to β4 integrin in a second biological sample obtained from the subject during or after treatment with the OCP therapeutic, wherein a decrease in the concentration of autoantibodies that specifically bind β4 integrin in the second biological sample relative to the first biological sample indicates that the subject is responding to treatment with the OCP therapeutic. In some embodiments, the concentration of

autoantibodies that bind specifically to β4 integrin is assayed in two or more biological samples collected from the subject during the course of treatment with an OCP therapeutic.

[091 ] 5. Methods of Treating Ocular Cicatricial Pemphigoid (OCP)

[092] The level or concentration of autoantibodies that bind specifically to β4 integrin can be used to select a subject for treatment with an OCP therapeutic. Accordingly, in one embodiment, the method provides a method of treating OCP in a subject, by selecting a subject having an increased concentration of autoantibodies that bind specifically to β4 integrin in a biological sample obtained from the subject, relative to that in comparable sample obtained from a normal subject; and administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an OCP therapeutic, thereby treating OCP in the subject.

[093] In some embodiments, an increase in the concentration of autoantibodies that bind to β4 integrin, e.g., autoantibodies that bind to the cytoplasmic domain of β4 integrin, in a subject sample relative to a suitable control identifies the subject as a candidate for treatment with an OCP therapeutic as described herein. In some embodiments of the invention, an increase in the concentration of autoantibodies that bind to the cytoplasmic domain of β4 integrin in a subject is predictive of an OCP disease state in the subject, and therefore signals to a medical professional, e.g. , a doctor or clinician, that the subject should be treated with one or more OCP therapeutic agents. In some embodiments, a subject with an increased concentration of autoantibodies that bind to β4 integrin, relative to a control subject, is selected for OCP therapy. In some embodiments, at least one OCP therapeutic agent is administered to a subject with an increased concentration of autoantibodies relative to a control.

[094] In one embodiment, the invention provides a method of diagnosing and treating OCP in a subject. The method can include detecting the level or concentration of autoantibodies that bind to β4 integrin in a blood-derived sample obtained from the subject, e.g. , a serum or plasma sample; diagnosing the subject with OCP when an elevated level or concentration of the autoantibodies are detected, relative to the level or concentration in a comparable sample obtained from normal subject; and administering an effective amount of an OCP therapeutic to the subject diagnosed with OCP. In one embodiment, the method can further include obtaining the blood-derived sample from the subject. In some embodiments, the subject is suspected of having OCP, for example, because the subject exhibits clinical symptoms suggestive of OCP, as described herein. In exemplary embodiments, the level or concentration of β4 integrin-binding autoantibodies are detected in the subject using an enzyme-linked immunosorbent assay (ELISA), as described herein.

[095] In an exemplary embodiment, the invention provides a method of diagnosing and treating OCP in a subject by performing an ELISA to detect the level or concentration of autoantibodies that specifically bind to β4 integrin in a blood-derived sample obtained from the subject, e.g. , a serum or plasma sample; diagnosing the subject as having OCP when an elevated level or concentration of the autoantibodies are detected, relative to the level or concentration present in a comparable sample obtained from a normal subject; and

administering an effective amount of an OCP therapeutic to the subject diagnosed with OCP. In one embodiment, the ELISA uses a capture reagent that comprises a β4 integrin

polypeptide, or a fragment thereof comprising all or a part of the β4 integrin cytoplasmic domain. In one embodiment, the method can further include obtaining the blood-derived sample from the subject.

[096] In one embodiment, the invention provides a method of treating Ocular

Cicatricial Pemphigoid (OCP) in a subject by selecting a subject having an increased serum or plasma concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin relative to that in a normal subject, and administering to the subject a

pharmaceutical composition comprising a therapeutically effective amount of an OCP therapeutic, thereby treating OCP in the subject.

[097] In one embodiment, the OCP therapeutic agent is a chemo therapeutic agent. In one embodiment, the OCP therapeutic is an immune suppressive agent (e.g. , a corticosteroid), or a systemic immunomodulator. Chemo therapeutic and immune suppressive therapeutics for treating or impro ving symptoms of OCP are known in the art, and include, for example, mitomycin C, methotrexate, mycophenolate mofetil (CellCept), or cyclo hosphamide (Cytoxan). Immune suppressive agents useful for treating OCP include, for example, rituximab, intravenous immunoglobulin (IVIg), azathioprine (Imuran), a TNF-cx inhibitor, cyclosporine, prednisone, diaminodiphenylsulfone (Dapsone).

[098] The OCP therapeutic can comprise the therapeutic agent alone, or the therapeutic can be formulated in a composition comprising a pharmaceutically acceptable carrier, excipient, flow agent, processing aid, diluent or a combination thereof. A pharmaceutical composition generally comprises a therapeutically effective amount of an agent together with suitable di luents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers known in the art. In some embodiments, the OCP therapeutic can be in solid or liquid form, including as tablets, powders, capsules, pellets, solutions, suspensions, elixirs, emulsions, gels, creams, or suppositories, including rectal and urethral suppositories. Pharmaceutically acceptable carriers include gums, starches, sugars, cellulosic materials, and mixtures thereof.

[099] In some embodiments, the OCP therapeutic can be suitable for oral, intravenous, intraaorterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, or topical (e.g., ocular) administration.

[0100] Generally, the OCP therapeutic agent is administered to a subject diagnosed as having OCP at a therapeutically effective amount, which is adjusted specifically to the disease severity sought to be treated. A "therapeutically effective amount" as used herein refers to that amount which provides a therapeutic effect for a given condition (e.g. , an amount sufficient to treat or reduce symptoms associated with OCP). For example, a therapeutically effective amount can be an amount that provides an observable therapeutic benefit compared to baseline clinically observable signs and symptoms of ocular cicatricial pemphigoid (OCP), e.g. , by reducing dry, irritated and/or blistering eyes. Alternatively, a therapeutically effective amount can be an amount that leads to an observable decrease in the concentration of autoantibodies that bind to β4 integrin, as detected by a diagnostic test described herein.

[0101] The methods described herein can further comprise, in some embodiments, administering a therapeutically effective amount of an OCP therapeutic to a subject diagnosed as having OCP. In one embodiment, the method includes administering a therapeutically effective amount of a chemotherapeutic agent, as described herein, to the subject. In one embodiment, the method includes administering a therapeutically effective amount of an immune suppressive agent, as described herein, to the subject. In one embodiment, the method includes administering a therapeutically effective amount of one or more chemotherapeutic agents in combination with one or more immune suppressive agents to the subject.

[0102] The therapeutically effective dosage of chemotherapeutic agents, immune suppressive agents, or combinations thereof, will vary somewhat from subject to subject, and will depend upon factors such as the age, weight, and condition of the subject and the route of delivery. Therapeutically effective dosages and dosing regimens can be determined in accordance with procedures known to those skilled in the art.

[0103] In one embodiment, a therapeutically effective amount of a therapeutic agent for OCP reduces the concentration of autoantibodies that bind to β4 integrin in the serum of a patient by at least 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, or 50-fold. In one embodiment, a therapeutically effective amount of a therapeutic agent for OCP reduces the concentration of autoantibodies that bind to the β4 integrin in the serum of a patient by 1.5- fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13- fold, 14-fold, 15-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more. In one embodiment, a therapeutically effective amount of a therapeutic agent for OCP reduces the concentration of autoantibodies that bind to β4 integrin in the serum of a patient by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater. In one embodiment, a therapeutically effective amount of a therapeutic agent for OCP reduces the concentration of autoantibodies that bind to β4 integrin in the serum of a patient to 100 nM or less, optionally 80 nM or less, optionally 50 n or less, optionally 10 nM or less. In one embodiment, a therapeutically effective amount of a therapeutic agent for OCP reduces the concentration of autoantibodies that bind to β4 integrin in the serum of a patient to less than 100 ng/mL (e.g. , less than about 90 ng/mL, 50 ng/mL, 25 ng/mL, 10 ng/mL, 5 ng/mL, 1 ng/mL, 0.1 ng/mL, or 0.01 ng/mL).

[0104] One or more therapeutic agents can be administered in combination to a subject that is diagnosed as having treat OCP, as described herein. In one embodiment, more than one chemotherapeutic agent is administered in combination to a subject diagnosed as having OCP. In one embodiment, more than one immune suppressive agent is administered in combination to a subject diagnosed as having OCP. In one embodiment, a chemotherapeutic agent and an immune suppressive agent can be administered in combination to a subject diagnosed as having OCP. [0105] The term "combination" as in the phrase "a first agent in combination with a second agent" includes co-administration of a first agent and a second agent, which for example may be dissolved or intermixed in the same pharmaceutically acceptable carrier, or administration of a first agent, followed by the second agent, or administration of the second agent, followed by the first agent. The present invention, therefore, includes methods of combination therapeutic treatment and combination pharmaceutical compositions. OCP combination therapies may be executed step-wise by the same actor or by different actors. For example, one actor may administer to a subject a first agent and a second actor may administer to the subject a second agent. The administering steps may be executed at the same time, or nearly the same time, or at distant times.

[0106] 6. Kits for Determining the Autoantibody Concentration

[0107] The invention also provides kits for diagnosing OCP, and/or monitoring disease progression in a subject having OCP. These kits include means for determining the concentration of autoantibodies that bind to β4 integrin in a biological sample obtained from a subject and instructions for use of the kit.

[0108] The kits of the invention may optionally comprise additional components useful for performing the methods of the invention. By way of example, the kits may comprise means for obtaining a biological sample from a subject, a control sample, e.g., a sample from a subject having OCP and/or a subject not having OCP, one or more sample compartments, an instructional material which describes performance of a method of the invention and tissue specific controls/standards .

[0109] The means for determining the concentration of autoantibodies that bind to β4 integrin can include, for example, buffers or other reagents, such as detection reagents, for use in an assay for evaluating β4 integrin-binding autoantibody concentrations. The instructions can be, for example, printed instructions for performing the assay for detecting and quantifying the concentration of autoantibodies.

[0110] The kits of the invention can include, in one embodiment, an ELISA plate and a capture reagent, e.g., a capture antigen. In one embodiment, the capture antigen is bound to the ELIS plate, in another embodiment, the capture antigen is not bound to the ELISA plate. In this embodiment, the kit may optionally include instructions for binding the capture antigen to the ELISA plate. [0111] The capture antigen can include any β4 integrin polypeptide or β4 integrin polypeptide fragment described herein. For example, the capture antigen can comprise a β4 integrin polypeptide or β4 integrin polypeptide fragment that is at least 80%, e.g. , at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: l, or a fragment thereof. In one embodiment, the capture antigen can comprise a β4 integrin polypeptide or β4 integrin polypeptide fragment is at least 80%, e.g. , at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:3, or a fragment thereof. In another embodiment, the capture antigen can comprise a β4 integrin polypeptide or β4 integrin polypeptide fragment is at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:4, or a fragment thereof. In another

embodiment, the capture antigen can comprise a β4 integrin polypeptide or β4 integrin polypeptide fragment is at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:5, or a fragment thereof. In another embodiment, the capture antigen can comprise a β4 integrin polypeptide or β4 integrin polypeptide fragment is at least 80%, e.g. , at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:6, or a fragment thereof. In another embodiment, the capture antigen can comprise a β4 integrin polypeptide or β4 integrin polypeptide fragment is at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:7, or a fragment thereof.

[0112] In exemplary embodiments, kits of the invention include an ELISA plate useful for diagnosing OCP in a subject, wherein the ELISA plate is coated with at least one β4 integrin protein. In one embodiment, the ELISA plate is coated with full-length β4 integrin, comprised of SEQ ID NO: 1, or a fragment thereof. In one embodiment, the ELISA plate is coated with the cytoplasmic domain of β4 integrin, comprising SEQ ID NO: 3, or a fragment thereof. In one embodiment, the ELISA plate is coated with a β4 integrin fragment comprising at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In one embodiment, the ELISA plate is coated with a β4 integrin fragment comprising at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO: 3. In one embodiment, the ELISA plate is coated with a β4 integrin fragment comprising residues 1489- 1510 of β4 integrin (SEQ ID NO:4). In another embodiment, the ELISA plate is coated with a β4 integrin fragment comprising residues 1489- 1572 of β4 integrin (SEQ ID NO:5). In another embodiment, the ELISA plate is coated with a β4 integrin fragment comprising residues 1489- 1822 of β4 integrin (SEQ ID NO:6). In another embodiment, the ELISA plate is coated with a β4 integrin fragment comprising residues 1689- 1702 of β4 integrin (SEQ ID NO:7). **

[0113] An ELISA plate included in the kit may, in some embodiments, be coated with numerous different β4 integrin proteins, and fragments thereof, such that autoantibodies predictive of OCP may bind to some β4 integrin fragments, but not others. In some embodiments, the fragments overlap in amino acid sequence. In other embodiments, the fragments do not overlap in amino acid sequence. For example, an ELISA plate can be coated with fragments comprising various portions of the cytoplasmic domain, including, e.g. , amino acid residues 756 to 777 of SEQ ID NO: 3.

[0114] The kits of the invention can optionally contain reagents for detecting

autoantibodies bound to the capture antigen. Any such detection reagents described herein are suitable for inclusion in the kits of the invention.

[0115] The kits of the invention can optionally contain means for obtaining or isolating a biological sample (e.g. , blood, serum, or plasma) from a subject known or suspected of having OCP. The means for isolating a biological sample from a subject can comprise one or more reagents that can be used to obtain a fluid or tissue sample from a subject, such as means for obtaining a sample that contains circulating autoantibodies. The means for obtaining a biological sample from a subject may also comprise means for isolating a blood, blood-derived, plasma, or serum sample from a subject.

[0116] In one embodiment, a kit of the invention includes means for obtaining a biological sample from a subject, e.g., serum, means for determining the concentration of autoantibodies that bind to β4 integrin, e.g., the cytoplasmic domain of β4 integrin, and instructions for use of the kit.

[0117] The instructions may include suitable operational parameters in the form of a label or product insert. For example, the instructions may include information or directions regarding how to collect a sample, how to determine the concentration of autoantibodies in a sample, and/or how to correlate the autoantibody concentrations in a sample with the OCP status of a subject.

[0118] Preferably, the kits are designed for use with a human subject.

[0119] The present invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference in their entirety.

Examples

[0120] Example 1

[0121] ELISA tests were performed at the Massachusetts Eye Research and Surgery Institution (MERSI). An indirect ELISA method was used to test patient sera for the presence and concentration of autoantibodies indicative of OCP. Specifically, ELISA was used to determine the concentration of autoantibodies that bind to full-length β4 integrin (SEQ ID NO: 1) or to a segment of β4 integrin comprising an epitope localized within the cytoplasmic domain, spanning amino acids 1689-1702 of SEQ ID NO: 1, in serum samples obtained from test subjects. β4 integrin polypeptides, primary antibodies, and secondary antibodies were obtained from Abeam. The β4 integrin antigen and the antibodies were tested using different dilutions in order to build a standard curve (prior to serum testing) according to the following protocol.

[0122] The β4 integrin polypeptide was diluted to a final concentration of 20 μg/ml in PBS, and the wells of a multi-well plate were coated by pipetting 50 μΐ of the β4 integrin dilution into the top of each well. The plate was covered with an adhesive plastic and incubated at 4°C overnight. Then the plate was washed three times by filling the wells with 200 μΐ PBS. After that, the coated wells were blocked by adding 200 μΐ blocking buffer (5% serum in /PBS). Next, the plate was covered with an adhesive plastic and was incubated overnight at 4°C. The plate was then washed twice with PBS. 100 μΐ of serial dilutions of primary antibody was added to the plate, and the plate was covered with an adhesive plastic and incubated for 2 hours at room temperature. After this incubation, the plate was washed four times with PBS. 100 μΐ of conjugated secondary antibody, diluted according to manufacturer instructions in blocking buffer immediately before use, was applied to each well. The plate was covered with an adhesive plastic and incubated for 1 to 2 hours at room temperature. The plate was then washed 4 times with PBS.

[0123] A detection step was conducted by adding 100 μΐ of the substrate solution to each well with a multichannel pipet or a multi-pipet. After sufficient color development, 100 μΐ of stop solution was added, and the optical density (OD450) of each well was recorded and analyzed using a plate reader. [0124] FIG. 1 depicts exemplary standard curves produced during development of the ELISA.

[0125] The ELISA was used to test patient serum for the presence and concentration of autoantibodies that bind to β4 integrin. Results are provided in Table 1. A subset of patients were known or suspected to have OCP as a result of prior clinical diagnosis. Some of these patients had received or were receiving therapeutic treatment for the disease. In addition, a subset of patients were also known to not have OCP, and therefore served as negative controls for the ELISA test.

[0126] As depicted in Table 1, sera from patients having OCP, and in particular, OCP that was not in remission, exhibited ELISA OD450 values of 1.19525 and above, while the sera from patients without OCP exhibited ELISA OD450 values of 0.32315 or less. In some cases, the ELISA was performed on samples collected from a patient before and after treatment with an OCP therapeutic. For example, an ELISA was performed on 2 samples collected from patient number 6 (sample number 6A and 6B in Table 1), where patient sample 6A was collected before treatment, and patient sample 6B was collected after treatment. In this case, treatment with an OCP therapeutic (CellCept) resulted in a drop in the OD450 reading relative to the OD450 reading of the sample collected before treatment, indicating that the serum concentration of β4 integrin-binding autoantibodies decreased following treatment.

[0127] Table 1: Results of an ELISA detecting β4 integrin-binding autoantibodies in patient sera

A 3/20/2007 OCP relapse None (prior 0.15125 Negative

CellCept

treatment)

B 12/17/2014 OCP IV-Ig, rituxan, 0.15125 Negative remission CellCept

6/4/2015 OCP Methotrexate 0.15075 Negative remission (treatment

completed)

2005 None IV-Ig 0.32315 Negative

(negative

control)

0 12/13/2014 None None 0.21475 Negative

(negative

control)

1 1/5/2015 None None 0.24235 Negative

(negative

control)

2 1/29/2015 None None 0.18205 Negative

(negative

control)

3 1/29/2015 None None 0.26195 Negative

(negative

control)

4 1/29/2015 None None 0.20175 Negative

(negative

control)

5 5/6/2015 None None 0.27405 Negative

(negative

control)

6 6/19/2015 None None 0.13865 Negative

(negative

control)

6 12/3/2015 Unknown IV-Ig, 0.17545 Negative

CellCept

7 4/25/2008 None IV-Ig 0.24765 Negative8 4/25/2008 None None 0.19835 Negative

(negative

control)

9 1/12/2009 None None 0.23845 Negative

(negative

control)

Claims

CLAIMS We claim:

1. A method of diagnosing Ocular Cicatricial Pemphigoid (OCP) in a subject, comprising:

determining the concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin in a plasma or serum sample from the subject, wherein an increase in the concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin relative to that in a normal subject is indicative of OCP in the subject.

2. The method of claim 1, wherein the concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin is determined using an ELISA assay.

3. The method of claim 1, wherein an increase in the concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin of 2-fold or more relative to a normal subject is indicative of OCP.

4. The method of claim 1, wherein a concentration of autoantibodies that specifically bind to the cytoplasmic domain of β4 integrin of 100 ng/mL or more is indicative of OCP in the subject.

5. The method of claim 1, wherein the autoantibodies are detected using a β4 integrin fragment comprising at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO: 3.

6. The method of claim 1, wherein the autoantibodies are selected from the group consisting of IgG, IgM, IgA, or combinations thereof.

7. The method of claim 1, further comprising administering to the subject a

therapeutically effective amount of an agent for treating OCP.

8. The method of claim 6, wherein the agent is a chemotherapeutic agent or an immune suppressive agent, or a combination thereof.

9. The method of claim 6, wherein the agent is mitomycin C, methotrexate, mycophenolate mofetil, cyclophosphamide, rituximab, intravenous immunoglobulin (IVIg), azathioprine, a TNF-a inhibitor, cyclosporine, prednisone, diammodiphenylsulfone, or any combination thereof.

10. A method of diagnosing Ocular Cicatricial Pemphigoid (OCP) in a subject, comprising:

contacting a plasma or serum sample from the subject with a β4 integrin peptide bound to a solid support;

incubating the plasma or serum sample with the solid support for a period of time sufficient to allow antibodies in the plasma or serum sample to specifically bind the β4 integrin peptide;

washing the solid support to remove components of the plasma or serum sample that do not specifically bind the β4 integrin peptide;

contacting the solid support with a detection antibody, or antigen-binding fragment thereof, that specifically binds the antibodies in the plasma or serum sample bound to the β4 integrin peptide, wherein the detection antibody is coupled to a detectable label;

determining the level of the antibodies in the plasma or serum sample that specifically bind the β4 integrin peptide; and

comparing the level of the antibodies in the plasma or serum sample that specifically bind the β4 integrin peptide to a suitable control, wherein the suitable control is indicative of the level of antibodies that specifically bind the β4 integrin peptide in a plasma or serum sample from a normal subject;

wherein an increase in the level of antibodies that specifically bind the β4 integrin peptide in the plasma or serum sample relative to the suitable control indicates that the subject has OCP.

11. The method of claim 10, wherein the β4 integrin peptide comprises amino acid residues 1489-1510 of human β4 integrin (SEQ ID NO:4).

12. The method of claim 10, wherein the β4 integrin peptide comprises amino acid residues 1489-1572 of human β4 integrin (SEQ ID NO:5).

13. The method of claim 10, wherein the β4 integrin peptide comprises amino acid residues 1489-1822 of human β4 integrin (SEQ ID NO:6).

14. The method of claim 10, wherein the β4 integrin peptide comprises amino acid residues 1689-1702 of human β4 integrin (SEQ ID NO:7).

15. The method of claim 10, wherein the β4 integrin peptide comprises SEQ ID NO: l.

16. The method of any one of claims 1-15, wherein the detection antibody specifically binds the constant region of one or more immunoglobulins selected from the group consisting of lgG, IgM, IgA, IgD, and IgE.

17. The method of claim 16, wherein the detection antibody specifically binds the constant region of an IgG immunoglobulin and/or the constant region of an IgM

immunoglobulin.

18. The method of any one of claims 10-17, wherein the solid support is a microtiter plate.

19. The method of any one of claims 10-18, wherein the detectable label is an enzymatic label, a fluorophore label, a chromophore label, or a radio label.

20. The method of any one of claims 10-19, further comprising administering to the subject a therapeutically effective amount of an agent for treating OCP.

21. The method of claim 20, wherein the agent is a chemotherapeutic agent or an immune suppressive agent, or a combination thereof.

22. The method of claim 21, wherein the agent is mitomycin C, methotrexate, mycophenolate mofetil, cyclophosphamide, rituximab, intravenous immunoglobulin (IVIg), azatbioprine, a T F- inhibitor, cyclosporine, prednisone, diammodiphenylsulfone, or any combination thereof.

The method of any one of claims 1-22, wherein the sample is a serum sample

24. The method of any one of claims 1-22, wherein the sample is a plasma sample.

25. A method of treating Ocular Cicatricial Pemphigoid (OCP) in a subject comprising: selecting a subject having an increased plasma or serum concentration of

autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin relative to that in a normal subject; and administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an OCP therapeutic,

thereby treating OCP in the subject.

26. The method of claim 25, wherein the plasma or serum concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin is determined using an ELISA assay.

27. The method of claim 25, wherein a subject having a plasma or serum concentration of autoantibodies that specifically bind to the cytoplasmic domain of β4 integrin of 100 ng/mL or more is selected for treatment with the OCP therapeutic.

28. The method of claim 25, wherein the autoantibodies specifically bind a β4 integrin peptide fragment comprising at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO: 3.

29. The method of claim 25, wherein the OCP therapeutic is selected from the group consisting of mitomycin C, methotrexate, mycophenolate mofetii, cyclophosphamide, rituximab, intravenous immunoglobulin (IVIg), azatliioprine, a TNF-a inhibitor,

cyclosporine, prednisone, diaminodiphenylsulfone, or any combination thereof.

30. A method of monitoring the course of Ocular Cicatricial Pemphigoid (OCP) in a subject, comprising:

determining a concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin in a first plasma or serum sample obtained from the subject prior to treatment with an OCP therapeutic; and determining a concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin in a second plasma or serum sample obtained from the subject during or after treatment with the OCP therapeutic;

wherein a decrease in the concentration of autoantibodies that specifically bind the cytoplasmic domain of β4 integrin in the second plasma or serum sample relative to the first plasma or serum sample indicates that the subject is responding to treatment with the OCP therapeutic.

31. The method of claim 30, wherein the decrease in the concentration of autoantibodies that specifically bind the cytoplasmic domain of β4 integrin is a decrease of at least 2-fold.

32. The method of claim 30, wherein a concentration of 100 ng/mL or less in the second plasma or serum sample obtained during or after treatment with the OCP therapeutic indicates that the subject has entered remission from OCP.

33. The method of claim 30, wherein the concentration of autoantibodies that specifically bind the cytoplasmic domain of β4 integrin is determined using an ELISA assay.

34. A method of diagnosing Ocular Cicatricial Pemphigoid (OCP) in a subject, comprising:

performing an ELISA assay to determine the concentration of autoantibodies that bind to the cytoplasmic domain of β4 integrin in a plasma or serum sample from the subject, wherein a concentration of 100 ng/mL or more is indicative of OCP in the subject.

35. The method of claim 34, wherein the autoantibodies are detected using a β4 integrin fragment comprising at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO: 3.

36. The method of claim 1, wherein an increase in the concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin of 5-fold or more relative to a normal subject is indicative of OCP.

37. The method of claim 1, wherein an increase in the concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin of 10-fold or more relative to a normal subject is indicative of OCP.

38. The method of claim 1, wherein an increase in the concentration of autoantibodies that bind specifically to the cytoplasmic domain of β4 integrin of 100-fold or more relative to a normal subject is indicative of OCP.

39. The method of claim 1, wherein a concentration of autoantibodies that specifically bind to the cytoplasmic domain of β4 integrin of 200 ng/mL or more is indicative of OCP in the subject.

40. The method of claim 1, wherein a concentration of autoantibodies that specifically bind to the cytoplasmic domain of β4 integrin of 500 ng/mL or more is indicative of OCP in the subject.

41. The method of claim 1, wherein a concentration of autoantibodies that specifically bind to the cytoplasmic domain of β4 integrin of 1000 ng/mL or more is indicative of OCP in the subject.