Treatment and diagnosis of autoantibody-mediated eye diseases

Abstract

The present disclosure provides ophthalmic formulations comprising one or more pharmaceutically acceptable excipients and a pharmaceutically active compound capable of reducing the amount or adverse effects of autoantibodies, such as IgG, on the ocular surface. In particular, the present disclosure provides ophthalmic formulations capable of treating a clinical condition selected from the group consisting of inflammatory, infectious, and immunological ocular surface or intraocular diseases that can cause symptoms of ocular discomfort, keratitis, dry eye disease, symblepheron formation, fornix foreshortening, eyelid margin / conjunctival keratosis, subconjunctival fibrosis, retinal gliosis, and glaucoma.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 757,641, filed November 8, 2018, and U.S. Provisional Patent Application No. 62 / 855,253, filed May 31, 2019, the entire contents of which are hereby incorporated by reference in their entireties.

[0002] Statement regarding federally funded research This disclosure was made with U.S. Government support under Grant Number R01EY024966 awarded by the National Eye Institute (NEI) and the National Institutes of Health (NIH). The U.S. Government has certain rights in this disclosure.

[0003] The present disclosure relates to ophthalmic formulations that can reduce the concentration or deleterious effects of autoantibodies on the ocular surface and / or within the eye to prevent or treat inflammatory, immunological, allergic, infectious, and traumatic ocular surface and / or intraocular eye diseases. [Background technology]

[0004] Inflammatory, immunological, allergic, infectious, and traumatic ocular surface and intraocular eye diseases can cause ocular discomfort, ocular redness, dry eye syndrome, conjunctivitis, keratitis, symblepheron formation, fornix foreshortening, marginal / conjunctival keratitis, and subconjunctival fibrosis. More specific types of ocular surface diseases include ocular graft-versus-host disease (oGVHD), Steven Johnson syndrome, and others. syndrome), Ocular Cicatricial Penphygoid (OCP), Mild, Moderate and Severe Aqueous Deficiency Dry Eye Disease (DED), Meybomian Gland Disease, Ocular Rosacea, Stye, Blepharitis, Superior Limbal Keratoconjunctivitis (SLK), Aqueous Sufficiency DED, Floppy Eyelid Syndrome, Neurotrophic Eye Disease, Symptom-Sign Disconnection (Discordant DED), Neuropathic Pain, Thyroid Eye Disease (Grave Ophthalmopathy), Rheumatoid Arthritis-Related Eye Disease, Lupus-Related Eye Disease, Sjogren's Syndrome, Secondary Symptoms Inflammatory, infectious or immunological eye diseases include Egren's syndrome, ocular rosacea, allergic keratoconjunctivitis (vernal), viral keratoconjunctivitis (adenovirus EKC, herpes virus), Thygeson's keratitis, aniridia, keratitis or post-operative / post-traumatic eye conditions, peripheral ulcerative keratitis, keratitis, episcleritis, scleritis, retinal gliosis, uveitis (anterior or posterior) and glaucoma (open angle or angle closure or normal tension).

[0005] Conventional treatments for some of these ocular surface diseases, particularly dry eye syndrome, include (i) instillation of artificial tears to replenish and stimulate tears, and (ii) the use of anti-inflammatory drugs to reduce inflammation of the ocular surface. In general, current dry eye treatments include topical application of artificial tear products / lubricants, tear pool management, stimulation of tear secretion, topical application of antibiotics (e.g., erythromycin or bacitracin ointment), oral administration of tetracyclines (e.g., tetracycline, doxycycline, or minocycline), application of anti-inflammatory compounds and corticosteroids. Conventional treatments for some intraocular diseases include (i) treatments to reduce intraocular pressure and (ii) intraocular steroid injections. These treatments are often time-consuming, frustrating, and often ineffective or of variable effectiveness. Furthermore, while conventional treatments are effective in reducing the symptoms of dry eye syndrome to some extent, they have many undesirable side effects, such as burning and stinging sensations. Reducing local side effects and increasing patient comfort and response to treatment are among the goals of this disclosure.

[0006] Another drawback of conventional therapies in treating ocular surface disease is the inconsistent uptake of active pharmaceutical ingredients into corneal cells and their residence time in the cornea to effectively treat ocular surface disease syndromes without continuous application. Although ointment or cream formulations may allow for a longer residence time, such formulations may not disrupt the stratum corneum (the superficial keratinized layer of the skin) and may not reach the blood vessels and nerves present deeper in the eyelid tissue.

[0007] Yet another problem associated with conventional treatments is that the underlying cause is not directly addressed. Discovering the cause of ocular surface disease would allow for more effective treatment. Conventional treatments, for the most part, only treat the symptoms of ocular surface disease.

[0008] Thus, there is a continuing need for diagnostic kits, compositions, and methods for the effective treatment of clinical conditions associated with ocular surface and intraocular disease. Further, there is a need to address the underlying causes of ocular surface and intraocular disease. Summary of the Invention [Means for solving the problem]

[0009] Some embodiments of the present disclosure provide ophthalmic formulations comprising (a) one or more pharma- ceutically acceptable ophthalmic excipients, and (b) immunoglobulin G (IgG) or a fragment thereof.

[0010] Some embodiments of the present disclosure provide ophthalmic formulations comprising (a) one or more pharma- ceutically acceptable ophthalmic excipients and (b) pooled human immunoglobulin G (IgG) derived from pooled plasma.

[0011] Additionally, the present disclosure provides an ophthalmic formulation comprising (a) one or more pharma- ceutically acceptable ophthalmic excipients, and (b) an ophthalmic pharma- ceutically active compound for the treatment of a clinical condition selected from the group consisting of inflammatory, infectious, immunological, allergic, and / or traumatic ocular surface or intraocular diseases, wherein the pharma- ceutically active compound comprises IgG.

[0012] In various embodiments, the present disclosure provides ophthalmic formulations comprising pharma- ceutically active compounds capable of reducing the amount of autoantibodies on or within the eye, such autoantibodies being generated in response to citrullinated proteins (herein referred to as "ACPA autoantibodies", which are generated in response to citrullination of proteins), or autoantibodies being generated in response to homocitrullinated proteins (herein referred to as "anti-CarP antibodies", which are generated in response to carbamylation of proteins), or native autoantibodies being generated in response to native proteins. Citrullination and carbamylation are two types of post-translational modifications (PTMs) of proteins. In related embodiments, the pharma-ceutically active compounds comprise immunoglobulin G (IgG) or fragments thereof. These ophthalmic formulations may also comprise one or more pharma- ceutically acceptable ophthalmic excipients.

[0013] In various embodiments, the disclosed ophthalmic formulations include one or more of the following pharma- ceutically acceptable ophthalmic excipients. For example, pharma-ceutically acceptable ophthalmic excipients include cyclodextrin, carbopol or carbomer or acrylic acid polymers, poloxamer, xyloglucan, methylcellulose, hydroxypropylmethylcellulose, ethyl(hydroxyethyl)cellulose, pseudolatex, cellulose acetate phthalate, gellan gum, alginate, carrageenan, hyaluronic acid, sodium acetate, disodium edetate, hypromellose, acetic acid, alcohol, alginic acid, Amerchol-cab (Amerchol-cab), glycerol, ... b), antipyrine, benzalkonium chloride, benzododecinium bromide, boric acid, caffeine, calcium chloride, carbomer 1342, carbomer 934P, carbomer 940, carbomer homopolymer type B (allyl pentaerythritol crosslinked), sodium carboxymethylcellulose, castor oil, cetyl alcohol, chlorobutanol, citric acid, citric acid monohydrate, creatinine, divinylbenzenestyrene copolymer, ethylene vinyl acetate copolymer, gellan gum (low acyl), glycerin, glyceryl stearate Lyceryl, hypromellose, lanolin, lauralkonium chloride, lauroyl sarcosine, magnesium chloride, methylparaben, mineral oil, nonoxynol-9, octoxynol-40, petrolatum, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric nitrate, polydonium chloride, poloxamer 188 or 407, polycarbophil, polyethylene glycol 400 or 8000, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 40 stearate, polypropylene glycol Cole, polysorbate 20, polyvinyl alcohol, potassium chloride, potassium sorbate, povidone K29 / 32, povidone K30, povidone K90, povidone, propylene glycol, propylparaben, soda ash, sodium acetate, sodium bisulfate, sodium borate, sodium borate decahydrate, sodium carbonate, sodium chloride, sodium citrate, sodium metabisulfate, sodium nitrate, sodium sulfate, sodium sulfite, sodium thiosulfate, sorbic acid, sorbitol, stabilized oxychloro complex, sulfuric acid,Thimerosal, titanium dioxide, tocophersolan, trisodium citrate dihydrate, tromethamine, tyloxapol, xanthan gum, zinc chloride, or combinations thereof.

[0014] Yet another aspect of the present disclosure provides an ophthalmic formulation comprising: (a) a pharma- ceutically acceptable ophthalmic excipient; (b) a therapeutically effective amount of a pharma- ceutically active compound comprising pooled human immunoglobulin G (IgG); and / or (c) a therapeutically effective amount of pooled human plasma proteins, pooled human plasma lipids, or a combination thereof. As used herein, the term "therapeutically effective" includes having an immunomodulatory effect or the ability to neutralize autoantibodies.

[0015] In any of the disclosed ophthalmic formulations, the pharma- ceutically active compound may include autologous IgG purified from autologous plasma / serum, multimerized IgG1Fc molecules, IgG1Fc hexamers, IgG2a Fc multimers, stradomers, multivalent Fc structures, glycoengineered sialylated IgG, IgG-Fc glycosylation, synthetic IgG and fragments thereof, or combinations thereof.

[0016] In various embodiments, any of the disclosed ophthalmic formulations may be administered with or without the use of any of the following agents: anti-inflammatory agents such as steroids, methylprednisone, prednisone, dexamethasone, cyclosporine, the nonsteroidal anti-inflammatory drug Lifitegrast®; mucolytic agents such as N-acetylcysteine, nasylcysteine, N-acetylglucosamine; PAD enzyme inhibitors (paclitaxel, glucocorticoids, or Cl-amidine) or NET degraders (DNase or heparin); targeted Fab antibody fragments; Fc receptor blocking peptides; Fc receptor blocking antibodies; recombinant peptides containing pathogenic epitopes; traditional synthetic DMARDs (methotrexate, leflunomide / teriflunomide, sucrose ... The therapeutic agent may further comprise a second pharmacokinetically active compound selected from a medicament for treating rheumatoid arthritis (rheumatoid arthritis), ...

[0017] In some embodiments, the concentration or amount of IgG present in the ophthalmic formulation is, for example, in the range of about 0.01 mg / mL to about 1 g / mL (1000 mg / mL), or about 0.05 mg / mL to about 1 g / mL, or about 0.1 mg / mL to about 1 g / mL, or about 0.1 mg / mL to about 0.5 mg / mL, or 0.05 mg / mL to about 0.5 mg / mL, or 0.01 mg / mL to about 0.1 mg / mL. In certain embodiments, the concentration or amount of IgG may be about 0.01 mg / mL, and in other embodiments, the concentration or amount of IgG may be about 0.05 mg / mL. In certain embodiments, the concentration or amount of IgG may be about 1 mg / mL, and in other embodiments, the concentration or amount of IgG may be about 10 mg / mL.

[0018] In various embodiments, the concentration or amount of IgG can be about 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 350 mg / mL, 400 mg / mL, 450 mg / mL, 500 mg / mL, 550 mg / mL, 600 mg / mL, 650 mg / mL, 700 mg / mL, 750 mg / mL, 800 mg / mL, 850 mg / mL, 900 mg / mL, 950 mg / mL, or 1000 mg / mL.

[0019] In various exemplary embodiments, the amount of IgG is about 10 mg / mL or less. In certain embodiments, the ophthalmic formulation can include a concentration or amount of IgG that is about 0.1 mg / mL or less, about 0.15 mg / mL or less, 0.2 mg / mL, about 0.25 mg / mL or less, about 0.3 mg / mL or less, about 0.35 mg / mL or less, about 0.4 mg / mL or less, about 0.45 mg / mL or less, about 0.5 mg / mL or less, about 0.55 mg / mL or less, about 0.6 mg / mL or less, about 0.65 mg / mL or less, about 0.7 mg / mL or less, about 0.75 mg / mL or less, about 0.8 mg / mL or less, about 0.85 mg / mL or less, about 0.9 mg / mL or less, about 0.95 mg / mL or less, or about 1.0 mg / mL or less.

[0020] The term "pooled human immunoglobulin G from pooled plasma" refers to immunoglobulin G isolated by subsequent fractionation of unfractionated plasma pooled from several donors. The present disclosure provides an ophthalmic composition comprising pooled plasma from several donors that is dispensed unfractionated into single-use droppers or multi-dose bottles at various dilutions based on IgG concentration (0.01% to 10%). Pooling of plasma can also be performed prior to the processing step. This pooled plasma and immunoglobulin (PPIG) preparation differs from conventional plasma products (e.g., PRP) in that conventional plasma products are prepared from the blood of one subject, whereas PPIG preparations are made from pooled plasma of hundreds to thousands of healthy subjects. The final product contains pooled IgG and plasma proteins with or without platelets and platelet activation products. Pooled plasma-derived IgG may also contain plasma lipids.

[0021] In yet another iteration, pooled human immunoglobulin G (at concentrations of 0.01% to 10%) derived from pooled plasma from hundreds to thousands of healthy subjects is formulated into plasma or serum (allogeneic or autologous) with ophthalmic excipients. This ophthalmic formulation provides a combination of pooled IgG neutralizing autoantibodies and ocular surface regeneration promoting serum / plasma proteins, cytokines, and growth factors.

[0022] In various embodiments, the IgG present in any of the disclosed ophthalmic formulations comprises serum / plasma derived pooled immunoglobulin G (OSIG: ocular surface immunoglobulin), multimerized IgG1 Fc molecules (IgG1 Fc hexamers), IgG2a Fc multimers (stradomers), multivalent Fc structures, or combinations thereof.

[0023] In various embodiments, the IgG present in any of the disclosed ophthalmic formulations is an antigen-binding fragment in which one or more antibody domains are truncated or absent, an engineered antibody or protein-binding fragment thereof, a single chain antibody, or an antibody capable of binding to more than one epitope or capable of binding to one or more different antigens. For example, the fragment of IgG is an antigen-binding fragment, a single chain antibody, an Fv fragment, a Fab fragment, a Fab' fragment, or a F(ab)2 fragment.

[0024] In various embodiments, the IgG present in any of the disclosed ophthalmic formulations comprises IgG1, IgG2, IgG3, and IgG4, or a combination thereof. Additionally or alternatively, the IgG may comprise or be derived from any suitable source, such as an IgG-containing stem cell preparation, manufactured IgG or IgG stem cells, glycoengineered sialylated IgG, IgG-Fc glycosylation, and the like, or a combination thereof.

[0025] In various embodiments, the IgG present in any of the disclosed ophthalmic formulations comprises a preselected concentrate of autologous IgG purified from autologous plasma / serum using a separation process such as affinity chromatography using Protein A beads or another IgG separation process.

[0026] In various embodiments, the IgG present in any of the disclosed ophthalmic formulations neutralizes autoimmune antibodies or antibodies that specifically bind to citrylated proteins.

[0027] In various embodiments, the pH of any of the disclosed ophthalmic formulations is about pH 6 to about pH 8, or about pH 6.2 to about pH 7.2, about pH 6.4 to about pH 7.4, or about pH 6.5 to about pH 7.5, about pH 6.6 to about pH 7.6, or about pH 6.8 to about pH 7.8. For example, the pH is about 6.0, or about 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6, or about 6.7, or about 6.8, or about 6.9, or about 7.0, or about 7.1, or about 7.2, or about 7.3, or about 7.4, or about 7.5, or about 7.6, or about 7.7, or about 7.8, or about 7.9, or about 8.0.

[0028] In various embodiments, any of the disclosed ophthalmic formulations comprise one or more pharma- ceutically acceptable excipients including polyvinyl alcohol, povidone, hydroxypropyl methylcellulose, poloxamer, polyol, carbopol, Pluronic®, carbomer, carboxymethylcellulose, hydroxyethylcellulose, cyclodextrin, phosphate buffer, citrate buffer, Tris buffer, sodium chloride, potassium chloride, polysorbate 80, vegetable oil, a preservative, or a combination thereof.

[0029] Any of the disclosed ophthalmic formulations can be used to treat inflammatory, infectious, or immune diseases at the mucosa or to exhibit an immunomodulatory effect at the mucosa. The ophthalmic formulations may treat inflammatory, infectious, or immune diseases or have an immunomodulatory effect at the mucosa of the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and cavities, synovial (joint) cavities, vaginal cavity, or on the skin.

[0030] In various embodiments, the disclosed ophthalmic formulations comprise multi-dose vials with preservatives or single-dose sterile containers without preservatives.

[0031] In various embodiments, the present disclosure provides a method of treating a clinical condition in a patient in need thereof, comprising administering to the patient an ophthalmic formulation as disclosed herein, the clinical condition being an inflammatory ocular surface or intraocular ocular disease, an infectious ocular surface or intraocular ocular disease, and / or an immunological ocular surface or intraocular ocular disease. In various embodiments, the present disclosure provides a method of treating an inflammatory, infectious, and / or immunological ocular disease in a patient in need thereof, comprising administering to the patient an ophthalmic formulation as disclosed herein.

[0032] In various embodiments, the present disclosure provides a method for alleviating, alleviating, or preventing ocular discomfort in a patient suffering from a clinical condition, comprising administering to the patient an ophthalmic formulation disclosed herein, the clinical condition being an inflammatory ocular surface or intraocular disease, an infectious ocular surface or intraocular disease, and / or an immunological ocular surface or intraocular disease. In any of these methods, the ocular discomfort comprises one or more of foreign body sensation, photosensitivity, stinging, irritation, pain, dryness, burning, redness, itching, or tingling.

[0033] The present disclosure provides a method of treating a clinical condition in a patient in need thereof, the clinical condition being an inflammatory, infectious and / or immune disease of the mucous membranes of the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and cavities, synovial (joint) cavity, vaginal cavity, or on the skin, comprising administering to the patient an ophthalmic formulation as disclosed herein.

[0034] The present disclosure also provides a method of inducing an immunomodulatory effect in a mucosa of a patient in need thereof, the mucosa being on the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and cavities, synovial (joint) cavities, vaginal cavity or skin, comprising administering to the patient an ophthalmic formulation as disclosed herein.

[0035] The present disclosure also provides the use of any ophthalmic formulation for preparing a medicament for treating a clinical condition in a patient's need, wherein the clinical condition is an inflammatory or intraocular ocular disease, an infectious or intraocular ocular disease, and / or an immunological or intraocular ocular disease.In various embodiments, the present disclosure provides the use of any ophthalmic formulation for preparing a medicament for reducing, alleviating, or preventing the ocular discomfort of a patient suffering from a clinical condition, wherein the clinical condition is an inflammatory or intraocular ocular disease, an infectious or intraocular ocular disease, and / or an immunological or intraocular ocular disease.In any of these applications, the ocular discomfort comprises one or more of foreign body sensation, pain, photosensitivity, stinging, irritation, pain, dryness, burning, redness, itching, or stinging.

[0036] The present disclosure also provides the use of an ophthalmic formulation disclosed herein for the preparation of a medicament for the treatment of a clinical condition in a patient in need thereof, the clinical condition being an inflammatory, infectious and / or immune disease of the mucosa in the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and cavities, synovial (joint) cavity, vaginal cavity, or on the skin. Further, the present disclosure provides the use of an ophthalmic formulation disclosed herein for the preparation of a medicament for inducing an immunomodulatory effect in the mucosa in a patient in need thereof, the mucosa being on the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and cavities, synovial (joint) cavity, vaginal cavity, or on the skin.

[0037] The present disclosure also provides a composition comprising any of the ophthalmic formulations described herein for use in treating clinical conditions.In various embodiments, the composition can be used to treat inflammatory, infectious, and / or immunological eye diseases in patients in need.In various embodiments, the composition comprising any of the ophthalmic formulations described herein can be used to reduce, alleviate, or prevent eye discomfort in patients suffering from clinical conditions, where the clinical conditions are inflammatory ocular surface or intraocular eye diseases, infectious ocular surface or intraocular eye diseases, and / or immunological ocular surface or intraocular eye diseases.In related embodiments, the eye discomfort comprises one or more of foreign body sensation, pain, photosensitivity, stinging, irritation, pain, burning, dryness, redness, itching, or stinging.

[0038] The present disclosure provides compositions for treating a clinical condition in a patient in need thereof, the composition comprising an ophthalmic formulation as disclosed herein, the clinical condition being an inflammatory, infectious and / or immune disease in the mucosa of the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and cavities, synovial (joint) cavities, vaginal cavity, or on the skin. Additionally, the present disclosure provides compositions for inducing an immunomodulatory effect in the mucosa of a patient in need thereof, the composition comprising an ophthalmic formulation as disclosed herein, the mucosa being in the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and cavities, synovial (joint) cavities, vaginal cavity, or on the skin.

[0039] Exemplary clinical conditions treatable in accordance with the exemplary methods, uses, and compositions herein include, but are not limited to, immune-mediated eye disease, metabolic eye disease, allergic eye disease, traumatic eye disease, infectious eye disease, and genetic eye disease, such as, for example, ocular graft-versus-host disease (oGVHD), Stevens-Johnson syndrome, ocular cicatricial pemphigoid (OCP), mild, moderate, and severe aqueous deficient dry eye disease (DED), Meybomian gland disease, styes, ocular rosacea, blepharitis, superior limbic keratoconjunctivitis (SLK), aqueous sufficient DED, floppy eyelid syndrome, neurotrophic eye disease, symptom-symptom amputation (discordant DED), neuroendocrinopathy, ... These conditions include, but are not limited to, myopathic pain, thyroid eye disease (Graves' ophthalmopathy), rheumatoid arthritis associated eye disease, lupus associated eye disease, Sjogren's syndrome, secondary Sjogren's syndrome, ocular rosacea, allergic keratoconjunctivitis (vernal), viral keratoconjunctivitis (adenovirus EKC, herpes virus), Thygeson's keratitis, retinal gliosis, viral keratoconjunctivitis (adenovirus EKC, herpes virus), Thygeson's keratitis, aniridia, keratitis or post-operative / post-traumatic eye conditions, peripheral ulcerative keratitis, keratitis, episcleritis, scleritis, uveitis (anterior or posterior), and glaucoma (open angle or narrow angle or normal tension). For example, the exemplary methods herein can be used to treat diseases resulting from a variety of conditions, such as keratitis due to sterile inflammation (e.g., PUK) or due to viral (e.g., adenoviral subepithelial infiltration, herpes virus dendritic epithelial ulcer), bacterial (e.g., staphylococcus, pseudomonas) or fungal (e.g., candida, acanthamoeba) infection. Additionally, the exemplary methods herein can be used to treat post-operative / post-traumatic ocular conditions or ocular conditions associated with ocular surface reconstruction surgery, application of metabolites to the ocular surface, pterygium surgery, glaucoma surgery, cataract surgery, refractive surgery (LASIK, LASEK, or PRK), keratoprosthetic surgery, vitreoretinal surgery, or radiation or chemical (alkaline or acidic) or traumatic injury.

[0040] In various embodiments, the patient has an autoantibody in the biological sample. In a related embodiment, the autoantibody present in the biological sample is an anti-citrullinated protein antibody. In a particular embodiment, the biological sample is an ocular fluid. In various embodiments, the ocular fluid is tears, eyewash, anterior aqueous humor or posterior vitreous humor.

[0041] The present disclosure provides a method for treating an ocular disease comprising administering a therapeutically effective amount of an allogeneic or autologous IgG ophthalmic formulation to a patient in need of such treatment. The present disclosure also provides a use of a therapeutic amount of an allogeneic or autologous IgG ophthalmic formulation for the preparation of a medicament for the treatment of an ocular disease in a patient in need of such treatment. Additionally, the present disclosure provides a composition for treating an ocular disease comprising a therapeutically effective amount of an allogeneic or autologous IgG ophthalmic formulation to a patient in need of such treatment.

[0042] In any of the disclosed methods, uses, or compositions, the ophthalmic formulation is administered as an eye drop formulation, a topical liquid, a gel formulation, an emulsion formulation, a suspension formulation, an ointment formulation, or an injectable formulation. The ophthalmic formulation, agent, or composition can be administered to the subject at least once daily. In various embodiments, the IgG ophthalmic formulation is administered to the subject once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times daily. In embodiments, the IgG ophthalmic formulation is administered to the subject at least once every 1 to 3 weeks. In various embodiments, one or more doses of the IgG ophthalmic formulation are administered weekly, biweekly, monthly, or bimonthly. In various aspects, the concentration of IgG is defined by a 5%, 10%, or 20% Ocular Surface Immunoglobulin (OSIG) solution. In various embodiments, the concentration of IgG administered is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% OSIG solution. In yet another embodiment, the IgG ophthalmic formulation is administered intraocularly as an intraocular injection. In an embodiment, the IgG ophthalmic formulation is administered to the surface of the eye as eye drops or ointments.

[0043] In certain embodiments, pooled IgG ophthalmic preparations (modified or unmodified) are produced using blood from human subjects and then administered to the ocular surface or intraocularly in the same subjects at specific concentrations. Subconjunctival or sub-Tenon injections can be performed or OSIG preparations can be modified (using nanotechnology such as polymer / biological spheres, particles or membranes (with or without surface modification)) to allow delivery of OSIG throughout the tissue. Other methods to enhance penetration of OSIG include using penetrating agents such as benzalkonium, charge-based strategies such as iontophoresis, or increasing residence time at the ocular surface, such as by increasing viscosity or adsorption on contact lenses or punctal plugs. OSIG eye drops can be formulated as microemulsions. The contact time with the cornea and the bioavailability of OSIG eye drops can be increased by using highly polymerized IgG that does not diffuse through biological membranes and forms a three-dimensional network in water. Viscosity may be increased by using low molecular weight hydrophilic polymers to increase viscosity. Examples of such polymers include polyvinyl alcohol, poloxamer, hyaluronic acid, carbomer, carbopol, and polysaccharides, i.e., cellulose derivatives, gellan gum, and xanthan gum. Corneal and intraocular penetration of OSIG eye drops can be enhanced by modifying the continuity of the corneal epithelial structure using chelating agents, preservatives (such as benzalkonium chloride), surfactants, and bile salts, or by using cyclodextrin complexes. For intraocular delivery or to the cornea, subconjunctiva, or lacrimal gland, OSIG's polymeric, solid, multicompartment drug delivery systems are used.

[0044] Nanoparticles are polymeric carriers constructed from biodegradable, biocompatible, natural, or synthetic polymers, often with mucoadhesive properties. The components used in their development are poly(cyanoacrylate alkyl), polylactic acid, poly(epsilon-caprolactone), poly(lactic-co-glycolic acid), chitosan, gelatin, sodium alginate, and albumin, for the purpose of ocular application. These forms can be divided into nanospheres, solid monolithic spheres constructed from a dense polymer matrix in which OSIG is scattered, and nanocapsules that constitute a reservoir constructed from a polymer membrane that surrounds OSIG in solid or liquid form. Liposomes are also used to deliver OSIG throughout ocular tissues. Liposomes are phospholipid drug carriers that are usually composed of phosphatidylcholine, stearylamine, and varying amounts of cholesterol or lecithin with α-L-dipalmitoylphosphatidylcholine. Niosomes, discosomes, and dendrimers are also used to deliver OSIG throughout ocular tissues.

[0045] In another embodiment, the present disclosure provides a method for detecting the presence of autoantibodies in a subject, the method comprising detecting the level of autoantibodies in a sample of ocular fluid obtained from a subject, the subject suffering from or at risk of developing ocular surface disorder.In a related aspect, the method further comprises comparing the level of autoantibodies in a sample obtained from the subject with the level of autoantibodies of a control.For example, the ocular fluid is tear fluid, eyewash, anterior chamber aqueous humor or posterior chamber vitreous humor.

[0046] The present disclosure also provides a method for diagnosing, determining the risk of developing, or monitoring ocular surface disorder in a subject, comprising detecting the level of an autoantibody in a sample obtained from a subject, and comparing the level of the antibody in the sample obtained from the subject with a control level of the autoantibody, and the control level of the autoantibody is used to diagnose, determine the risk of developing, or monitor ocular surface disorder in the subject. In a related embodiment, the autoantibody is a native autoantibody, an anti-CarP autoantibody, or an ACPA antibody. In various embodiments, the autoantibody comprises multiple autoantibodies. In a related embodiment, the antigen for detecting the autoantibody comprises a citrullinated protein, a citrullinated peptide sequence, or a combination thereof. In a related embodiment, the antigen for detecting the autoantibody comprises a carbamylated protein, a carbamylated peptide sequence, or a combination thereof.

[0047] In any of the disclosed methods, the sample is an ocular fluid, which may be tears, eyewash, anterior chamber aqueous humor, or posterior chamber vitreous humor.

[0048] In any of the disclosed methods, the level of autoantibody is detected by enzymatic, spectroscopic, chromatographic, immunological, or combinations thereof. In an exemplary embodiment, the method can include determining the progression of ocular surface disorder, or determining the effectiveness of treatment in a subject suffering from, suspected of suffering from, or predisposed to ocular surface disorder. In any of the disclosed methods, the control level of autoantibody includes the level of said autoantibody in the subject before treatment begins, the level of said autoantibody in the subject at an early stage of treatment, or a combination thereof.

[0049] The present disclosure also provides diagnostic kits comprising a panel of antigens for detecting autoantibodies comprising citrullinated proteins, citrullinated peptide sequences, or combinations thereof, which can be used to carry out any of the methods described herein.

[0050] In an exemplary embodiment, the Fc receptor blocking composition can be formulated for ocular use. For example, the Fc receptor blocking composition can be further defined by an Fc receptor that blocks ocular peptide concentration, and optionally a suitable carrier. In another embodiment, the Fc receptor blocking composition can be further defined by an Fc receptor that blocks ocular antibody concentration, and optionally a suitable carrier. Furthermore, the Fc blocking composition can be used to treat inflammatory, infectious, or immune diseases in mucous membranes, or to exert immunomodulatory effects in mucous membranes. The Fc blocking composition can be used to treat inflammatory, infectious, or immune diseases in mucous membranes, or to exert immunomodulatory effects in mucous membranes in the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and cavities, synovial (joint) cavities, vaginal cavities, or on the skin.

[0051] The present disclosure also provides a concentrated ocular or mucosal IgG composition. In accordance with the principles of the present disclosure, a concentrated ocular IgG composition is provided. The composition may further include an anti-inflammatory agent.

[0052] The present disclosure also provides a therapeutic dose for the treatment of an ocular immune disease, which is indicated by a level of anti-citrullinated protein autoantibodies determined from ocular fluid removed from a treatment site, and the therapeutic dose can be formulated using a concentrated ocular IgG composition. In certain embodiments, the therapeutic dose can be determined based on tears removed from a subject when the subject suffers from an immune disease, such as dry eye disease. In certain embodiments, the therapeutic dose can include a composition further defined by a concentrated amount of ACPA antibodies and / or a concentration of the concentrated ocular IgG composition to reduce the effect via application at the treatment site. For example, the therapeutic dose can include a concentrated ocular IgG composition ranging from about 0.01 mg / mL to about 1 g / mL by weight.

[0053] In some embodiments, the diagnostic kit comprises a test device for determining the concentration of anti-citrullinated protein autoantibodies in an ocular fluid, such as tears, and a dosing device for indicating a therapeutic treatment dose and regimen sufficient to treat and / or reduce the damaging effects of anti-citrullinated protein autoantibody concentrations in the ocular fluid. In related aspects, the ocular fluid is tears, eyewash, anterior aqueous humor, or posterior vitreous humor.

[0054] In an exemplary embodiment, a method of creating a therapeutic dose for immune damage detected in ocular fluid removed from a treatment site includes providing a concentration of IgG configured to reduce the level and / or effect of anti-citrullinated protein autoantibodies when administered to the treatment site, and adding a carrier to the concentration of IgG, thereby forming a therapeutic dose configured to enable delivery of the IgG to the treatment site.

[0055] In another exemplary embodiment, the method of preparing a therapeutic dose may include providing an OSIG solution in the range of about 0.01% to about 20% ocular surface immunoglobulin (OSIG), or in the range of about 0.1% to about 0.5% OSIG, or in the range of about 0.4% to about 1% IgG. For example, an OSIG solution in the range of about 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% IgG. In one embodiment, the formulation was made using OSIG Phlebo Gamma 5% DIF to test its toxicity to human cells (Figure 19). A concentration of 4 mg / mL of Flebogamma 5% DIF is non-toxic to human epithelial cells and is therefore a suitable exemplary embodiment. OSIG solutions can be manufactured using commercially available IgG or pooled plasma IgG. Other embodiments formulated with non-toxic concentrations of OSIG solutions using commercially available preparations are also suitable examples according to the principles herein. In a related aspect, the method further comprises periodically delivering a therapeutic dose to the treatment site at least twice a day and twice a month, or any other suitable time, during the treatment period defined by the response detected by the diagnostic kit, or for a period to achieve improvement in immune or inflammatory conditions, or until the test device indicates that the concentration is within normal range.

[0056] In embodiments, the B cell targeted ocular composition can include a suitable B cell composition, such as rituximab, formulated as eye drops or other suitable delivery methods for treating ocular or other mucosal diseases. For example, the B cell targeted ocular composition is immunomodulatory in mucosa, such as the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and cavities, synovial (joint) cavities, vaginal cavity, or on the skin.

[0057] In addition to applying the compositions described herein to the eye, these IgG compositions can be applied to other sites, such as mucous membranes in the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and cavities, synovial (joint) cavities, vaginal cavities, and on the skin. The IgG compositions can be used to treat inflammatory and / or immune diseases at these mucosal sites. Specifically, the IgG compositions can be formulated for delivery to mucous membranes at various sites, such as the eye, oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and cavities, synovial (joint) cavities, vaginal cavities, and on the skin, to treat inflammatory and / or immune diseases at these mucosal sites.

[0058] In yet another exemplary embodiment, the B cell targeted therapy (rituximab, ofatumumab, belimumab, atacicept, tabalumab) may form a composition for treating immune mediated eye disease, metabolic eye disease, allergic eye disease, traumatic eye disease, infectious eye disease and genetic eye disease, including, for example, Stevens-Johnson syndrome, ocular cicatricial pemphigoid (OCP), mild, moderate and severe aqueous deficient dry eye disease (DED), Meybomian gland disease, superior limbic keratoconjunctivitis (SLK), aqueous sufficient DED, floppy eyelid syndrome, neurotrophic eye disease, and others. Inflammatory, infectious or immunological eye diseases, including, but not limited to, eye diseases, symptom-sign amputation (discordant DED), neuropathic pain, thyroid eye disease (Graves' eye disease), rheumatoid arthritis-related eye disease, lupus-related eye disease, Sjogren's syndrome, secondary Sjogren's syndrome, ocular rosacea, allergic keratoconjunctivitis (vernal), viral keratoconjunctivitis (adenovirus EKC, herpes virus), Thygeson's keratitis, retinal gliosis, aniridia, keratitis or postoperative / post-traumatic eye conditions, peripheral ulcerative keratitis, keratitis, episcleritis, scleritis, uveitis and glaucoma. For example, rituximab can be formulated as eye drops or other suitable delivery methods to treat eye diseases. [Brief description of the drawings]

[0059] [Figure 1] FIG. 1 is a box plot showing data regarding the presence of ACPAs in ocular surface irrigation fluids from healthy subjects. [Diagram 2] FIG. 1 is a box plot showing data regarding the presence of ACPA autoantibodies in ocular surface washes of several patients with ocular surface disease. [Diagram 3] Graph showing the percentage of all patients with specific ocular surface diseases who tested positive for ACPAs on ocular surface washes. [Figure 4] FIG. 1 is a box plot showing data on ACPA levels in tears of patients with Sjogren's syndrome before and after initiation of treatment. [Diagram 5] FIG. 1 is a box plot showing data on ACPA levels in tears of ocular GVHD patients before and after initiation of treatment. [Figure 6] Photographs of immunofluorescence staining of impression cytology specimens from the ocular surface showing the presence of citrullinated proteins in non-epithelial cells of patients with Sjögren's syndrome. Sjögren's-cit / k14 or cit / NE → N=5; healthy-cit / k14 → N=2. [Figure 7] Photographs of immunofluorescence staining of impression cytology specimens from the ocular surface showing the presence of citrullinated proteins in neutrophils from patients with Sjögren's syndrome. cit / NE→N=2. [Figure 8] Photographs of immunofluorescence staining of impression cytology specimens from the ocular surface showing the presence of citrullinated proteins in epithelial cells of patients with ocular GVHD (oGVHD). Def.oGVHD, cit / k14 → N = 4; No oGVHD, cit / k14 → N = 2. [Figure 9] Photographs of immunofluorescence staining of ocular surface mucoid films showing the presence of inducible Fc-γ receptors on neutrophils. Isolated neutrophils N=1; Pt mucoid N=2 [Figure 10] Photographs of immunofluorescence staining of impression cytology specimens from the ocular surface showing the presence of Fc-gamma receptors on non-epithelial cells (neutrophils and mononuclear cells). FcR+NE: N=5 different individuals, 4 different diseases. [Figure 11] Photograph of a dot blot analysis and graph showing that ACPAs in tears react with NET citrullinated proteins. [Figure 12]1 is a photograph of a dot blot analysis and a graph showing that ACPA-positive tears demonstrate autoantibody polyclonality. [Figure 13] FIG. 1 is a photograph of immunofluorescence staining of ocular surface mucoid film showing the presence of the citrullinated protein H4R3cit in the patient's mucosal cell aggregates. [Figure 14] Figure 1 shows a photograph of a mouse cornea showing ocular surface staining with fluorescein staining indicative of corneal epithelial disease. The data indicate that ACPA antibodies can cause corneal epithelial disease, although not all ACPA antibodies are capable of doing so. [Figure 15] Photographs of immunofluorescent staining of impression cytology specimens from the ocular surface of mouse corneas showing that ACPA antibody #1 causes NETosis and induces citrullination in mouse corneal epithelium. [Figure 16-1] Photographs of mouse corneas showing ocular surface staining with fluorescein staining indicative of corneal epithelial disease. Data indicate that ACPA-induced pathological effects on the cornea are mediated through Fc receptors. Mouse IgG competes with ACPA antibodies for binding to Fc receptors, and Fc receptor blockers block all Fc receptors. This competitive peptide blockade may abolish the pathological effects of ACPAs on the cornea, suggesting the use of methodologies to prevent ACPA antibody interaction with Fc receptors, which may be a potential therapeutic strategy in treating citrullination-associated eye diseases. [Figure 16-2] Photographs of mouse corneas showing ocular surface staining with fluorescein staining indicative of corneal epithelial disease. Data indicate that ACPA-induced pathological effects on the cornea are mediated through Fc receptors. Mouse IgG competes with ACPA antibodies for binding to Fc receptors, and Fc receptor blockers block all Fc receptors. This competitive peptide blockade may abolish the pathological effects of ACPAs on the cornea, suggesting the use of methodologies to prevent ACPA antibody interaction with Fc receptors, which may be a potential therapeutic strategy in treating citrullination-associated eye diseases. [Figure 17]Photographs of immunofluorescence staining of neutrophils showing that ACPA-H4R3cit induces NETosis in vitro. [Figure 18] 1 shows data demonstrating the cytotoxicity of OSIG on human corneal epithelial cells. OSIG concentrations from 1 mg / ml to 4 mg / ml do not cause cytotoxicity as evidenced by the lack of an increase in LDH levels. [Figure 19] Exemplary images of eyes with high autoantibodies (ACPA) in tears and severe ocular surface disease are provided. [Figure 20] Exemplary images are provided of eyes with high autoantibodies (ACPA) in tears but no systemic autoimmune disease, and seronegative for ACPA autoantibodies and rheumatoid arthritis. [Figure 21] 1 provides exemplary images of eyes with asymmetric eye disease, with eyes with more severe ocular surface disease having higher levels of autoantibodies (ACPAs) in tears. [Figure 22] We provide images from a 56-year-old female with severe aqueous tear deficiency and severe ocular surface disease due to graft-versus-host disease (Case Study 1). These images demonstrate that OSIG treatment reduced the signs and symptoms of severe dry eye and reduced levels of inflammatory biomarkers in tears. [Figure 23] We provide images of a 31-year-old woman with severe aqueous tear deficiency and severe ocular surface disease due to graft-versus-host disease (Case No. 2). These images demonstrate that OSIG treatment reduced the symptoms of neurotrophic keratitis. [Figure 24] We present images of a 74-year-old male with neurotropic keratitis due to a history of previous LASIK eye surgery in the left eye (Case Study 3). These images demonstrate that OSIG treatment reduced the signs and symptoms of severe dry eye, as well as reduced levels of inflammatory biomarkers in tears. [Diagram 25] We provide images of a 29-year-old man with aqueous tear deficiency and severe ocular surface disease due to Stevens-Johnson Syndrome (Case Study 6). These images demonstrate that OSIG treatment reduced the signs and symptoms of severe dry eye after Stevens-Johnson Syndrome. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] Following the principles herein, autoantibodies such as anti-citrullinated protein antibodies (ACPAs) were found to be present in the tears of patients with ocular diseases such as dry eye disease (DED), Sjögren's syndrome, Meybomian gland disease, superior limbal keratoconjunctivitis (SLK), ocular cicatricial pemphigoid, Stevens-Johnson syndrome, graft-versus-host disease, peripheral ulcerative keratitis, scleritis, sclerodermatitis and such autoimmune diseases. Surprisingly, ACPA autoantibodies were present in the tears of patients even though ACPAs and rheumatoid factor were not detectable in serum, suggesting that tear autoantibodies are produced locally in / around the ocular tissues. This result was surprising as we did not expect to see ACPA autoantibodies in the tears of patients with ocular disease, as there was no ACPA or rheumatoid factor in the serum and no evidence of systemic autoimmune disease. Patients with unilateral disease had higher levels of ACPAs in the tears of the more severe eye. Furthermore, eyes with very high ACPA levels in tears frequently showed evidence of severe disease, such as corneal melting and scarring, which was poorly controlled despite aggressive treatment. The study described herein also identified citrullinated proteins on the ocular surface of these patients, as well as the presence of Fc receptors. Laboratory experiments confirmed that exposure of mouse corneas to ACPAs can cause ocular surface disease, and that targeting their interaction with ocular tissues by using IgG or Fc receptor blockade reduces the deleterious effects of ACPAs on ocular surface disease. Taken together, pooled IgG and / or its synthetic variants are suitable forms of treatment for ocular diseases caused or affected by autoantibodies. Furthermore, the polyclonal nature of autoantibodies was confirmed in tears (e.g., the presence of cit histones, cit alpha enolase, cit fibrinogen, etc. in tears, which makes pooled IgG an attractive treatment, as targeting individual antibodies would be unattractive and laborious.

[0061] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0062] In an exemplary embodiment, the ophthalmic formulation comprises (a) a pharma- ceutically active compound capable of reducing the amount of autoantibodies on or in the eye, where such autoantibodies are generated in response to citrullinated proteins (ACPA autoantibodies due to citrullination of proteins) or in response to homocitrullinated proteins (anti-CarP antibodies due to carbamylation of proteins) or in response to native proteins, (b) optionally a second pharma-ceutically active compound selected from the group consisting of PAD enzyme inhibitors, NET disintegrators and Fc receptor blockers, and combinations thereof, and (c) a pharma-ceutically acceptable ophthalmic excipient. Unless otherwise required by context, the term "pharma-ceutically active compound" refers to a compound that is pharma-ceutically active when applied topically to the ocular surface or injected into the eye. Thus, for example, the term pharma-ceutically active NSAID refers to an NSAID that is pharma-ceutically active when applied topically to the ocular surface or injected into the eye.

[0063] Yet another aspect of the present disclosure provides an ophthalmic formulation comprising (a) a pharma- ceutically acceptable ophthalmic excipient, (b) a therapeutically effective amount of a pharma- ceutically active compound comprising immunoglobulin G (IgG), and (c) optionally a second pharma- ceutically active compound selected from the group consisting of steroids, anti-inflammatory agents, mucolytic agents, PAD enzyme inhibitors, NET disintegrators and Fc receptor blockers, and combinations thereof. The pharma-ceutically active compound comprising immunoglobulin G (IgG) can treat clinical conditions selected from the group consisting of inflammatory and immunological ocular surface diseases that may cause symptoms of ocular discomfort, dry eye syndrome, keratitis, blepharophore formation, conjunctival fornix shortening, lid margin / conjunctival keratosis, and subconjunctival fibrosis. By "therapeutically effective amount" is meant an amount of a compound sufficient to effect such treatment of a disease or clinical condition when administered to a mammal to treat such disease or clinical condition. A "therapeutically effective amount" will vary depending on the compound, the disease or clinical condition, and the severity, age, weight, etc., of the mammal being treated. "Treatment" or "treatment" of a clinical condition or disease includes (1) preventing the clinical condition or disease, i.e., not causing clinical symptoms of the condition or disease to develop in a mammal that may be susceptible to the clinical condition or disease but has not yet experienced or exhibited symptoms of the clinical condition or disease, (2) inhibiting the clinical condition or disease, i.e., arresting or reducing the onset of the clinical condition or disease or its symptoms, or (3) relieving the clinical condition or disease, i.e., causing regression of the clinical condition or disease or its symptoms.

[0064] Yet another aspect of the present disclosure provides an ophthalmic formulation comprising: (a) a pharma- ceutically acceptable ophthalmic excipient; (b) a therapeutically effective amount of a pharma- ceutically active compound comprising pooled human immunoglobulin G (IgG); and / or (c) a therapeutically effective amount of pooled human plasma proteins (at a dilution ranging from 0.1% to 99% of human plasma proteins) and pooled human plasma lipids (at a dilution ranging from 0.1% to 99%).

[0065] The ophthalmic preparations of the present disclosure are useful for treating various clinical conditions related to inflammatory and immunological ocular surface diseases.The exemplary clinical conditions that can be treated by the ophthalmic preparations of the present disclosure include, but are not limited to, ocular surface diseases that can cause symptoms of ocular discomfort, dry eye syndrome, mucous cell aggregates / debris of tear film, synechiae formation, conjunctival fornix shortening, eyelid margin / conjunctival keratinization and subconjunctival fibrosis. More specific types of ocular surface disease include ocular graft versus host disease (oGVHD), Stevens-Johnson syndrome, ocular cicatricial pemphigoid (OCP), mild, moderate and severe aqueous deficient dry eye disease (DED), Meybomian gland disease, superior limbic keratoconjunctivitis (SLK), aqueous sufficient DED, floppy eyelid syndrome, neurotrophic eye disease, symptom-sign disconnection (discordant DED), neuropathic pain, thyroid eye disease (Grave's ophthalmopathy), rheumatoid arthritis associated eye disease, lupus associated eye disease, Sjogren's syndrome, secondary Sjogren's syndrome, ocular rosacea, allergic keratoconjunctivitis (vernal), aniridia, sterile inflammation or keratitis due to viral, bacterial or fungal infection, post-operative / post-traumatic eye conditions, peripheral ulcerative keratitis, episcleritis, scleritis, uveitis and glaucoma. Exemplary post-operative / post-traumatic ocular conditions that can be treated using the ophthalmic formulations of the present disclosure include, but are not limited to, after ocular surface reconstruction surgery, application of metabolites to the ocular surface, pterygium surgery, glaucoma surgery, cataract surgery, laser vision correction surgery (LASIK, LASEK, or EPI-LASIK), keratoprosthetic surgery, and radiation injury.

[0066] Other ocular clinical conditions that may be treated using the ophthalmic formulations of the present disclosure include dry eye syndrome (keratoconjunctivitis sicca), Sjogren's syndrome, congenital anlacrimation, xerophthalmia (dry eye due to vitamin A deficiency), keratomalacia, thyroid eye disease, ocular rosacea, eyelid disorders, meibomian gland disease, meibomian gland dysfunction, ectropion, blepharitis, cutis blepharochalasis, sarcoidosis, styes, styes, chalazions, ptosis, pterygium, eyelid edema, eyelid dermatitis, trichiasis. , shedding, eyelid edema, eyelid dermatitis, trichiasis, trichilidae, dacryoadenitis, Stevens-Johnson syndrome, ocular graft-versus-host disease, dacryocystitis, conjunctivitis, keratoconjunctivitis, blepharoconjunctivitis, allergic conjunctivitis, vernal conjunctivitis, conjunctival congestion, conjunctival chalazion, subconjunctival hemorrhage, pterygium, pinguecula, conjunctival edema, iritis, iridocyclitis, anterior uveitis, glaucoma, red eye, keratitis, sclerodermatitis, episcleritis, peripheral ulcerative keratitis, neurotrophic keratitis, neurotrophic eye disease, corneal ulcer , ulcerative keratitis, corneal abrasion, photokeratitis, ultraviolet keratitis, exposure keratitis, superficial penetrating keratitis, Cygeson's superficial penetrating keratitis, herpes simplex keratitis, post-operative herpes zoster keratitis, rosacea, post-operative inflammation after ophthalmic surgery (i.e. eyelid surgery, cataract surgery, corneal surgery, refractive surgery including photorefractive keratectomy, glaucoma surgery, lacrimal gland surgery, conjunctival surgery, ocular muscle surgery), ocular surface conditions caused by chemical burns, thermal burns or physical trauma, autoimmune or Eye conditions caused by vascular disorders: including, but not limited to, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, Reiter's syndrome, enteropathic arthritis, psoriatic arthritis, discoid and systemic erythematous lupus, multiple sclerosis, Graves' disease, antiphospholipid syndrome, sarcoidosis, Wegener's granulomatosis, Behcet's syndrome, polyarteritis nodosa, Takayasu's arteritis, dermatomyositis, psoriasis, relapsing polychondritis, vasculitis, and sickle cell anemia.

[0067] In some embodiments, the ophthalmic formulations of the present disclosure are used to treat dry eye syndrome. There are two major classes of dry eye syndrome: (i) aqueous deficient dry eye (ADDE) and (ii) evaporative dry eye (EDE). There may also be mixed mechanism dry eye (i.e., both ADDE and EDE). ADDE is primarily due to failure of tear secretion. ADDE can be further classified as Sjogren's syndrome dry eye (where lacrimal and salivary glands are targeted by autoimmune processes such as rheumatoid arthritis) and non-Sjogren's syndrome dry eye (where lacrimal gland dysfunction is present but the systemic autoimmune features of Sjogren's syndrome are excluded, e.g., age-related dry eye). In contrast, EDE is primarily due to excessive water loss from the exposed ocular surface in the presence of normal tear secretion function. The causes can be extrinsic (e.g., ocular surface disorders due to some exogenous exposures, contact lens wear or vitamin A deficiency) or intrinsic (e.g., meibomian gland dysfunction and impaired eyelid opening). The meibomian glands secrete a mixture of lipids and other components that form the outer layer of the preocular tear film. This lipid layer serves to reduce evaporation of the tear film. Meibomian gland dysfunction (MGD) causes evaporative dry eye disease. One of the best recognized clinical findings in MGD is the presence of numerous telangiectatic blood vessels running across the margin of the eyelid. MGD may also be accompanied by aqueous deficient dry eye disease, as seen in ocular graft-versus-host disease (oGVHD).Other specific dry eye syndromes that may be treated using the compositions of the present disclosure include keratoconjunctivitis, dry eye caused by conjunctivitis, dry eye caused by allergic conjunctivitis, dry eye caused by blepharitis, dry eye caused by keratitis, dry eye caused by dacryoadenitis, dry eye caused by ocular rosacea, dry eye caused by Boehm's syndrome, dry eye caused by conjunctivochalasis, dry eye caused by blepharoconjunctivitis, dry eye caused by blepharoconjunctivitis, dry eye caused by superficial penetrating keratitis, and dry eye caused by ocular rosacea. Dry eye due to EYE, Thygeson's superficial penetrating keratopathy, dry eye caused by oGVHD, Sjogren's dry eye syndrome, dry eye due to Stevens-Johnson syndrome, MGD, dry eye due to Meybomian gland disease, dry eye induced by vitamin A deficiency, pharmacologically induced dry eye (hormone replacement therapy, blood pressure medications, antihistamines, antidepressants, anticholinergics, glaucoma medications, antihypertensives, diuretics, sedatives, isotretinoin, nasal decongestants, oral contraceptives, beta blockers) dry eye caused by ophthalmic drugs, such as ophthalmic gels, ...

[0068] In some specific embodiments, the ophthalmic formulations of the present disclosure are used to treat meibomian gland dysfunction (MGD). In yet other embodiments, the ophthalmic formulations of the present disclosure are used to treat aqueous tear deficiency dry eye (ADDE). In some cases, the method for treating ADDE includes treating a patient in need of treatment for Sjogren's dry eye syndrome, ocular graft-versus-host disease (oGVHD), or non-Sjogren's dry eye syndrome. In yet other embodiments, the method for treating dry eye syndrome includes treating a patient in need of treatment for evaporative dry eye (EDE). In yet other embodiments, the method of the present disclosure includes treating a patient in need of treatment for mixed mechanism dry eye consisting of ADDE and EDE. In yet other embodiments, the method of the present disclosure includes treating a patient suffering from dry eye syndrome due to complications of refractive eye surgery or due to one or more of the following causes: vitamin A deficiency, ocular surface disorder, allergy, aging, contact lens use, drug use, or eyelid disorder.

[0069] Citrullination and carbamylation, two forms of post-translational modifications (PTMs), generate two non-canonical amino acids in polypeptides, citrulline and homocitrulline, respectively, which are highly chemically related. There is growing evidence for the pathophysiological role of protein citrullination and carbamylation, including the role of autoantibodies against these proteins in rheumatoid arthritis. However, the role of citrullination and carbamylation and ACPAs in the context of ocular surface disease has not been described previously. The studies provided herein identify the presence of ACPAs in ocular surface fluids.

[0070] In some embodiments, the ophthalmic formulation comprises immunoglobulin G (IgG) as a pharma- ceutical active ingredient. Types of IgG that can be used in the ophthalmic formulations of the present disclosure include, but are not limited to, (i) serum / plasma derived pooled immunoglobulin G (OSIG), (ii) autologous IgG purified from autologous plasma / serum, (iii) multimerized IgG1 Fc molecules (IgG1 Fc hexamers), (iv) IgG2a Fc multimers (stradomers), (v) multivalent Fc structures, (vi) glycoengineered sialylated IgG, (vii) IgG-Fc glycosylation, or combinations thereof. In some embodiments, commercially available OSIG preparations are formulated with ophthalmic excipients (diluted or concentrated as needed) to achieve an IgG concentration that is 3-6 times the concentration of IgG normally (physiologically or pathologically) present at the site, and then dispensed as eye drops, injections, or other appropriate delivery methods. Examples of commercially available IgG solutions include, but are not limited to, Bivigam 10%, Cuvitru (20%), Flebogamma DIF 5%&10%, Gammagard Liquid 10%, Gammagard S / D 5%&10%, Gammaked 10%, Gammaplex 5%&10%, Gamunex-C 10%, Hizentra 20%, HYQVIA 10%, Octagam 5%&10%, Privigen 10%. These commercially available IgG solutions can be formulated with ophthalmic excipients to the desired IgG concentration, e.g., 4 mg / ml, and a desired pH of at least 6.0 to produce the disclosed OSIG compositions.

[0071] In some embodiments, the ophthalmic formulations consist of functional equivalents of whole IgG antibodies, such as antigen-binding fragments in which one or more antibody domains have been truncated or are absent (e.g., Fv, Fab, Fab', or F(ab)2 fragments), as well as engineered antibodies or protein-binding fragments thereof, including single chain antibodies or antibodies capable of binding to more than one epitope (e.g., bispecific antibodies), or antibodies capable of binding to one or more different antigens (e.g., bispecific or multispecific antibodies), may also be used in the present disclosure.

[0072] Immunoglobulins are a group of closely related glycoproteins composed of 82%–96% protein and 4%–18% carbohydrate. These glycoproteins, of approximately 150 kDa, are present in plasma at an average concentration of 7–12 g / L, depending on individual differences and the level of environmental exposure to antigens. Immunoglobulin G (IgG), the main effector molecule of the humoral immune response in men, accounts for approximately 75% of the total Ig in the plasma of healthy individuals. The basic Ig molecule has a four-chain structure, including two identical heavy (H) chains and two identical light (L) chains linked to each other by interchain disulfide bonds. IgG has a dual function characterized by its ability to recognize and specifically react to antigens and to perform a series of nonspecific effector functions that render the antigen harmless and ultimately eliminate it. This functional dichotomy of IgG is reflected in the structure of the molecule, which contains two variable regions (Fab) involved in antigen binding and a constant region (Fc or crystallizable fragment) that mediates specific effector functions. The presence of complex oligosaccharide structures modulates IgG functions, particularly complement activation and binding to FcγR. In some embodiments, the ophthalmic formulations of the present disclosure consist of immunoglobulin G (IgG) with enhanced sialylation (sialylated IgG), where such sialylation is achieved using standard chemical processes.

[0073] In some embodiments, the concentration of IgG in the disclosed ophthalmic formulations is defined by a 10% Ocular Surface Immunoglobulin (OSIG) solution, or any other suitable concentration. OSIG is a therapeutic preparation of pooled normal multispecific human IgG obtained from the serum / plasma of a large number of healthy donors. This preparation contains antibodies against microbial antigens, autoantigens (natural autoantibodies), and anti-idiotypic antibodies that recognize other antibodies. The plasma used to manufacture OSIG comes from two sources, approximately 20% from blood donors and the remaining 80% from plasma donors. Individual plasma is pooled, with pool sizes ranging from a minimum of 1000 donors, but can be as large as 100,000 donors. The thousands of donors contributing to a typical pool of plasma used for isolation of immunoglobulins represent a wide range of antibody specificities against infectious agents such as bacteria, viruses, etc., as well as a large number of autoantigens that reflect the cumulative exposure of the donor population to the environment. Preparations of OSIG consist of intact IgG molecules with a distribution of IgG subclasses (IgG1, IgG2, IgG3, and IgG4) that corresponds to that of normal serum. The primary purification processes for OSIG production include (i) fractionation, e.g., polyethylene glycol (PEG) is a synthetic polymer also used to separate proteins by fractional precipitation by exclusion mechanisms from natural mixtures such as plasma, and (ii) chromatography, e.g., anion exchange chromatography, hydrophobic charge induction chromatography, or size exclusion chromatography. The biological effects of OSIG that may contribute to the efficacy of the disclosed ophthalmic formulations include (i) functional blockade of Fc receptors. Saturation of Fc receptors by OSIG leads to reduced cell destruction as a result of Fc-mediated biological effects. (ii) Neutralization of autoantibodies and inhibition of autoantibody production. OSIG preparations contain anti-idiotypic antibodies, i.e., antibodies that can specifically interact with the variable regions (antigen recognition sites) of autoantibodies. This interaction may neutralize autoantibodies and prevent their production via binding to autoreactive B lymphocytes. (iii) Complement inhibition.The Fc portion of OSIG can bind to the C3b and C4b fragments of complement, thereby inhibiting their tissue deposition and the generation of C5 convertase; (iv) regulation of cytokines (including IL-1, -2, -3, -4, -5, -10, TNF-α, and GM-CSF) and cytokine antagonist production (IL-1 receptor antagonist); and (v) signaling through the inhibitory Fc receptor, Fc gamma RIIB.

[0074] The amount of IgG in the ophthalmic formulation of the present disclosure may range from about 0.01 mg / mL to about 1 g / mL, typically from about 1 mg / mL to about 10 mg / mL. In certain embodiments, the concentration or amount of IgG may be about 0.01 mg / mL, and in other embodiments, the concentration or amount of IgG may be about 0.05 mg / mL. In certain embodiments, the concentration or amount of IgG may be about 1 mg / mL, and in other embodiments, the concentration or amount of IgG may be about 10 mg / mL. In various embodiments, the concentration or amount of IgG can be 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, or 10 mg / mL.

[0075] The present disclosure encompasses the presence of autoantibodies on the ocular surface (tears) or within the eye (aqueous and vitreous) in various ocular diseases, of which anti-citrullinated protein antibodies (ACPA) are the prototypical example. Autoantibodies that may be present and are encompassed by this disclosure include antibodies to the following proteins: beta2-glycoprotein, C1q, CENP-B (centromere protein B), CENP-A (centromere protein A), Jo-1, Ku, Mi-2, myeloperoxidase (MPO), PCNA (proliferating cell nuclear antigen A), PL-12 (alanyl-tRNA synthetase), PM / Scl 100, proteinase 3, RNP (ribonucleoprotein), RNP / Smith (RNP / Sm), ribosomal P, Scl-70, Sm, SSB / La (Sjogren's syndrome associated antigen B / La), SSA / Ro60 (Sjogren's syndrome associated antigen A / Ro60 kDa), SSA / Ro52 (Sjogren's syndrome associated antigen A / Ro52 kDa). While not designed to be exhaustive / complete, the autoantibodies (ACPA or non-ACPA) covered in this disclosure may be derived from the proteins listed in Table 1 or their fragments (citrullinated or native). [Table 1]

[0076] "Pharmaceutically acceptable excipient" or "Pharmaceutically acceptable ophthalmic excipient" refers to an excipient useful for preparing a pharmaceutical composition of the present disclosure. Such excipients are generally considered by those skilled in the art to be safe, non-toxic, and not biologically active or undesirable, and include excipients that are acceptable for veterinary and human pharmaceutical use. As used herein and in the claims, "a pharmaceutically acceptable excipient" includes both one and more than one such excipient. Exemplary pharmaceutically acceptable excipients include salts (such as sodium chloride) or isotonicity agents, gums, resins, solvents such as water, non-aqueous solvents (such as alcohols, oils, buffers to maintain pH, etc.), pH modifiers (e.g., bases such as sodium hydroxide, and acids such as hydrochloric acid), emulsifiers, thickeners, micro- or nano-emulsion formers, preservatives, surfactants, and the like. Exemplary pharma- ceutically acceptable excipients that can be used in the ophthalmic formulations of the present disclosure include, but are not limited to, water, benzyl alcohol, sodium hydroxide, hydrochloric acid, Castrol oil, citrate buffer, Tris buffer, phosphate buffer, and other excipients known to those of skill in the art.

[0077] In some embodiments, the ophthalmic formulations of the present disclosure may also include a salt, such as sodium chloride, hi yet other embodiments, the ophthalmic formulations of the present disclosure may also include a non-aqueous solvent, such as benzyl alcohol, ethanol, or other non-aqueous solvents known to those of skill in the art.

[0078] In yet other embodiments, the pH of the ophthalmic formulations of the present disclosure is adjusted to a pH of about 5.0 to about 8.5, typically a pH of about 5.0 to about 8.0, and often a pH of about 5.0 to about 7.5. Additional exemplary pH ranges are about pH 6 to about pH 8, or about pH 6.2 to about pH 7.2, about pH 6.4 to about pH 7.4, or about pH 6.5 to about pH 7.5, about pH 6.6 to about pH 7.6, about pH 6.8 to about pH 7.8. For example, the pH is about 5.0, about 5.1, or about 5.2, or about 5.3, or about 5.4, or about 5.5, or about 5.6, or about 5.7, or about 5.8, or about 5.9, about 6.0, or about 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6, or about 6.7, or about 6.8, or about 6.9, or about 7.0, or about 7.1, or about 7.2, or about 7.3, or about 7.4, or about 7.5, or about 7.6, or about 7.7, or about 7.8, or about 7.9, or about 8.0. The pH of the ophthalmic formulations of the present disclosure can be adjusted, for example, using sodium hydroxide and / or hydrochloric acid as needed to achieve the desired pH level.

[0079] Ophthalmic formulations can be formulated as eye drops, topical liquids, ointments, emulsions, suspensions, or gels (e.g., sodium IgG in hydrogels). Ophthalmic formulations can also be formulated as nanoemulsions of oils or suspensions. Additionally, ophthalmic formulations can be formulated as injectable formulations.

[0080] In some embodiments, the ophthalmic formulation of the present disclosure does not contain preservatives and is formulated in single-use or multi-dose vials.When preservatives are used, suitable preservatives include, but are not limited to, benzalkonium, purite, chlorobutanol, sodium perborate, stabilized oxychloro complex (SOC), polyquaternium-1 (polyquad, PQ-1), thimerosal, benzyl alcohol, sorbic acid, methyl / propyl paraben, chlorhexidine, disodium EDTA, sofZia, and other preservatives known to those skilled in the art of ophthalmic or ophthalmic formulation chemistry.

[0081] When present, the second pharma- ceutical active compound is selected from the group consisting of steroids, anti-inflammatory agents, mucolytic agents, and combinations thereof. Exemplary steroids suitable for the ophthalmic formulations of the present disclosure include, but are not limited to, methylprednisone, prednisone, dexamethasone, loteprednol etabonate, fluocinolone, difluprednate, fluorometholone, medrysone, fluocinolone, rimexolone triamcinolone, and combinations thereof. When a steroid is used in the ophthalmic formulations of the present disclosure, the amount of steroid present in the formulation is about 0.01% w / w to 2% w / w, typically about 0.05% w / w to 1% w / w, and often in the range of about 0.1% w / w to about 0.3% w / w. It is to be understood that the scope of the present disclosure is not limited to these particular ranges of the amount of steroid. In particular, the amount of steroid present in the ophthalmic formulations of the present disclosure generally depends on the steroid used. For example, the amount of steroid present can vary depending on the activity of the particular steroid used, the molecular weight of the steroid, as well as the purpose for which the steroid is used in the ophthalmic formulations of the present disclosure.

[0082] Exemplary anti-inflammatory agents suitable for the ophthalmic formulations of the present disclosure include, but are not limited to, cyclosporine, lifitegrast®, tacrolimus, interleukin 1 receptor antagonists (anakinra), ketorolac, diclofenac, other NSAIDs such as flurbiprofen, bromfenac, nepafenac, and combinations thereof. When an anti-inflammatory agent is used in the ophthalmic formulations of the present disclosure, the amount of the anti-inflammatory agent present in the formulation is about 0.01% w / w to 2% w / w, typically about 0.05% w / w to 1% w / w, and often in the range of about 0.1% w / w to about 0.3% w / w. It is understood that the scope of the present disclosure is not limited to these specific ranges of the amount of anti-inflammatory agent. In particular, the amount of anti-inflammatory agent present in the ophthalmic formulations of the present disclosure will generally depend on the particular anti-inflammatory agent used, such as the activity of the particular anti-inflammatory agent used, the molecular weight of the anti-inflammatory agent, and the like.

[0083] Exemplary mucolytic agents useful in the ophthalmic formulations of the present disclosure include, but are not limited to, N-acetylcysteine, nasistellin, dextran, DNase I (dornase alpha), gelsolin, thymosin β4, 14 and 15 membered macrolide antibiotics (e.g., erythromycin, non-antibacterial derivatives of erythromycin (e.g., EM703 and EM900), clarithromycin, roxithromycin, fidaxomicin, telithromycin and azithromycin), and combinations thereof. It is understood that when an antibiotic is used, it is used primarily for its mucolytic activity. When a mucolytic agent is used in the ophthalmic formulations of the present disclosure, the amount of mucolytic agent present in the formulation ranges from about 0.01% w / w to 2% w / w, typically from about 0.05% w / w to 1% w / w, and often from about 0.1% w / w to about 0.3% w / w. It is to be understood that the scope of the present disclosure is not limited to these particular ranges of the amount of mucolytic agent In particular, the amount of mucolytic agent present in the ophthalmic formulations of the present disclosure will generally depend on the particular mucolytic agent used, such as the activity of the mucolytic agent, the molecular weight of the mucolytic agent, etc.

[0084] The ophthalmic formulations of the present disclosure may be homogeneous or heterogeneous. In some embodiments, the ophthalmic formulations of the present disclosure include oils or fatty acid esters. Fatty acid esters have the meaning commonly understood in the art and are esters formed between alcohols and fatty acids. Exemplary fatty acid esters useful in the formulations of the present disclosure include, but are not limited to, triglyceride esters, commonly known as vegetable oils, mono- and diglyceride esters of fatty acids, fatty acid methyl esters, and other fatty acid esters known to those skilled in the art. It should be understood that fatty acid esters can be mixtures of several compounds or essentially pure compounds. Typically, fatty acid esters are vegetable oils. Specific examples of vegetable oils that can be used include, but are not limited to, castor oil, sesame oil, soybean oil, cottonseed oil, olive oil, peanut oil, safflower oil, sunflower oil, palm oil, palm kernel oil, canola oil, and Miglyol oil®.

[0085] Various vehicles can be used in the ophthalmic formulations of the present disclosure.These vehicles include, but are not limited to, purified water (water), polyvinyl alcohol, povidone, hydroxypropylmethylcellulose, poloxamer, carboxymethylcellulose, hydroxyethylcellulose, polyol, sodium hyaluronate, Pluronic®, Corbopol, cyclodextrin, and mixtures of two or more.Vehicles are used in the formulation in the amount required to provide the concentration of active compounds disclosed herein.In one particular embodiment, vehicle comprises water.

[0086] In some embodiments of the present disclosure, emulsion stabilizing polymers are used. Without intending to limit the scope of the present disclosure, emulsion stabilizing polymers generally contain hydrophilic groups such as cellulose, sugar, ethylene oxide, hydroxide, carboxylic acid or other polyelectrolytes. Without being bound by theory, it is believed that these polymers help stabilize the emulsion by increasing the viscosity of the formulation as well as by reducing the interfacial tension. Surfactants such as polysorbate 80 or other ophthalmically acceptable surfactants can be used to stabilize the emulsion. Some examples of emulsion stabilizing polymers useful in the present disclosure include, but are not limited to, carbomer, Pemulen®, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, povidone, polyvinyl alcohol, polyethylene glycol, and mixtures of two or more thereof.

[0087] The ophthalmic formulation of the present disclosure can be packaged in various package formats known in the field of topical ophthalmology. In one particular embodiment, the ophthalmic formulation is packaged in a sterile, preservative-free, single-use pack, or in a vial or container (i.e., unit dose vial). Each vial can be made of low density polyethylene, for example, as small as 0.9 mL, and contain a small amount of formulation, for example, 0.4 mL for a single use. Thus, when the ophthalmic formulation is sterile and contained in a disposable, single-dose container for topical use in drop form, multiple vials in the form of a set of 30 vials, 60 vials, etc. can be packaged in a tray with a lid, for example, a polypropylene tray with an aluminum peelable lid. The entire contents of each tray can be dispensed intact, with one vial or pack being used each time and discarded immediately after each use. For example, plastic ampoules, vials, or containers can be manufactured using blow-fill-seal (BFS) technology. The BFS process may include plastic extrusion, molding, aseptic filling, and hermetic sealing in one continuous operation, and these processes are known in the art. In another embodiment, the formulation is packaged in a multi-dose vial using a special container / closure that maintains the integrity of the sterility so that the material can be dispensed as sterile every time. In yet another embodiment, the ophthalmic formulation is packaged in a conventional vial / container as a sterile product.

[0088] In some embodiments, the dosage form of the present disclosure is a heterogeneous aqueous eye drop, ophthalmic solution formulation.

[0089] In one particular embodiment, ophthalmic formulations comprising IgG(OSIG) are formulated as eye drops and used to treat inflammatory and immune-mediated ocular surface diseases that may result in symptoms of ocular discomfort, mucosal cell aggregates / debris in the tear film, synechiae formation, conjunctival fornix shortening, lid margin / conjunctival keratinization, or subconjunctival fibrosis. Specific clinical conditions that may be treated using the ophthalmic formulations of the present disclosure include ocular graft versus host disease (oGVHD), Stevens-Johnson syndrome, ocular cicatricial pemphigoid (OCP), mild, moderate and severe aqueous deficient dry eye disease (DED) (secondary to Sjogren's syndrome, non-Sjogren's syndrome, idiopathic and other causes), meibomian gland disease (dysfunction or atrophy), superior limbic keratoconjunctivitis (SLK), sufficient tear DED (congruent or incongruent), floppy eyelid syndrome, neurotrophic eye disease, aniridia, and post-operative / post-traumatic conditions, such as following ocular surface reconstructive surgery, application of antimetabolites to the ocular surface (such as mitomycin-C, 5-fluorouracil) or artificial corneal surgery or radiation injury.

[0090] In yet another aspect of the present disclosure, a method is provided for treating an ocular disease in a subject in need of such treatment. Such a method generally comprises administering to a subject a therapeutically effective, sub-anticoagulant amount of an IgG ophthalmic formulation to treat the ocular disease. In some embodiments, the IgG ophthalmic formulation is administered to the subject at least twice a day. In yet other embodiments, the IgG ophthalmic formulation is administered to the subject at least twice a month. The ophthalmic formulation can be an aqueous solution, an aqueous suspension, a gel, or the like.

[0091] Another aspect of the present disclosure provides a method for diagnosing or monitoring ocular surface disorder in a subject.The method includes comparing the level of autoantibody from a sample obtained from a subject with a control level of said autoantibody to diagnose or monitor ocular surface disorder in a subject.As used herein, the term "control level" of autoantibody refers to the level that can be compared with the level of autoantibody in a test sample, including (i) that of a subject without ocular surface disorder, (ii) that of a subject with ocular surface disorder, (iii) that of the same subject before treatment, immediately after starting treatment, or before showing symptoms of ocular surface disorder.

[0092] In some embodiments, the control level can be a normal level, i.e., the level in a sample from a normal patient, i.e., a subject without ocular surface disorder. This control level can be more specifically called a "negative control". This allows a determination based on the control level of autoantibodies, and the sample evaluated for ocular surface disease has a measurable difference or substantially no difference in autoantibody level compared to the control level.

[0093] In another embodiment, the control level can be the level of autoantibodies established in a sample from a subject or from a population of individuals believed to have ocular surface disease. This can be more specifically referred to as a "positive control level." The term "positive control" as used herein refers to a level of autoantibody expression or biological activity established in a sample from a subject, from another individual, or from a population of individuals, where the sample is believed to have disease based on data from that sample.

[0094] In other embodiments, a control level can be established from a previous sample from the subject being tested such that the subject's disease progression or regression can be monitored over time and / or the effectiveness of treatment can be assessed.

[0095] Unless otherwise stated or required by context, the term "monitoring" refers to determining the progression of ocular surface disease or determining the effectiveness of a particular treatment protocol or drug. The term "diagnosis" refers to the process of determining the presence or absence of ocular surface disease in a subject. It can also include determining which particular ocular surface disease is present in a subject.

[0096] The autoantibodies can be used to diagnose ocular surface disease and initiate appropriate therapy or treatment, for example, with IgG eye drops or another appropriate pharmaceutical agent.

[0097] Changes in the levels of autoantibodies can be used to assess response to or efficacy of treatment. Treatment intensity can also be titrated to autoantibody levels.

[0098] This method can be used to provide a rapid-result in-house test for diagnosing and managing ocular surface diseases and for initiating / titrating IgG eye drop therapy for such diseases, either as a single agent or in combination with other active agents. The test can be designed to measure components of tears and other ocular fluids. As discussed in connection with FIG. 1, concentrations below 5 have not been considered noteworthy thus far. However, levels above 5 may prove to be indicative of a treatable condition and are contemplated within the scope of this disclosure.

[0099] The IgG composition is stable for long periods of time, so that the treatment can be long-term. The treatment can be designed to be delivered at least twice a day or at least twice a month, or according to other suitable treatment regimes, provided that the treatment is sufficient to either reduce the level of, or reduce the adverse biological effects resulting from, the autoantibodies present in the ocular fluid.

[0100] The disclosed method of utilizing autoantibody can also be used to diagnose or monitor ocular surface disorders such as oGVHD or Sjogren's syndrome and other dry eye diseases.Such methods typically include measuring the level of one or more autoantibodies present in a biological sample taken from a test subject (e.g., tears, blood / serum, or ocular surface cells) and comparing it with a control level.The control level can be the subject's previous autoantibody level, the autoantibody level of a healthy subject, or the autoantibody level of a subject with ocular surface disease.

[0101] Another aspect of the present disclosure provides sensors, biosensors, multi-analyte panels, arrays, assays and kits for determining the level of one or more autoantibodies obtained from a subject.Autoantibodies and the methods they are used can be used to aid in diagnosis and evaluate the onset and development of ocular surface disorders.The present disclosure also relates to the use of autoantibodies in clinical screening, prognosis evaluation, treatment evaluation, for drug screening and drug development.

[0102] In certain embodiments, the present disclosure provides a method of diagnosing or monitoring an ocular surface disorder in a subject, comprising obtaining a biological sample from the subject and comparing the level of one or more autoantibodies in the biological sample to a control level.

[0103] The level of autoantibody can be easily determined by any of the conventional methods available to those skilled in the art.Exemplary methods include, but are not limited to, direct or indirect methods such as combined or uncombined enzyme methods, electrochemistry, spectroscopy (e.g., spectrophotometry, such as using UV / VIS spectrometer, fluorimetry, luminometric methods, polarimetry, etc.), chromatographic methods (e.g., HPLC, gas chromatography, MPLC, LPLC, etc.), ELISA, immunological methods such as DOT-BLOT analysis, etc.

[0104] Yet another aspect of the present disclosure provides a method for diagnosing or monitoring ocular surface disorder or predisposition thereto.Such method comprises comparing the level of one or more autoantibodies present in the biological sample taken from test subject, and comparing it with a control level.In one particular embodiment, the method of the present disclosure is used to monitor the effectiveness of treatment (e.g., therapeutic substance) in subjects who have, suspect, or are predisposed to have ocular surface disorder.

[0105] Yet another aspect of the present disclosure provides a multi-analyte panel or array that can detect one, two, three, four, or more (hundreds) of the autoantibodies of the present disclosure. A multi-analyte panel can detect many different analytes. The array can detect a single analyte in many samples, or as a multi-analyte array, can detect many different analytes in a sample. A multi-analyte panel or multi-analyte array according to the present disclosure can detect one or more autoantibodies as described herein, and can detect autoantibodies in addition to those specifically described herein. Detection methods may include bead-based (e.g., Luminex platform) or Phadia ImmunoCap.

[0106] Also provided are diagnostic or monitoring test kits suitable for carrying out the methods according to the present disclosure, optionally together with instructions for use of the kit. The diagnostic or monitoring kits may include one or more biosensors according to the present disclosure, and a single sensor, or a biosensor, or a combination of sensors and / or biosensors may be included in the kit. The diagnostic or monitoring kits may include a panel or array according to the present disclosure. The diagnostic or monitoring kits may include an assay or combination of assays according to the present disclosure.

[0107] Further provided is the use of the method, sensor, biosensor, multi-analyte panel, array or kit according to the present disclosure to identify substances that can regulate or treat ocular surface disorders.Substances that can regulate or treat ocular surface disorders can be anti-inflammatory, immunomodulatory, immunosuppressive, antibiotic or palliative measures (artificial tears, contact lenses, or punctal plugs) that are useful for treating ocular surface disorders.

[0108] As used throughout this specification and the appended claims, the terms "treating" or "treatment" of a disease include (1) preventing the disease, i.e., not causing the disease to develop in a subject who may be susceptible to the disease but has not yet experienced or exhibited symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the onset of the disease or its symptoms; or (3) ameliorating the disease, i.e., causing regression of the disease or its symptoms.

[0109] A higher level of autoantibody in the test biological sample compared to a negative control level (e.g., the level of the biomarker in a subject without ocular surface disease) indicates the presence of an ocular surface disorder, such as oGVHD, Sjogren's syndrome and other dry eye diseases.

[0110] The disclosed monitoring, risk determination, and diagnostic methods are useful for confirming the presence of or predisposition to ocular surface disorders, for monitoring the onset of ocular surface disorders by assessing onset and progression, or for assessing the improvement or regression of disorders. The monitoring and diagnostic methods are also useful in methods for clinical screening, prognosis, treatment selection, evaluation of treatment benefits, i.e., drug screening and evaluation of drug development. Efficient diagnostic and monitoring methods provide a very powerful "patient solution" that may improve prognosis by establishing the correct diagnosis, allowing rapid identification of the most appropriate treatment (thus reducing unnecessary exposure to harmful drug side effects), and reducing "downtime" and recurrence rates.

[0111] Methods for monitoring therapeutic efficacy can be used to monitor the therapeutic efficacy of existing and new treatments in human subjects and non-human animals (e.g., animal models). These monitoring methods can be incorporated into screening of new drug substances and substance combinations. Modulation of protein biomarker levels is useful as an indicator of the state of, or predisposition to, ocular surface disorders. An increase in the level of autoantibodies over time indicates onset or progression, i.e., worsening of the disorder, whereas a decrease in the level of protein biomarkers indicates improvement or remission of the disorder. Identification of autoantibodies against ocular surface disorders allows for the integration of diagnostic procedures and treatment plans.

[0112] Currently, there is no methodology available to determine effective treatments, and thus far no rapid assessment of drug response has been possible. Traditionally, many ocular surface disease treatments have required treatment trials lasting weeks to months for a given treatment approach. Detection of autoantibodies of the present disclosure can be used to screen subjects prior to participation in clinical trials. Autoantibodies provide a means to indicate treatment response, failure to respond, unfavorable side effect profile, degree of medication compliance, and achievement of adequate serum drug levels. Autoantibodies can be used to provide warning of adverse drug reactions, a major problem encountered with all ocular surface disease treatments. Autoantibodies aid in the development of individualized ocular surface disease treatments, as response assessments can be used to fine-tune dosage, minimize the number of formulation drugs, reduce delays in achieving effective treatment, and avoid adverse drug reactions. Thus, by monitoring the autoantibodies of the present disclosure, patient care can be precisely tailored to match the needs dictated by the disorder and the patient's pharmacogenomic profile, and thus autoantibodies can be used to titrate optimal doses, predict positive treatment responses, and identify patients at high risk for severe side effects.

[0113] The measurement of autoantibodies can be performed by direct or indirect detection methods.Biomarkers can be detected directly or indirectly through interaction with ligands such as enzymes, binding receptor or transporter proteins, antibodies, peptides, aptamers, or oligonucleotides, or any synthetic chemical receptors or compounds that can specifically bind biomarkers.Ligands can have detectable labels such as luminescent, fluorescent or radioactive labels and / or affinity tags.

[0114] In one particular embodiment, the autoantibodies of the present disclosure are detected and measured using mass spectrometry-based techniques, chromatography-based techniques, enzyme detection systems (by direct or indirect measurement), or sensors, for example, using sensor systems with amperometric, potentiometric, conductometric, impedance, magnetic, optical, acoustic, or thermal transducers. The sensors may incorporate physical, chemical, or biological detection systems. Examples of sensors are biosensors, i.e., sensors with biological recognition systems based on nucleic acids, such as oligonucleotide probes or aptamers, or proteins, such as enzymes, binding proteins, receptor proteins, transporter proteins, or antibodies. Biosensors can incorporate immunological methods, electrical, thermal, magnetic, optical (e.g., holographic) or acoustic techniques for the detection of biomarkers. Such biosensors can be used to detect target autoantibodies at the expected concentrations found in biological samples. The methods of the present disclosure are suitable for clinical screening, evaluation of prognosis, monitoring of treatment results, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development, and to help identify new targets for drug treatment. Identification of key disease-specific autoantibodies is central to integrating diagnostic procedures and treatment regimens.

[0115] The disclosed methods involving the detection and / or quantification of autoantibodies can be carried out using benchtop equipment or can be incorporated into disposable, diagnostic, or monitoring platforms that can be used in non-laboratory environments, such as clinics or at the patient's bedside.Suitable sensors or biosensors for carrying out the disclosed methods include "credit" cards equipped with optical or acoustic readers.Sensors or biosensors can be configured to allow collected data to be electronically transmitted to physicians for interpretation, thus forming the basis of electronic medicine.

[0116] The foregoing discussion of the present disclosure has been presented for purposes of illustration and description. The foregoing is not intended to limit the present disclosure to one or more forms disclosed herein. The description of the present disclosure includes one or more embodiments and descriptions of certain variations and modifications, but other variations and modifications are within the scope of the disclosure, for example, as may be within the skill and knowledge of a person skilled in the art after understanding the present disclosure. It is intended to acquire the right to include alternative embodiments to the extent permitted, including alternative, interchangeable and / or equivalent structures, functions, scopes or steps to those claimed, without intending to publicly dedicate patentable subject matter, whether such alternative, interchangeable and / or equivalent structures, functions, scopes or steps are disclosed herein or not.

[0117] In addition to the examples below, clinically significant results relevant to the present disclosure are also provided in Kwon et al., Pathological consequences of anti-citrullinated protein antibodies in tear fluid and therapeutic potential of pooled human immune globulin -eye drops in dry eye disease, Ocul Surf. 2019 Oct 10 (pii: S1542-0124(19)30377-5. doi:10.1016 / j.jtos.2019.10.004), available at https: / / doi.org / 10.1016 / j.jtos.2019.10.004. Kwon et al. is incorporated herein by reference in its entirety. EXAMPLES

[0118] Example 1: Autoantibody (ACPA) levels in healthy subjects and ocular surface disease The presence of autoantibodies (ACPA) was demonstrated in ocular surface washes of healthy subjects (Figure 1) and a cut-off was established for diagnosing autoantibody ACPA positivity in subjects. Ocular surface washes were performed using 50 μl of artificial tears in healthy subjects (without aqueous deficient dry eye (DED)) and 10 μl of collected washes were analyzed for ACPA levels by ELISA using a commercial plate (CCP3). All negative values ​​were considered 0. The mean + 3SD was used to establish the threshold for positive values ​​of ACPA. The mean of ACPA, all negative = 0 was 0.57, 3SD 1.2 = 4.17. Thus, a cut-off of 5.0 was established.

[0119] The presence of autoantibodies (ACPA) was also demonstrated in ocular surface washes of patients with ocular surface disease (Figures 2 and 3). As shown in Figure 2, ACPA levels were measured in ocular surface washes of patients with ocular surface disease. These patients were treated with appropriate topical eye drops (artificial tears, Restasis, Xidra or steroids). Tear ACPA levels were positive (i.e., ACPA >5.0) in several ocular autoimmune diseases such as Sjögren's syndrome, aqueous tear-deficient dry eye disease, mixed mechanism DED, ocular cicatricial pemphigoid (OCP), Stevens-Johnson syndrome, and ocular graft-versus-host disease (oGVHD). Patients with evaporative DED such as meibomian gland dysfunction also had high ACPA levels. Superior limbal keratoconjunctivitis (SLK) and neurotrophic conjunctivitis also had high ACPA levels. Surprisingly, patients with symptomatic amputation (discordant DED) also had high ACPA levels. Therefore, all of these eye diseases, including other intraocular autoimmune / inflammatory eye glaucoma, would benefit from OSIG eye drop treatment to reduce the contribution of autoantibodies to the signs and symptoms of eye disease. The data in Figure 3 show the percentage of all patients with a particular eye disease who had positive ACPAs on ocular surface washes.

[0120] Example 2: Poor Effect of Conventional Treatments on ACPA Levels Experiments were performed to show the insufficient effect of conventional treatment on the levels of ACPAs in ocular surface washes (Figures 4 and 5). Figure 4 shows ACPA levels in tears of Sjogren's syndrome patients before and after the start of conventional treatment. Compared to ACPA levels in Sjogren's patients who have already been treated, patients who have not yet been treated (Sjogren's pre-Rx) have significantly higher ACPA levels. Levels after treatment initiation (Sjogren's post-Rx) are lower, but still significantly higher compared to healthy subjects. This data shows that even when Sjogren's patients are treated, ACPA levels in tears are high, and therefore the contribution of ACPAs to ocular surface disease can be reduced if ACPAs are specifically targeted, such as with OSIG therapy.

[0121] ACPA levels in tears of oGVHD patients before and after initiation of treatment. ACPA levels in oGVHD patients not yet on treatment (oGVHD Pre-Rx) are significantly higher than ACPA levels after initiation of treatment (oGVHD Post-Rx) (Figure 5). ACP levels one month after initiation of treatment are significantly higher than in healthy subjects. This data indicates that ACPA levels are higher in tears even when oGVHD patients are on treatment, and therefore the contribution of ACPAs to ocular surface disease can be reduced when specifically targeting ACPAs, such as with OSIG therapy.

[0122] Example 3: Citrullinated Proteins Are Present on the Ocular Surface of Patients with Ocular Surface Disease We next demonstrated that citrullinated proteins are present on the ocular surface of patients with ocular surface disease. As shown in Figure 6, citrulline is expressed in nonepithelial cells of Sjögren's syndrome. The purpose of this experiment was to determine whether cells of the ocular surface of Sjögren's patients are citrullinated. Approval for the study was obtained from the Institutional Review Board of the University of Illinois at Chicago (UIC). Informed consent was obtained from all participants after the nature and possible outcomes of the study were explained. The study was conducted in accordance with the requirements of the Health Insurance Portability and Accountability Act (HIPAA) and the tenets of the Declaration of Helsinki.

[0123] Antibiotics were applied to the patient's eyes and filter paper (PALL, #60298) was applied to the temporal conjunctiva and lower eyelid to remove the superficial layer of the ocular surface epithelium. The filter paper was fixed with 4% paraformaldehyde (PFA) in a Petri dish for 20 minutes, followed by washing with 1XPBS for 5 minutes and mounting to a microscope slide via adhesive tabs. A water-resistant barrier was drawn around the sample with a Pap pen, followed by permeabilization with PBS-T (0.025% Triton in 1X PBS) for 1 hour. With gentle shaking, the filter paper was washed once with 1XPBS for 5 minutes and then blocked with 2.5% donkey serum (secondarily produced host species) and 1% BSA in 1XPBS for at least 1 hour. The filter paper was then incubated with primary antibodies overnight at 4°C. The primary antibodies used were mouse monoclonal anti-citrulline antibody (1:1000; Millipore, MABN328) and rabbit polyclonal anti-cytokeratin 14 antibody (1:1000; BioLegend, #905301).

[0124] The next day, the filters were washed three times with 1XPBS for 5 min with gentle shaking, followed by secondary antibody incubation at room temperature for 1 h. The secondary antibodies used were: AlexaFluor594 donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch Lab, #715-585-150) and Alexa Fluor 488 donkey anti-rabbit IgG (1:1000; Jackson ImmunoResearch Lab, #711-546-152). After incubation, the filters were washed three times with 1XPBS for 5 min with gentle shaking in the dark. Slides were dried and mounted with ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, P36931). Coverslips were placed and sealed with nail polish. Slides were allowed to dry completely before imaging. Images were captured at 63x magnification using a Zeiss LSM 710 confocal microscope (Leica, UIC Ophthalmology Core facility) and analyzed with Zeiss LSM image software. Impression cytology was performed in a patient with Sjögren's syndrome and found that citrulline was expressed in non-epithelial cells (neutrophils). The non-epithelial cells may be neutrophils, which is supported by the presence of NETs. These data demonstrated that non-epithelial cells in Sjögren's patients are citrullinated.

[0125] Example 4: Citrullinated Proteins Are Not Present in Healthy Subjects In Sjogren's, citrullinated proteins appear to be predominantly neutrophilic (Figures 6 and 7). The purpose of this experiment was to determine whether the citrullinated non-epithelial cells were neutrophils (Figure 7). Impression cytology was performed as described in Example 3 above.

[0126] The primary antibodies used were mouse monoclonal anti-citrulline antibody (1:1000; Millipore, MABN328) and rabbit monoclonal anti-neutrophil elastase antibody (1:500; Abcam, ab131260). The next day, the filters were washed three times with 1XPBS for 5 min with gentle shaking, followed by secondary antibody incubation for 1 h at room temperature. The secondary antibodies used were: AlexaFluor594 donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch Lab, #715-585-150) and Alexa Fluor 488 donkey anti-rabbit IgG (1:1000; Jackson ImmunoResearch Lab, #711-546-152). After incubation, the filters were washed three times with 1XPBS for 5 min with gentle shaking in the dark. Slides were dried and mounted with ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, P36931). Coverslips were placed and sealed with nail polish. Slides were allowed to dry completely before imaging. Images were captured at 63x magnification using a Zeiss LSM 710 confocal microscope (Leica, UIC Ophthalmology Core facility) and analyzed with ZeissLSM image software.

[0127] The citrulline-positive cells were neutrophils, as suggested by their multilobed nuclei. This means that one major source of citrulline on the ocular surface was neutrophils. Citrullinated neutrophil proteins may be responsible for the ACPA response. This was verified by the finding that ACPA-positive tears reacted with citrullinated neutrophil proteins in the dot blot assay. These data suggest that neutrophils are the source of citrullination and that citrullinated neutrophil proteins may be responsible for the ACPA response.

[0128] Example 5: In oGVHD, citrullinated proteins appear to be predominantly epithelial We next demonstrated that citrulline is expressed in epithelial cells in definite oGVHD. The purpose of this experiment was to compare citrulline expression between Sjögren's and definite oGVHD patients (Figure 8). Antibiotics were applied to the patient's eyes and filter paper (PALL, #60298) was applied to the temporal conjunctiva and lower eyelid to remove the superficial layer of the ocular surface epithelium. The filter papers were fixed with 4% paraformaldehyde (PFA) in a Petri dish for 20 minutes. They were then washed in 1XPBS for 5 minutes and mounted on microscope slides via adhesive tabs. A water-resistant barrier was drawn around the samples with a Pap pen, followed by permeabilization with PBS-T (0.025% Triton in 1XPBS) for 1 hour. With gentle shaking, the filter papers were washed once for 5 minutes in 1XPBS and then blocked with 2.5% donkey serum (secondarily produced host species) and 1% BSA in 1XPBS for at least 1 hour. The filters were then incubated with primary antibodies overnight at 4°C. The primary antibodies used were mouse monoclonal anti-citrulline antibody (1:1000; Millipore, MABN328) and rabbit polyclonal anti-cytokeratin 14 antibody (1:1000; BioLegend, #905301). The next day, the filters were washed three times with 1X PBS for 5 min with gentle shaking, followed by secondary antibody incubation for 1 h at room temperature. The secondary antibodies used were: AlexaFluor594 donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch Lab, #715-585-150) and Alexa Fluor 488 donkey anti-rabbit IgG (1:1000; Jackson ImmunoResearch Lab, #711-546-152).

[0129] After incubation, the filter papers were washed 3 times for 5 min in 1XPBS with gentle shaking in the dark. Slides were dried and mounted with ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, P36931). Coverslips were placed and sealed with nail polish. Slides were allowed to dry completely before imaging. Images were captured at 63x magnification using a Zeiss LSM 710 confocal microscope (Leica, UIC Ophthalmology Core facility) and analyzed with ZeissLSM image software. Unlike Sjögren's syndrome, where neutrophils were the primary source of citrulline, definite oGVHD patients have citrullinated total epithelial cells, which correlate with high levels of citrulline and PAD4 enzyme. These data indicated that strong citrullination of epithelial cells supports high levels of citrulline and PAD4 enzyme. Among the patients tested, colocalization of citrulline with K14 was not observed in two different Sjögren's patients, but was present in a patient with oGVHD. Furthermore, colocalization of citrulline with neutrophils was observed in two different Sjögren's patients.

[0130] Example 6: Inducible Fc receptors expressed on neutrophils Citrullinated proteins were not found in patients without oGVHD. These findings demonstrate that one source of autoantibody production is citrullinated neutrophils and / or ocular surface cells in patients with DED. We also demonstrated that Fc receptors (receptors for IgG) are present on the ocular surface and that ACPA antibodies interact with Fc receptors to produce surface disease. It has been hypothesized that Fc receptors are not expressed on naive neutrophils but are inducible. Fc receptors are expressed on neutrophils within mucosal cell aggregates.

[0131] The aim of this experiment was to support the hypothesis of inducible Fc receptors on neutrophils. Peripheral blood was collected by venipuncture into BD Vacutainer sodium heparin tubes (BD Biosciences, #367878) and immediately transported to the laboratory for neutrophil isolation. Neutrophils were isolated by immunomagnetic depletion of non-target cells using MACSxpress beads (MACSxpress Neutrophil Isolation Kit, Miltenyi Biotech, #130-104-434) according to the manufacturer's instructions. Residual red blood cells were removed using a MACSxpress Red Blood Cell Depletion Kit (Miltenyi Biotec, #130-094-183). Isolated neutrophils were resuspended in 3 mL of serum-free phenol red-free RPMI-1640 medium (GIBCO, #11835-030). After cell counting, 50,000 cells / 200 μL of neutrophils were loaded onto an EZ Single Cytofunnel (Thermo Scientific, #A78710003). Patient mucoid samples were prepared with 4 U / mL DNaseI and 1xBuffer in 100 μL solution and incubated at 37 °C for 30 min to achieve a single cell suspension. Mucoid samples diluted 10-fold were loaded onto an EZ Single Cytofunnel (Thermo Scientific, #A78710003). Samples were centrifuged at 1000 rpm for 5 min by Cytospin4 (Thermo Scientific, Kalamazoo, MI) to achieve a monolayer of cell deposition in a defined area of ​​a cytoslide (Thermo Scientific, #5991056). After centrifugation, slides were air-dried for 5 min and fixed in 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA, #15710) solution for 20 min. A water-resistant barrier was drawn around the samples with a Pap pen, followed by permeabilization with PBS-T (0.025% Triton in 1X PBS) for 1 h. Samples were washed once with 1XPBS for 5 min with gentle shaking, and then blocked with 2.5% donkey serum (secondarily produced host species) and 1% BSA in 1X PBS for at least 1 h. Samples were then incubated with primary antibodies overnight at 4 °C.The primary antibodies used were mouse monoclonal anti-CD64 antibody (1:1000; Santa Cruz, sc-1184), mouse monoclonal anti-IgMκ antibody (1:2500; Novus Biologicals, NBP1-96975), rabbit monoclonal anti-neutrophil erstase antibody (1:500; Abcam, ab131260) and rabbit monoclonal IgG antibody (0.03ug / mL; Abcam, ab172730).

[0132] The next day, samples were washed 3 times for 5 min with 1XPBS with gentle shaking, followed by secondary antibody incubation at room temperature for 1 h. Secondary antibodies used were: AlexaFluor594 donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch Lab, #715-585-150) and Alexa Fluor 488 donkey anti-rabbit IgG (1:1000; Jackson ImmunoResearch Lab, #711-546-152). After incubation, samples were washed 3 times for 5 min with 1XPBS with gentle shaking in the dark. Slides were dried and mounted with ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, P36931). Coverslips were placed and sealed with nail polish. Slides were allowed to dry completely before imaging. Images were captured at 63x magnification using a Zeiss LSM 710 confocal microscope (Leica, UIC Ophthalmology Core facility) and analyzed with Zeiss LSM image software.

[0133] As shown in Figure 9, Fc receptors were only expressed on neutrophils stimulated with PMA and on mucoids from patients, meaning that the neutrophils were activated. The staining was confirmed to be positive with isotype and negative controls. In summary, naïve neutrophils do not express Fc receptors, but activated neutrophils do, confirming that the expression of Fc receptors is inducible.

[0134] Additional experiments were performed to confirm the presence of Fc receptor-positive neutrophils on the ocular surface. Impression cytology was performed as described in Example 3 above. The primary antibodies used were mouse monoclonal anti-CD64 antibody (1:1000; Santa Cruz, sc-1184), and rabbit polyclonal anti-cytokeratin 14 antibody (1:1000; BioLegend, #905301). The next day, the filter paper was washed three times for 5 min with 1XPBS with gentle shaking, followed by secondary antibody incubation for 1 h at room temperature. The secondary antibodies used were: AlexaFluor594 donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch Lab, #715-585-150) and Alexa Fluor 488 donkey anti-rabbit IgG (1:1000; Jackson ImmunoResearch Lab, #711-546-152). After incubation, the filter papers were washed 3 times for 5 min with 1XPBS with gentle shaking in the dark. Slides were dried and mounted with ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, P36931). Coverslips were placed and sealed with nail polish. Slides were allowed to dry completely before imaging. Images were captured at 63x magnification using a Zeiss LSM 710 confocal microscope (Leica, UIC Ophthalmology Core facility) and analyzed with Zeiss LSM image software. After immunofluorescence staining, the same filter papers were stained with hematoxylin and eosin. Filter papers were stained with hematoxylin (Fisher Scientific, SH26-500D), rinsed with acid, immersed in bluing solution, and counterstained with eosin (Thermo Scientific, Waltham, MA). Slides were examined using an upright Axioscope 100 microscope (Carl Zeiss Meditec GmbH, Hamburg, Germany), imaged using a Zeiss MRc color camera, and analyzed using Zeiss Axiovision.

[0135] Figure 10 shows that Fc receptors are expressed on K14 negative cells (non-epithelial cells) and intermixed with epithelial cells. Some of these non-epithelial cells have multilobed nuclei (white boxes) reminiscent of neutrophils, which is further confirmed by H&E staining. Other cells are large mononuclear cells, which may be T cells. K14 positive cells do not show Fc receptors, which is confirmed in the literature.

[0136] Further experiments were performed to determine whether ACPAs in tears react with NET citrullinated proteins. Dot blot assays were performed using a Bio-Dot microfiltration device (Bio-Rad, #170-6545) with a nitrocellulose membrane (Company, cat#). The overall protocol followed the manufacturer's instructions. The membrane was pre-wetted with TBS. It was rehydrated as necessary. 1 μg / 100 μL of total protein from the lysate was applied to the membrane and incubated for 1 h with the vacuum chamber open to air to allow the protein to pass through the membrane by gravity. 200 μL of blocking solution (1% BSA-TBS) was added to each well and filtered by gravity, followed by washing. 200 μL of washing solution (0.05% Tween-TBS) was added, aspirated, and repeated once more. 100 μL of 1:80 diluted patient tear fluid (diluted in 1% BSA-TTBS) was added and allowed to filter by gravity, after which the membrane was washed with 200 μL of TTBS. The membrane was incubated with 100 μL of HRP-conjugated human IgG antibody (#A112P, EMD). The blot was incubated with enhanced chemiluminescence SuperSignalWest Femto maximum sensitivity substrate (34095, Thermo Fisher) and the signal was detected with an ImageQuantLAS 4000 system (GE Lifesciences Inc). The intensity of the dot blot was determined using ImageJ software.

[0137] Neutrophils were simulated for 3 h with calcium ionophore, to induce hypercitrullination and RPMI for the same period served as a control. After stimulation, neutrophils were lysed to extract total proteins, which were immobilized on membranes. As shown in Figure 11, ACPAs in tears were found to react with NET proteins, confirming that citrullinated proteins in neutrophils may be one source of ACPA stimulation. Results were expressed as a fold calculated using the following formula: fold = intensity of citrullinated protein / intensity of wild-type protein. ACPA-positive tears show greater reactivity to stimulated NET proteins (92.8 ± 7.85) than unstimulated NET proteins (54.1 ± 5.46), whereas ACPA-negative tears showed no difference (45.0 ± 3.19 vs. 49.3 ± 4.79). In summary, ACPAs in tears react to NET proteins.

[0138] Another experiment was performed to determine whether polyclonal ACPAs are present in tears. Dot blot assays were performed using Direct Detect assay free cards (#DDAC00010-GR) obtained from Millipore (Burlington, MA). 2 μL of recombinant protein (1 μg) was applied to the membrane and allowed to dry for 20 min at room temperature (RT). The membrane was then blocked with 10% BSA and 0.05 Tween-20 prepared in 1XPBS (pH 7.4) for 30 min at RT. The membrane was rinsed with rinse buffer (1% BSA and 0.05% Tween 20 in 1XPBS) and probed with a patient tear sample (1:40 dilution, diluted in sterile water), applied to the membrane, and incubated for 30 min at RT. The membrane was washed three times with rinse buffer and then incubated with HRP-conjugated human IgG antibody (#A112P, EMD) for 30 min at RT. The membrane was washed three times with rinse buffer. Blots were incubated with enhanced chemiluminescence SuperSignalWestFemto maximum sensitivity substrate (34095, Thermo Fisher) and signals were detected with an ImageQuantLAS 4000 system (GE Lifesciences Inc). The intensity of the dot blots was determined using ImageJ software. The results shown in Table 2 are expressed as a fold calculated using the following formula: fold = intensity of citrullinated protein / intensity of wild-type protein (Table 2).

[0139] As shown in Figure 12, ACPA-positive tears indicate the presence of polyclonality, and ACPAs react with several citrullinated proteins. The fold change of citrullinated fibrinogen or enolase over wild type was greater than 1.5 in both Sjogren's and definite oGVHD patients. In Sjogren's patients, citrullinated vimentin was greater than 1.5-fold change over wild type, whereas in definite oGVHD, citrullinated histone H4 was greater than wild type. Tears from two diseased patients demonstrated polyclonality of ACPAs in their tears, whereas neurotropic and healthy tears are negative for ACPAs. In summary, ACPA-positive tears show polyclonality. (+):>1.5. [Table 2]

[0140] Example 7: ACPA H4R3cit antisera in ocular surface disease We next investigated whether a specific autoantibody (ACPA (H4R3cit antiserum)) causes ocular surface disease in an experimental model. Specifically, this experiment examines whether fH4R3cit is present in mucosal cell aggregates from patients. Mucous cell aggregates (MCAs) were collected from the patient's eyes using sterile jeweler's forceps and transferred to a sterile 0.2 mL PCR tube containing RefreshOptive and stored in an icebox. Fresh MCA samples were embedded in Tissue-Tek® OCT compound (Sakura Finetek, Torrance, CA, #4583) and flash frozen. Frozen sections were cut at 10 μm thickness using a cryostat (Thermo Scientific, CryoStar NX50, #957130). This particular MCA was collected from a patient diagnosed with definite oGHVD. For immunostaining, slides were air-dried and then fixed in 4% PFA for 20 min. Samples were then permeabilized with 0.025% Triton X-100 (Fisher Scientific, #BP151-100) and blocked with freshly prepared 10% donkey serum and 1% bovine serum albumin (Gemini Bio-Products, #700-100P) for 2 h. Samples were incubated with primary antibodies overnight at 4 °C. The following primary antibodies are used: mouse monoclonal anti-human neutrophil elastase (NE) (1:100; Dako, #M0752), rabbit polyclonal anti-histone H4 (citrulline R3) antiserum (1:1000; Abcam, ab81797) and rabbit polyclonal IgG (1:1000; Abcam, ab37415). Slides were washed three times for 5 min with 1XPBS with gentle shaking and incubated with secondary antibodies diluted in blocking buffer for 1.5 h at room temperature. The secondary antibodies used were: AlexaFluor594 donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch Lab, #715-585-150) and Alexa Fluor 488 donkey anti-rabbit IgG (1:1000; Jackson ImmunoResearch Lab, #711-546-152).Slides were washed twice with 1XPBS, counterstained with Hoechst 33342 (2 μl / mL) (Thermo Scientific, #62249) for 10 minutes, quickly washed with PBS, and covered with ProLong™ Gold antifade reagent (Invitrogen, #P36930). Images were captured at 100x magnification using a Zeiss LSM 710 confocal microscope (Leica, UIC Ophthalmology Core facility) and analyzed with ZeissLSM image software.

[0141] To confirm the presence of H4R3cit on the ocular surface of patients, MCA were collected from patients diagnosed with definite oGVHD and analyzed by immunofluorescence staining. Immunofluorescence stained cells showed numerous neutrophils with multilobed nuclei, which were confirmed by positive staining for neutrophil elastase. It is known in the art that H4R3cit is expressed around the nucleus (perinuclear), which can be seen in Figure 6. As shown in Figure 13, H4R3cit at H is immunolocalized on the ocular surface, and dot blot analysis using tears from oGVHD patients shows a reaction with H4cit. Figure 13 also shows the presence of H4R3cit in the mucosal cell aggregates of the patient. The staining was confirmed to be positive, as the isotype control showed negative staining. In summary, the mucosal cell aggregates of the patient have the H4R3cit antigen, which may be the cause of the ACPA response.

[0142] To investigate whether ACPA antibodies cause ocular surface disease, 8-10 week old Thy1-YFP mice were used for treatment experiments. Thy1-YFP mice (n=5 / group) were anesthetized by intraperitoneal injection of a combination of ketamine (20 mg / kg; Phoenix Scientific, St. Joseph, MO) and xylazine (6 mg / kg; Phoenix Scientific). To evaluate the pathological effects of ACPA, ACPA antibodies were applied to the corneas of mice and the extent of corneal surface disease induced by the antibodies was determined and compared with application of non-ACPA antibodies. The following conditions were used: 1) non-ACPA antibody: anti-histone H4 antibody (100ng / mL; Cell Signaling #2592, 2) ACPA antibody #1: anti-histone H4 (citrulline R3) (H4R3cit) antiserum (100ng / mL; Abcam #ab81797), 3) for 7 consecutive days, ACPA antibody #2: anti-histone H3 (citrulline R2+R8+R17) antiserum (100ng / mL; Novus Biologicals #NB100-57135SS) was applied to the surface of mouse corneas (10uL / cornea) and incubated for 40 minutes. Since the ACPA antibodies used were contained in rabbit antisera, normal rabbit serum application (100ng / mL; Abcam #ab7487) was used as a control. All antibody dilutions were performed in RefreshOptive. For fluorescein staining experiments, 10 μl of 0.2% fluorescein was infiltrated into the mouse cornea for 1 min and washed twice with 1XPBS. Fluorescein staining was detected and photographed with a slit lamp (Haag Streit, Bern, Switzerland) using cobalt blue illumination and a barrier yellow filter. Images from each group (n=5) were collected and the intensity of fluorescein staining was measured with a Metamorph imaging system (Metamorph, Universal Imaging, Downington, PA).

[0143] Application of H4R3cit antiserum to mouse corneas demonstrated a significant increase in ocular surface disease as evidenced by corneal fluorescein staining. Fluorescein staining was significantly increased in several controls (rabbit serum, H3cit antiserum, wild-type H4 antibody) (Figure 14). As shown in Figure 14, panels A1 and A5 show that seven consecutive days of non-ACPA antibody application on mouse corneas significantly reduced ocular surface disease as evidenced by the lack of fluorescein staining on day 7 (2.59x10 6 ±5.41x10 4 ), indicating that none of the ACPA antibodies produced corneal epithelial disease, as evidenced by a significant increase in corneal fluorescein staining on day 7. Of the two ACPA antibodies used, only one produced corneal epithelial disease, as evidenced by a significant increase in corneal fluorescein staining on day 7. The ACPA antibody that produced corneal epithelial disease was the only one that did not produce corneal epithelial disease, as evidenced by a significant increase in corneal fluorescein staining on day 0 (3.00x10 6 ±6.05x10 4 , P = 0.0006, Fig. 14, A2 and A6) compared with ACPA#1-H4R3cit serum (1.85x10 7 ±3.19x10 6 Fluorescence intensity of 3.36x10 6 ±3.05x10 5 , P = 0.97, Fig. 14, A3 and A7, compared with 3.34x10 6 ±4.90x10 5 Fluorescence intensity at day 7 (3.11x10 6 ±3.49x10 5 The vehicle control also did not cause corneal epithelial disease, as evidenced by the lack of fluorescein staining in Figure 14, A4 and A8. In summary, these data indicate that ACPA antibodies cause corneal epithelial disease, but not all ACPA antibodies are capable of doing so. [Table 3]

[0144] Experiments were also performed to determine whether ACPA antibodies could induce NETosis. Thy1-YFP mice aged 8–10 weeks were used for treatment experiments. Thy1-YFP mice (n=5 / group) were anesthetized by intraperitoneal injection of a combination of ketamine (20 mg / kg; Phoenix Scientific, St. Joseph, MO) and xylazine (6 mg / kg; Phoenix Scientific). Mice were divided into three groups: 1) RPMI (Gibco #11835030) treatment, which served as a control; 2) ACPA antibody #1:H4R3cit antiserum; or 3) ACPA antibody #2:H3cit antiserum treatment. 10 μL of each treatment was applied to the mouse cornea. Eyes were treated for 7 consecutive days. At the end of the experiment, impression cytology of mouse corneas was performed using filter paper staining. Microscope slides were attached with adhesive tabs (EMS, #76760). Filter paper (Millipore, #JHWP02500) was cut to the optimal size. While the mouse was under anesthesia, a drop of proparacaine was applied to the mouse's eye for 1 min and gently removed with a Kimwipe. The filter paper was placed on the cornea and gently pressed for 30 s. It was then gently lifted and placed on an adhesive tap, with the touched part facing up. All the following steps were performed in a humid chamber, except for the washing steps. A water-resistant barrier was drawn around the sample with a Pap pen, and the filter paper was fixed in 4% paraformaldehyde (PFA) for 20 min. With gentle shaking, the filter paper was washed in 1XPBS for 5 min, and then permeabilized in PBS-T (0.025% Triton in 1XPBS) for 10 min. With gentle shaking, the filter paper was washed once in 1XPBS for 5 min, and then blocked with 2.5% donkey serum (secondarily produced host species) and 1% BSA in 1XPBS for at least 1 h. The filters were then incubated with primary antibodies overnight at 4°C. The primary antibodies used were mouse monoclonal anti-citrulline antibody (1:1000; Millipore, MABN328) and rabbit polyclonal anti-histone H4 (citrulline R3) antiserum (1:1000; Abcam, ab81797). The next day, the filters were washed three times with 1XPBS for 5 min with gentle shaking, followed by secondary antibody incubation at room temperature for 1 h.The secondary antibodies used were: AlexaFluor594 donkey anti-mouse IgG (1:1000; Jackson Immu-noResearch Lab, #715-585-150), Alexa Fluor 488 donkey anti-rabbit IgG (1:1000; Jackson Immu- noResearch Lab, #711-546-152), Alexa Fluor 594 donkey anti-rabbit IgG (1:1000; Invitrogen, #A21207), and Alexa Fluor 488 donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch Lab, #715 -547-003). After incubation, the filters were washed three times for 5 min with 1XPBS in the dark with gentle shaking. Slides were dried and mounted with ProLong™ Gold Antifade Mountant with DAPI (Invitogen, P36931). A coverslip was placed and sealed with nail polish. Slides were allowed to dry completely before imaging. Images were captured at 63x magnification using a Zeiss LSM 710 confocal microscope (Leica, UIC Ophthalmology Core facility) and analyzed with Zeiss LSM imaging software. After 7 days of treatment with 1) RPMI, 2) ACPA#2-H3cit antiserum, or 3) ACPA#1-H4R3cit antiserum, mouse corneal epithelium was lifted onto filter paper to examine for other pathological effects.

[0145] As shown in Figure 15, NETosis was induced by ACPA-H4R3cit antiserum. The presence of neutrophils and neutrophil extracellular traps (NETs) was observed only in the ACPA#1-H4R3cit antiserum treatment group (Figure 15, A1), and NETs were confirmed by neutrophil elastase staining (Figure 15, A2). This suggests that H4R3cit antiserum induced NETosis. In summary, ACPA antibody #1 caused NETosis in mouse corneal epithelium and induced citrullination. This data suggests that ACPA antibodies contribute to a feed-forward cycle of citrullination to further amplify citrullination-associated eye diseases.

[0146] Example 8: Blocking interactions of ACPA(H4R3cit) - An Fc receptor blockade strategy abolished ACPA-H4R3cit-induced ocular surface disease. To determine whether the pathological effects of ACPA on the cornea are mediated through Fc receptors, 8-10 week old Thy1-YFP mice were used for treatment experiments. A peptide blocking all Fc receptors was used and ACPA antibodies were applied. 10 μL of 1) azide-free Fc receptor blocker (Innovex Biosciences #NB355) or 2) scrambled peptide was used and applied to the mouse cornea and incubated for 30 min. This preincubation with peptide was performed without anesthesia. Then, for 10 consecutive days, Thy1-YFP mice (n=5 / group) were anesthetized and 10 μL of ACPA#1-H4R3cit antiserum was applied to both the Fc receptor blocking group and the scrambled peptide blocking group and incubated for 40 min. Dilutions were performed in RefreshOptive. The mouse corneas were stained with fluorescein, monitored, and imaged with a slit lamp. Fluorescein intensity was measured by a Metamorph Imaging System. To investigate whether competitive blockade of Fc receptors reproduces the same results as peptide blockade of Fc receptors, mouse IgG was added together with ACPA antibodies. For IgG competition experiments, 10 μL of a mixture of ACPA antibodies (H4R3cit antiserum) and mouse IgG (Abcam#ab188776) was applied to mouse corneas in a 1:1 ratio and incubated for 40 min for 10 consecutive days. In control experiments, only ACPA antibodies were used. Mouse corneas were fluorescein stained, monitored, and imaged with a slit lamp. Fluorescein intensity was measured by a Metamorph imaging system. After 10 days, corneas were harvested, lysed, and subjected to Luminex for cytokine detection.

[0147] As shown in Figure 16, both strategies significantly reduced H4R3cit-induced ocular surface disease. Analysis of cytokines expressed in the cornea revealed that IL-2 was significantly increased in ACPA conditions but not in controls (Figure 16). Blocking Fc receptors with peptide blockers reproduced the inverse effect of H4R3cit antisera. Corneas were collected at the end of the experiment, lysed, and run for cytokine profiles. IL-2 levels were significantly reduced in both competitive and peptide-blocked mouse corneas than in unblocked ones. In summary, mouse IgG competes with ACPA antibodies for binding to Fc receptors, and Fc receptor blockers block all Fc receptors. This competitive and peptide blocking abolished the pathological effects of ACPA on the cornea, and using methods to prevent ACPA antibody interaction with Fc receptors may be a potential therapeutic strategy in the treatment of citrullination-associated eye diseases.

[0148] Example 9: ACPA-H4R3cit induces NETosis in vitro. To determine whether ACPAs can induce NETosis in vitro, peripheral blood was collected by venipuncture into BD Vacutainer sodium heparin tubes (BD Biosciences, #367878) and immediately transported to the laboratory for neutrophil isolation. Neutrophils were isolated by immunomagnetic depletion of non-target cells using MACSxpress beads (MACSxpress Neutrophil Isolation Kit, Miltenyi Biotech, #130-104-434) according to the manufacturer's instructions. Residual red blood cells were removed using a MACSxpress Red Blood Cell Depletion Kit (Miltenyi Biotec, #130-094-183). Isolated neutrophils were resuspended in 3 mL of serum-free phenol red-free RPMI-1640 medium (GIBCO, #11835-030). After cell count, 1.0x106 cells / mL were plated onto chamber glass slides (Millipore, #PEZGS0416) and incubated with the following antibodies along with controls: 1) RPMI as a negative control, 2) 1nM PMA as a positive control, 3) 100ng / mL normal rabbit serum (Abcam, ab7487), 4) 100ng / mL H3cit antiserum (Novusbio, NB100-57135SS), 5) 100ng / mL H4R3cit antiserum (Abcam, ab81797), 6) 100ng / mL citrullinated fibrinogen antibody (Cayman Chemical, 17088), 7) 100ng / mL citrullinated vimentin antibody (Cayman Chemical, 22054), 8) 100ng / mL citrullinated α-enolase antibody (Cayman 9) CCP antibody (Bioss, bs-1053R). Neutrophils were incubated overnight and stained with 1 μM Sytox Green (Molecular Probes, Invitrogen, Cat. No. S7020) and 5 μM Hoechst (Fisher Scientific, Pittsburgh, PA, #33342). They were directly imaged at 20x using a Zeiss Observer Z1.

[0149] As shown in Figure 17, in vitro experiments using isolated human neutrophils showed that ACPA-H4R3cit stimulation, but not the control (other ACPA), produced a significant increase in extracellular DNA strands. Only PMA stimulation, ACPA-H4R3cit stimulation, and CCP ACPA stimulation produced significant extracellular DNA strands. In summary, ACPA-H4R3cit induces NETosis in vitro.

[0150] Example 10: Determination of the cytotoxicity of ocular surface immunoglobulin (OSIG) towards human corneal epithelial cells under normal or stress conditions. Primary human corneal epithelial cells were used to determine the cytotoxicity of OSIG against human corneal epithelial cells under normal or stress conditions. Primary human corneal epithelial cells were purchased from EMD Millipore (EMD, #SCCE016). Cells were grown in EpiGRO™ Human Ocular Epithelia Complete MedUM (EMD, #SCMC001). The day before wound scratch, 20,000 cells / well were seeded into 96-well ImageLock plates (Essen Bioscience, #4379) and grown for 18 hours to achieve monolayer confluence. ImageLock plates are specially modified plates, and the technique is made possible by fiducial markers at the bottom of the plate that provide a point against which the image position can be precisely referenced. Wound scratches (700-800 μm wide) were created with an IncuCyte 96-pin wound maker (Essen Bioscience, #4493). After scratching, cells were washed twice with 100 μL of phenol red-free RPMI-1640 medium (Gibco #11835030). For dose-dependent experiments with OSIG (Flebogamma 5% DIF), the medium was replaced with 200 μL of the following conditioned medium: (1) EpiGRO, (2) 4 mg / mL OSIG, or (3) 8 mg / mL OSIG. OSIG dilutions were performed in EpiGRO. Plates were incubated in an IncuCyteZoom live cell analysis system (EssenBioscience). Images were captured every 3 hours. Relative wound density (%) was measured for 69 hours using IncuCyteZoom software. This metric relies on measuring the spatial cell density of the wound area relative to the spatial cell density outside the wound area at all time points. It is designed to be 0% at t=0 and 100% when the cell density within the wound is the same as the cell density outside the initial wound. It does not rely on finding the cell border. The cytotoxicity of HCE-T cells was determined by LDH (lactose dehydrogenase) cytotoxicity assay (Thermo Scientific, #88954). Cell culture supernatants were collected, 50 μL of the supernatant was mixed with 50 μL of the reaction mixture, incubated for 30 min, and the mixture was transferred to a 96-well flat-bottom plate.The absorbance at wavelengths (490-680 nm) was measured using a Cytation5 plate reader.

[0151] Human corneal epithelial (HCE-T) cell line was used for the epithelial scratch assay, which is a human corneal cell transformed with SV40-Adeno vector (RIKEN Cell Bank RCB2280, Tsukuba, Japan). Cells were grown in DMEM medium (GIBCO, #11965-092) supplemented with 1% antibiotic and 10% FBS (Invitrogen, #26140-079) and antimycotic solution containing 10,000 units / mL penicillin, 10,000 μg / mL streptomycin, and 25 μg / mL Gibco Amphotericin B (Thermo Fisher Scientific, #15240062) and incubated in a tissue culture incubator at 37 °C supplied with 5% CO2. The day before wound scratch, 30,000 cells / well were seeded into 96-well ImageLock plates (Essen Bioscience, #4379) and grown for 18 h to achieve monolayer confluence. ImageLock plates are specially modified plates whose technique is made possible by fiducial markers at the bottom of the plate that provide a point to which the image can be precisely referenced. Wound scratches (700-800 μm wide) were created with an IncuCyte 96-pin wound maker (Essen Bioscience, #4493). After scratching, cells were washed twice with 100 μL of phenol red-free RPMI-1640 medium (Gibco #11835030). For dose-dependent experiments with OSIG (Flebogamma 5% DIF), the medium was replaced with 200 μL of conditioned medium as follows: (1) Normal condition (Figure B): (i) Complete medium (CM), (ii) 4 mg / mL OSIG or (iii) 10 mg / mL OSIG, (2) Stress condition (Figure C): (i) RPMI; (ii) 4 mg / mL OSIG or (iii) 10 mg / mL. Plates were incubated in an IncuCyteZoom live cell analysis system (EssenBioscience). Images were captured every 3 hours. Relative wound density (%) was measured for 12 hours using IncuCyteZoom software.This metric relies on measuring the spatial cell density in the wound area relative to the spatial cell density outside the wound area at all time points. It is designed to be 0% at t=0 and 100% when the cell density inside the wound is the same as the cell density outside the initial wound. It does not rely on finding the cell boundary. The cytotoxicity of HCE-T cells was determined by LDH (lactose dehydrogenase) cytotoxicity assay (Thermo Scientific, #88954). Cell culture supernatants were collected, 50 μL of the supernatant was mixed with 50 μL of reaction mixture, incubated for 30 min, and the mixture was transferred to a 96-well flat-bottom plate. The absorbance at wavelengths (490-680 nm) was measured with a Cytation5 plate reader. 10 mg / mL OSIG was toxic in all three conditions, while 4 mg / mL was not toxic, which could serve as a working concentration (effective and non-toxic). As shown in Figure 18, toxicity did not change whether the cells were in a normal or stressed state. In summary, 4 mg / mL OSIG is the highest concentration without toxicity.

[0152] Example 11: ACPA responses at the ocular surface are active ocular antibody responses We investigated how many patients with high ocular surface disease had high ACPAs in their tears despite being found to have no ACPA antibodies in their serum and to be rheumatoid factor negative. Forty-nine percent of 166 patients were cyclic citrullinated peptide (CCP) negative in their serum but positive for ACPAs in their tears. Sixty-nine percent of these patients were rheumatoid factor negative. This suggests that the ocular surface ACPA response is an active local antibody production response, not a passive extravasation of ACPAs from the serum, and does not require a concomitant systemic immune disorder. [Table 4]

[0153] Example 12: Example of an ocular embodiment patient with high autoantibodies (ACPA) in tears and severe ocular surface disease The first patient was a 38-year-old woman with Sjögren's syndrome. As shown in Figure 19, the patient had high autoantibodies (ACPA) in her tears and corneal melt in her right eye. Despite treatment with steroid eye drops, serum tears and other conventional DED therapies, the patient remained severely symptomatic. This case demonstrates the association between high autoantibody levels and severe ocular surface disease. [Table 5]

[0154] The second patient was a 39-year-old woman who presented with severe ocular discomfort in May 2018. Examination revealed superior keratoconjunctivitis (SLK) 3+ in both eyes. She was started on methylprednisolone eye drops. At follow-up in June 2018, symptoms of ocular discomfort still persisted despite treatment. ACPA levels in tears were very high. Serum was negative for ACPA and rheumatoid arthritis. Subsequent ACPA levels measured in July 2018 were also high, and the patient still had severe symptoms. At testing in October 2018, ACPA levels had decreased but were still high, and the patient's symptoms had somewhat abated. As shown in Figure 20, this case demonstrates that autoantibodies (ACPA) were present on the eye, despite the absence of autoantibodies in the blood and the absence of a systemic autoimmune disease such as rheumatoid arthritis. This suggests that autoantibodies were produced locally in the ocular tissue. [Table 6]

[0155] The third patient was a 47-year-old woman with asymmetric ocular disease. This patient presented in February 2017 with severe dry eye and ocular discomfort. Examination revealed that the right eye had more severe ocular surface disease than the left eye. The right eye had much less tear secretion than the left eye (Schirmer I was 1 mm in the right eye and 12 mm in the left eye). As shown in Figure 21, the right corneal staining was 8 / 15 and the left eye was 2 / 15. The right eye conjunctival staining was 6 / 6 and the left eye was 3 / 6. ACPA in tears was high only in the right eye and not in the left eye. Eyes with more severe ocular surface disease have higher autoantibody (ACPA) levels in tears. This case shows that higher ACPA levels correlate with more severe ocular disease.

[0156] Example 13: Additional Patient Case Studies (Case Studies 1-6) In the following case studies, patients were administered commercially available IVIG (Flebogamma 10%) formulated to represent OSIG. The commercially available IVIG (10%) was diluted with an ophthalmic vehicle, NaCl solution, to a final concentration of 4 mg / ml IgG (0.4%) with a pH of at least 6.0. The resulting 0.4% IgG formulation (referred to herein as "OSIG") was administered to patients as eye drops using eye drops. OSIG was produced using commercially available IVIG (Flebogamma 10%) as it is not currently commercially available, however, OSIG formulations can be produced using commercially available IgG or pooled human immunoglobulin G derived from pooled plasma. These case studies provide clinically relevant results relevant to the ophthalmic formulations provided herein.

[0157] Case study 1 was a 56-year-old woman with severe aqueous tear deficiency and severe ocular surface disease due to ocular graft-versus-host disease (Figure 22). OSDI was 77.7 and corneal staining was 6 / 15 in the right eye. VBR was 70 in the right eye showing ocular redness. Corneal scarring was present without corneal neovascularization. OSIG eye drops 0.4% was initiated twice daily. The patient reported a significant reduction in ocular discomfort (OSDI reduced to 30.5), a significant reduction in corneal staining (2 / 15), and a significant reduction in VBR (50) after 2 weeks of treatment. Over the next 2 weeks of treatment, VBR reduced to 30 and corneal staining reduced to 1 / 15. Extracellular DNA in tears was 56 μg / mL in the right eye and reduced to 12.3 μg / mL after OSIG treatment. In this example, OSIG eye drops reduced the signs and symptoms of severe dry eye, as well as reduced inflammatory biomarkers in tears.

[0158] Case Study 2 was a 31-year-old female with severe aqueous tear deficiency and severe ocular surface disease due to ocular graft-versus-host disease (Figure 23). The patient had severe conjunctival keratinization outside the coverage area of ​​her PROSE contact lenses, but no corneal neovascularization, and severe symptoms of ocular discomfort. OSIG eye drops 0.4% twice daily were initiated. One month after treatment, conjunctival keratinization had decreased and the patient reported "much improved" subjective ocular symptoms. In this example, OSIG eye drops reduced keratinization and significantly reduced ocular discomfort due to severe dry eye.

[0159] Case study 3 was a 74-year-old male with neurotropic keratitis due to a surgical history of previous LASIK eye surgery in the left eye (Figure 24). The cornea showed staining of 2 / 15 in the left eye and the patient reported an intensity of ocular discomfort of 4 / 10. The cornea showed faint scarring at the edge of the LASIK flap without corneal neovascularization. Overall, the examination was consistent with a diagnosis of symptom-sign disconnection (discordant DED). OSIG eye drops 0.4% were started twice daily. One month after treatment, the patient was subjectively improved (SGA) and the symptom intensity decreased to 3. The corneal staining in the left eye had disappeared (0 / 10). In this instance, OSIG eye drops were beneficial for postoperative complications leading to symptom-sign disconnection.

[0160] Case study 4 was a 39-year-old male with severe aqueous tear deficiency and severe ocular surface disease. The corneas showed SPK staining of 1 / 15 in the right eye and 3 / 15 in the left eye, without corneal neovascularization. The patient reported an intensity of ocular discomfort of 3 / 10. OSIG eye drops 0.4% twice daily was initiated. After 1 month of treatment, the intensity of ocular discomfort decreased to 2 / 10 and corneal staining had disappeared in both eyes (0 / 10). This example demonstrates the beneficial effect of OSIG eye drops in ocular cicatricial pemphigoid.

[0161] Case Study 5 was a 33 year old diagnosed with symptomatic discontinuity (discordant DED) with severe aqueous tear deficiency and severe ocular discomfort. There was no corneal staining and no corneal neovascularization. The intensity of ocular discomfort was 6 / 10. OSIG eye drops 0.4% twice daily was initiated. After one month of treatment, the intensity of ocular discomfort decreased to 3 / 10 in the right eye and 4 / 10 in the left eye. This example illustrates the beneficial effect of OSIG eye drops in reducing ocular discomfort in a patient without any signs of the condition.

[0162] Case study 6 was a 29-year-old male with aqueous tear deficiency and severe ocular surface disease due to Stevens-Johnson syndrome (Figure 25). The OSDI was 54.7 and the symptom intensity was 8 / 10 (intense, terrible discomfort). OSIG eye drops 0.4% were initiated in both eyes 3 times daily. The patient reported a significant decrease in ocular discomfort (OSDI decreased to 14.2) and a decrease in symptom intensity to 0 / 10 (no discomfort) after 2 weeks of treatment. The light sensitivity and grittiness of the eye were present "all the time" prior to OSIG treatment. After OSIG treatment, the light sensitivity was only "part of the time" and the grittiness was completely absent. Ocular redness also decreased with OSIG treatment. In this instance, OSIG eye drops significantly reduced the signs and symptoms of dry eye after Stevens-Johnson syndrome. The present invention provides, for example, the following items. (Item 1) 1. An ophthalmic formulation comprising: (a) one or more pharma- ceutically acceptable ophthalmic excipients; (b) An ophthalmic preparation comprising immunoglobulin G (IgG) or a fragment thereof. (Item 2) 1. An ophthalmic formulation comprising: (a) one or more pharma- ceutically acceptable ophthalmic excipients; (b) An ophthalmic formulation comprising an ophthalmic pharmacologic compound for the treatment of a clinical condition selected from the group consisting of inflammatory, infectious and / or immunological ocular surface or intraocular diseases, wherein said pharmacologic compound comprises immunoglobulin G (IgG). (Item 3) Steroids, anti-inflammatory agents, mucolytic agents, PAD enzyme inhibitors (paclitaxel, glucocorticoids or Cl-amidine) or NET degraders (DNase or heparin), targeted Fab antibody fragments, Fc receptor blocking peptides, Fc receptor blocking antibodies, recombinant peptides containing pathogenic epitopes, conventional synthetic DMARDs (methotrexate, leflunomide / teriflunomide, sulfasalazine, chloroquine / hydroxychloroquine), TNF-α targeted therapies (infliximab, adalimumab, etanercept, golimumab, sertoli). 3. The ophthalmic formulation of item 1 or 2, further comprising a second pharmacologic active compound selected from: B-cell targeted therapy (rituximab, ofatumumab, belimumab, atacicept, tabalumab), T-cell targeted therapy (abatacept, belatacept), interleukin targeted therapy (tocilizumab, anakinra, canakinumab, rilonacept, secukinumab), growth and differentiation factors (denosumab, mavrilimumab), JAK pathway inhibitors (tofacitinib, baricitinib, filgotinib), and combinations thereof. (Item 4) 1. An ophthalmic formulation comprising a pharma- ceutically active compound capable of reducing the amount of or the deleterious biological effects of autoantibodies on the surface or in the eye, wherein the autoantibodies are produced in response to citrullinated proteins, homocitrullinated proteins or native proteins. (Item 5) 5. The ophthalmic formulation of item 4, wherein the pharma- ceutical active compound comprises immunoglobulin G (IgG) or a fragment thereof. (Item 6) 6. The ophthalmic formulation according to any one of items 1 to 5, wherein the pharma- ceutical active compound comprises pooled human immunoglobulin G derived from pooled plasma. (Item 7) 1. An ophthalmic formulation comprising: (a) a pharma- ceutically acceptable ophthalmic excipient; (b) a therapeutically effective amount of a pharma- ceutical active compound comprising pooled human immunoglobulin G (IgG), and (c) An ophthalmic formulation comprising a therapeutically effective amount of pooled human plasma proteins, pooled human plasma lipids, or a combination thereof. (Item 8) 8. The ophthalmic formulation according to any one of items 1 to 7, wherein said pharma- ceutical active compound comprises autologous IgG purified from autologous plasma / serum, multimerized IgG1 Fc molecules, IgG1 Fc hexamers, IgG2a Fc multimers, stradomers, multivalent Fc structures, glycoengineered sialylated IgG, IgG Fc glycosylation, synthetic IgG or fragments thereof, or combinations thereof. (Item 9) 9. The ophthalmic formulation according to any one of items 1 to 8, further comprising one or more pharma- ceutically acceptable ophthalmic excipients. (Item 10) One or more of the pharma- ceutically acceptable ophthalmic excipients may be selected from the group consisting of cyclodextrin, carbopol or carbomer or acrylic acid polymers, poloxamer, xyloglucan, methylcellulose, hydroxypropylmethylcellulose, ethyl(hydroxyethyl)cellulose, pseudolatex, cellulose acetate phthalate, gellan gum, alginate, carrageenan, hyaluronic acid, sodium acetate, disodium edetate, hypromellose, acetic acid, alcohol, alginic acid, Amerchol-cab, antipyri- yl . Ingredients: benzalkonium chloride, benzododecinium bromide, boric acid, caffeine, calcium chloride, carbomer 1342, carbomer 934P, carbomer 940, carbomer homopolymer type B (allyl pentaerythritol crosslinked), sodium carboxymethylcellulose, castor oil, cetyl alcohol, chlorobutanol, citric acid, citric acid monohydrate, creatinine, divinylbenzenestyrene copolymer, ethylene vinyl acetate copolymer, gellan gum (low acyl), glycerin, glyceryl stearate, hypromellose, lanolin, chloride Lauralkonium, lauroyl sarcosine, magnesium chloride, methylparaben, mineral oil, nonoxynol-9, octoxynol-40, petrolatum, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric nitrate, polydonium chloride, poloxamer 188 or 407, polycarbophil, polyethylene glycol 400 or 8000, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 40 stearate, polypropylene glycol, polysorbate 20, polyvinyl alcohol, potassium chloride, soybean oil, sorbitol, sorbitol 20 ... Potassium sorbate, Povidone K29 / 32, Povidone K30, Povidone K90, Povidone, Propylene glycol, Propylparaben, Soda ash, Sodium acetate, Sodium bisulfate, Sodium borate, Sodium borate decahydrate, Sodium carbonate, Sodium chloride, Sodium citrate, Sodium metabisulfate, Sodium nitrate, Sodium sulfate, Sodium sulfite, Sodium thiosulfate, Sorbic acid, Sorbitol, Stabilized oxychloro complex, Sulfuric acid, Thimerosal, Titanium dioxide, Tocophersolan, Trisodium citrate dihydrate, Tromethamine,10. The ophthalmic formulation according to item 9, wherein the active ingredient is selected from tyloxapol, xanthan gum, zinc chloride, or a combination thereof. (Item 11) Steroids, anti-inflammatory agents, mucolytic agents, PAD enzyme inhibitors (paclitaxel, glucocorticoids or Cl-amidine) or NET degraders (DNase or heparin), targeted Fab antibody fragments, Fc receptor blocking peptides, Fc receptor blocking antibodies, recombinant peptides containing pathogenic epitopes, conventional synthetic DMARDs (methotrexate, leflunomide / teriflunomide, sulfasalazine, chloroquine / hydroxychloroquine), TNF-α targeted therapies (infliximab, adalimumab, etanercept, golimumab, certolizumab, rituxim ... 11. The ophthalmic formulation of any one of items 1 to 10, further comprising a second pharmacologic active compound selected from an anti-inflammatory drug (AAG), B-cell targeted therapy (rituximab, ofatumumab, belimumab, atacicept, tabalumab), T-cell targeted therapy (abatacept, belatacept), interleukin targeted therapy (tocilizumab, anakinra, canakimumab, rilonacept, secukinumab), growth and differentiation factors (denosumab, mavrilimumab), JAK pathway inhibitors (tofacitinib, baricitinib, filgotinib), and combinations thereof. (Item 12) 12. The ophthalmic formulation according to any one of items 1 to 3 and 5 to 11, wherein the amount of IgG present in the ophthalmic formulation is in the range of about 0.01 mg / mL by weight to about 1 g / mL by weight. (Item 13) 13. The ophthalmic formulation of item 12, wherein the amount of IgG is about 10 mg / mL or less. (Item 14) 13. The ophthalmic formulation of item 12, wherein the amount of IgG is about 1 mg / mL or less. (Item 15) 15. The ophthalmic preparation according to any one of items 1 to 3 and 5 to 14, wherein the IgG fragment is an antigen-binding fragment, a single chain antibody, an Fv fragment, a Fab fragment, a Fab' fragment, or a F(ab)2 fragment. (Item 16) 16. The ophthalmic preparation according to any one of items 1 to 3 and 5 to 15, wherein the IgG comprises IgG1, IgG2, IgG3, and IgG4 or a combination thereof. (Item 17) 17. The ophthalmic formulation according to any one of items 1 to 3 and 5 to 16, wherein the IgG comprises a preselected concentration of autologous IgG purified from autologous plasma / serum using a separation process such as affinity chromatography using Protein A beads or another IgG separation process. (Item 18) 17. The ophthalmic formulation according to any one of items 1 to 3 and 5 to 16, wherein the IgG comprises glycoengineered sialylated IgG, IgG-Fc glycosylation, or a combination thereof. (Item 19) 19. The ophthalmic preparation according to any one of items 1 to 3 and 5 to 18, wherein the IgG neutralizes autoimmune antibodies or antibodies that specifically bind to citrullinated proteins. (Item 20) 20. The ophthalmic formulation according to any one of items 1 to 3 and 5 to 19, wherein the amount of IgG present in the ophthalmic formulation is in the range of about 0.01 mg / mL by weight to about 1 g / mL by weight. (Item 21) 21. The ophthalmic formulation according to any one of items 1 to 20, wherein the pH of the formulation is between pH 6 and pH 8. (Item 22) 22. The ophthalmic formulation of any one of items 1-21, comprising one or more pharma- ceutically acceptable excipients comprising polyvinyl alcohol, povidone, hydroxypropyl methylcellulose, poloxamer, polyol, carbopol, Pluronic®, carbomer, carboxymethylcellulose, hydroxyethylcellulose, cyclodextrin, phosphate buffer, citrate buffer, Tris buffer, sodium chloride, potassium chloride, polysorbate 80, vegetable oil, a preservative, or a combination thereof. (Item 23) 23. The ophthalmic formulation according to any one of items 1 to 22, wherein the formulation comprises a multi-dose vial with a preservative or a single-dose sterile container without a preservative. (Item 24) 24. A method of treating a clinical condition in a patient in need thereof, said method comprising the step of administering to said patient an ophthalmic formulation according to any one of items 1 to 23, wherein said clinical condition is an inflammatory ocular surface or intraocular eye disease, an infectious ocular surface or intraocular eye disease, and / or an immunological ocular surface or intraocular eye disease. (Item 25) 24. A method for treating an inflammatory, infectious and / or immunological eye disease in a patient in need thereof, comprising administering to the patient an ophthalmic formulation according to any one of items 1 to 23. (Item 26) 24. A method for reducing, alleviating or preventing ocular discomfort in a patient suffering from a clinical condition, comprising administering to the patient the ophthalmic formulation according to any one of items 1 to 23, wherein the clinical condition is an inflammatory ocular surface or intraocular eye disease, an infectious ocular surface or intraocular eye disease, and / or an immunological ocular surface or intraocular eye disease. (Item 27) 27. The method of claim 26, wherein the ocular discomfort comprises one or more of foreign body sensation, pain, photosensitivity, stinging, irritation, soreness, dryness, burning sensation, redness, itching or tingling. (Item 28) 24. Use of the ophthalmic formulation according to any one of items 1 to 23 for the preparation of a medicament for said treatment of a clinical condition in a patient in need thereof, wherein said clinical condition is an inflammatory ocular surface or intraocular eye disease, an infectious ocular surface or intraocular eye disease, and / or an immunological ocular surface or intraocular eye disease. (Item 29) 24. Use of an ophthalmic formulation according to any one of items 1 to 23 for the preparation of a medicament for said treatment of an inflammatory, infectious and / or immunological eye disease in a patient in need thereof. (Item 30) 24. Use of the ophthalmic formulation according to any one of items 1 to 23 for the preparation of a medicament for reducing, alleviating or preventing ocular discomfort in a patient suffering from a clinical condition, wherein the clinical condition is an inflammatory ocular surface or intraocular eye disease, an infectious ocular surface or intraocular eye disease, and / or an immunological ocular surface or intraocular eye disease. (Item 31) 31. The use according to item 30, wherein the eye discomfort comprises one or more of the following: foreign body sensation, stinging, irritation, pain, dryness, redness, itching or tingling. (Item 32) 24. A composition for treating a clinical condition in a patient in need thereof, said composition comprising the ophthalmic preparation according to any one of items 1 to 23, and said clinical condition is an inflammatory ocular surface or intraocular disease, an infectious ocular surface or intraocular disease, and / or an immunological ocular surface or intraocular disease. (Item 33) 24. A composition for treating an inflammatory, infectious and / or immunological eye disease in a patient in need thereof, said composition being an ophthalmic formulation according to any one of items 1 to 23. (Item 34) 24. A composition for reducing, alleviating or preventing ocular discomfort in a patient suffering from a clinical condition, comprising the ophthalmic preparation according to any one of items 1 to 23, wherein the clinical condition is an inflammatory ocular surface or intraocular eye disease, an infectious ocular surface or intraocular eye disease, and / or an immunological ocular surface or intraocular eye disease. (Item 35) 35. The composition according to item 34, wherein the ocular discomfort comprises one or more of foreign body sensation, pain, photosensitivity, stinging, irritation, soreness, burning sensation, dryness, redness, itching or tingling. (Item 36) 36. The method, use or composition according to any one of items 24 to 35, wherein the patient has autoantibodies present in a biological sample. (Item 37) 37. The method, use or composition according to item 36, wherein the autoantibodies present in the biological sample are anti-citrullinated protein antibodies. (Item 38) 38. The method, use or composition according to item 36 or 37, wherein said biological sample is an ocular fluid. (Item 39) 39. The method, use or composition according to item 38, wherein said ocular fluid is tears, eyewash, aqueous humor or vitreous humor. (Item 40) The clinical condition, inflammatory eye disease, infectious eye disease or immunological eye disease is ocular graft-versus-host disease (oGVHD), Steven Johnson Syndrome (Steven Johnson Syndrome), syndrome), Ocular Cicatricial Penphygoid (OCP), Mild, Moderate and Severe Aqueous Deficiency Dry Eye Disease (DED), Meybomian Gland Disease, Stye, Ocular Rosacea, Blepharitis, Superior Limbal Keratoconjunctivitis (SLK), Aqueous Sufficiency DED, Floppy Eyelid Syndrome, Neurotrophic Eye Disease, Symptom-Sign Amputation (Discordant DED), Neuropathic Pain, Thyroid Eye Disease (Grave's Ophthalmopathy), Rheumatoid Arthritis-Associated Eye Disease, Lupus-Associated Eye Disease, 40. The method, use or composition according to any one of items 24 to 39, comprising Sjogren's syndrome, secondary Sjogren's syndrome, ocular rosacea, allergic keratoconjunctivitis (vernal), viral keratoconjunctivitis (adenovirus EKC, herpes virus), Thygeson's keratitis, retinal gliosis, aniridia, keratitis or post-operative / post-traumatic eye conditions, peripheral ulcerative keratitis, keratitis, episcleritis, scleritis, uveitis and glaucoma. (Item 41) 41. The method of claim 40, wherein the keratitis is due to a sterile inflammation or due to a viral, bacterial, or fungal infection. (Item 42) 41. The method according to item 40, wherein said post-operative / post-traumatic ocular condition comprises after ocular surface reconstruction surgery, application of metabolites to the ocular surface, pterygium surgery, glaucoma surgery, cataract surgery, refractive surgery (LASIK, LASEK, or PRK), keratoprosthesis surgery or related ocular conditions after radiation or chemical (alkaline or acidic) or traumatic injury. (Item 43) A method for treating an ocular disease comprising administering a therapeutically effective amount of an allogeneic or autologous IgG ophthalmic preparation to a patient in need of such treatment. (Item 44) 2. Use of a therapeutic amount of an allogeneic or autologous IgG ophthalmic preparation for the preparation of a medicament for the treatment of an ocular disease in a patient in need of such treatment. (Item 45) A composition for treating said ocular disease comprising a therapeutically effective amount of an allogeneic or autologous IgG ophthalmic preparation to a patient in need of such treatment. (Item 46) 46. ​​The method, use or composition according to any one of items 24 to 45, wherein said ophthalmic formulation is administered as an eye drop formulation, a topical solution formulation, a gel formulation, an emulsion formulation, a suspension formulation, an ointment formulation or an injectable formulation. (Item 47) 47. The method, use or composition according to any one of items 24 to 46, wherein said ophthalmic preparation, medicament or composition is administered to said patient at least once a day. (Item 48) 47. The method, use or composition according to any one of items 24 to 46, wherein the ophthalmic preparation, medicament or composition is administered to the subject at least once every 1 to 3 weeks. (Item 49) 49. The method, use or composition according to any one of items 24 to 48, wherein said concentration of IgG is defined by a 5%, 10% or 20% Ocular Surface Immunoglobulin (OSIG) solution. (Item 50) 50. The method, use or composition according to any one of items 24 to 49, wherein said ophthalmic formulation is administered intraocularly as an intraocular injection. (Item 51) 51. The method, use or composition according to any one of items 24 to 50, wherein said autologous IgG ophthalmic preparation is prepared using blood from a human subject and then administered to the ocular surface or intraocularly in the same subject. (Item 52) 1. A method for detecting the presence of autoantibodies in a subject, the method comprising detecting a level of autoantibodies in a sample of ocular fluid obtained from the subject, the subject suffering from or at risk of developing an ocular surface disorder. (Item 53) 53. The method of claim 52, further comprising comparing the level of autoantibody in the sample obtained from the subject with a control level of autoantibody. (Item 54) A method for diagnosing, determining the risk of developing, or monitoring an ocular surface disorder in a subject, the method comprising detecting a level of an autoantibody in a sample obtained from the subject, and comparing the level of antibody in the sample obtained from the subject to a control level of the autoantibody, wherein the control level of the autoantibody is used to diagnose, determine the risk of developing, or monitor the risk of developing an ocular surface disorder in the subject. (Item 55) 55. The method according to any one of items 52 to 54, wherein the autoantibody is a native autoantibody, an anti-CarP autoantibody, or an ACPA antibody. (Item 56) 56. The method according to any one of items 52 to 55, wherein the autoantibody comprises a plurality of autoantibodies. (Item 57) 57. The method according to any one of items 52 to 56, wherein the antigen for detecting the autoantibody comprises a citrullinated protein, a citrullinated peptide sequence, or a combination thereof. (Item 58) 57. The method according to any one of items 52 to 56, wherein the antigen for detecting the autoantibody comprises a carbamylated protein, a carbamylated peptide sequence, or a combination thereof. (Item 59) 59. The method according to any one of items 52 to 58, wherein the sample is an ocular fluid. (Item 60) 60. The method of claim 59, wherein the ocular fluid is tears, eyewash, aqueous humor or vitreous humor. (Item 61) 61. The method of any one of items 52 to 60, wherein the level of autoantibodies is detected by a method selected from the group consisting of enzymatic methods, spectroscopic methods, chromatographic methods, immunological methods, or combinations thereof. (Item 62) 62. The method of any one of items 52 to 61, comprising determining the progression of the ocular surface disorder or determining the efficacy of a therapy in a subject suffering from, suspected of suffering from, or predisposed to an ocular surface disorder. (Item 63) 63. The method of any one of items 52-62, wherein the control level of an autoantibody comprises the level of the autoantibody in the subject before initiation of therapy, the level of the autoantibody in the subject at an earlier stage of therapy, or a combination thereof. (Item 64) The ocular surface disorder may be Ocular Graft Versus Host Disease (oGVHD), Stevens-Johnson Syndrome, Ocular Cicatricial Pemphigoid (OCP), Mild, Moderate and Severe Aqueous Deficiency Dry Eye Disease (DED), Meybomian Gland Disease, Superior Limbal Keratoconjunctivitis (SLK), Aqueous Sufficiency DED, Floppy Eyelid Syndrome, Neurotrophic Eye Disease, Symptom-Symptom Disconnection (Discordant DED), Neuropathic Pain, Thyroid Eye Disease (Grave Ophthalmopathy), Rheumatoid Arthritis-Associated Eye Disease, Lupus-Associated Eye Disease, 64. The method of any one of items 52 to 63, wherein the eye disease is an inflammatory, infectious or immunological eye disease, including Sjogren's syndrome, secondary Sjogren's syndrome, ocular rosacea, allergic keratoconjunctivitis (vernal), viral keratoconjunctivitis (adenovirus EKC, herpes virus), Thygeson's keratitis, retinal gliosis, aniridia, keratitis or post-operative / post-traumatic eye conditions, peripheral ulcerative keratitis, keratitis, episcleritis, scleritis, uveitis or glaucoma. (Item 65) A diagnostic kit comprising a panel of antigens for detecting autoantibodies selected from the group consisting of citrullinated proteins, citrullinated peptide sequences, or combinations thereof. (Item 66) A diagnostic kit comprising a panel of antigens for detecting autoantibodies for carrying out the method according to any one of items 24 to 27, 36 to 43, or 46 to 61. (Item 67) An Fc receptor blocking composition formulated for ocular or mucosal use. (Item 68) 68. The Fc receptor blocking composition of item 67, further defined by an Fc receptor blocking peptide concentration in the eye and optionally a suitable carrier. (Item 69) 69. An enriched ocular or mucosal IgG composition comprising the Fc receptor blocking composition of item 67 or 68. (Item 70) 70. The composition of claim 69, further comprising an anti-inflammatory agent. (Item 71) 71. A therapeutic dose for the treatment of an ocular immune disease, said ocular immune disease being indicated by a level of anti-citrullinated protein autoantibodies determined from ocular fluid removed from a treatment site, said therapeutic dose being capable of being formulated using the concentrated ocular IgG composition of item 69 or 70. (Item 72) 71. The therapeutic dose of item 70, wherein the ocular fluid is tears and the immune disease is dry eye disease. (Item 73) The therapeutic dose according to any one of items 70 to 72, wherein the composition further defined by the concentrated ocular IgG composition according to item 69 or 70 reduces ACPA enrichment and / or effect upon application at the treatment site. (Item 74) 74. The therapeutic dose of item 73, wherein the concentrated ocular IgG composition is in the range of about 0.01 mg / mL to about 1 g / mL by weight. (Item 75) A diagnostic kit comprising: A test device for determining the concentration of anti-citrullinated protein autoantibodies in ocular fluids; and A diagnostic kit comprising a dosing device for indicating a therapeutic treatment dose and regimen sufficient to treat and / or reduce the damaging effects of said anti-citrullinated protein autoantibody concentration in said ocular fluid. (Item 76) 76. The diagnostic kit of item 75, wherein the ocular fluid is tears, eyewash, aqueous humor or vitreous humor. (Item 77) 1. A method for producing a therapeutic dose of an immune injury detected in ocular fluid removed from a treatment site, comprising: providing a concentration of IgG configured to reduce the level and / or effects of anti-citrullinated protein autoantibodies when administered to the treatment site; and adding a carrier to said concentration of IgG, thereby forming a therapeutic dose configured to enable delivery of said IgG to said treatment site. (Item 78) 78. The method of claim 77, wherein the concentration of IgG is formulated in a 10% ocular surface immunoglobulin (OSIG) solution. (Item 79) 79. The method according to claim 77 or 78, further comprising the step of delivering the therapeutic dose to the treatment site periodically at least twice a day and twice a month for a treatment period defined by a response detected with the diagnostic kit of item 75 or 76, or any other suitable period to achieve an improvement in an immune or inflammatory condition, or until a test device indicates that the concentration is within a normal range. (Item 80) 80. The method according to any one of items 77 to 79, wherein the ocular fluid is tears, eyewash, aqueous humor or vitreous humor. (Item 81) A B-cell targeted ocular composition comprising rituximab formulated for treating an ocular disease. (Item 82) 82. The B cell targeted ocular composition of claim 81, wherein the composition is formulated as an eye drop. (Item 83) An ophthalmic formulation according to any one of items 1 to 23, or a B-cell targeted ophthalmic composition according to item 81 or 82, wherein the formulation or composition is formulated for treating an inflammatory, infectious or immune disease in the mucosa. (Item 84) 84. The ophthalmic formulation or B cell targeted ophthalmic composition of item 83, wherein the mucosa is on the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and cavities, synovial (joint) cavity, vaginal cavity, or skin.

Claims

1. 1. An ophthalmic formulation for treating an ophthalmic disease or condition associated with the action of autoantibodies on or within the eye of a subject, comprising: (a) i) one or more pharma- ceutically acceptable ophthalmic excipients, and ii) pooled human plasma; (b) i) one or more pharma- ceutically acceptable ophthalmic excipients, ii) pooled plasma-derived human immunoglobulin G (IgG), and iii) pooled human plasma proteins, pooled human plasma lipids, or a combination thereof; or (c) i) one or more pharma- ceutically acceptable ophthalmic excipients; and ii) pooled plasma-derived human immunoglobulin G (IgG).

1. An ophthalmic formulation comprising:

2. Steroids, anti-inflammatory drugs, mucolytics, PAD enzyme inhibitors (paclitaxel, glucocorticoids or Cl-amidine) or NET degraders (DNase or heparin), targeted Fab antibody fragments, Fc receptor blocking peptides, Fc receptor blocking antibodies, recombinant peptides containing pathogenic epitopes, conventional synthetic DMARDs (methotrexate, leflunomide / teriflunomide, sulfasalazine, chloroquine / hydroxychloroquine), TNF-α targeted therapies (infliximab, adalimumab, etanercept, golimumab, selenium), selenium phosphate dehydrogenase (selenium phosphate dehydrogenase ...

2. The ophthalmic formulation of claim 1, further comprising a pharmacologic agent selected from: B-cell targeted therapy (rituximab, ofatumumab, belimumab, atacicept, tabalumab), T-cell targeted therapy (abatacept, belatacept), interleukin targeted therapy (tocilizumab, anakinra, canakinumab, rilonacept, secukinumab), growth and differentiation factors (denosumab, mavrilimumab), JAK pathway inhibitors (tofacitinib, baricitinib, filgotinib), and combinations thereof.

3. One or more of the pharma- ceutically acceptable ophthalmic excipients may be selected from the group consisting of cyclodextrin, carbopol or carbomer or acrylic acid polymers, poloxamers, polyols, Pluronic®, xyloglucan, methylcellulose, hydroxypropylmethylcellulose, ethyl(hydroxyethyl)cellulose, pseudolatex, cellulose acetate phthalate, gellan gum, alginates, carrageenan, hyaluronic acid, sodium acetate, disodium edetate, hypromellose, acetic acid, alcohol, alginic acid, americhol, glycerol ... Amerchol-cab, antipyrine, benzalkonium chloride, benzododecinium bromide, boric acid, caffeine, calcium chloride, carbomer 1342, carbomer 934P, carbomer 940, carbomer homopolymer type B (allyl pentaerythritol crosslinked), sodium carboxymethylcellulose, hydroxyethylcellulose, castor oil, cetyl alcohol, chlorobutanol, citric acid, citric acid monohydrate, citrate buffer, creatinine, divinylbenzene styrene copolymer, ethylene vinyl acetate copolymer, gel Langum (low acyl), glycerin, glyceryl stearate, hypromellose, lanolin, lauralkonium chloride, lauroyl sarcosine, magnesium chloride, methylparaben, mineral oil, nonoxynol-9, octoxynol-40, petrolatum, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric nitrate, phosphate buffer, polydonium chloride, poloxamer 188 or 407, polycarbophil, polyethylene glycol 400 or 8000, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl Polysorbate 40 stearate, polypropylene glycol, polysorbate 20, polysorbate 80, polyvinyl alcohol, potassium chloride, potassium sorbate, povidone K29 / 32, povidone K30, povidone K90, povidone, preservatives, propylene glycol, propylparaben, soda ash, sodium acetate, sodium bisulfate, sodium borate, sodium borate decahydrate, sodium carbonate, sodium chloride, sodium citrate, sodium metabisulfate, sodium nitrate, sodium sulfate, sodium sulfite, sodium thiosulfate, sorbic acid,3. The ophthalmic formulation of claim 1 or 2, wherein the active ingredient is selected from sorbitol, stabilized oxychloro complexes, sulfuric acid, thimerosal, titanium dioxide, tocophersolan, trisodium citrate dihydrate, Tris buffer, tromethamine, tyloxapol, vegetable oils, xanthan gum, zinc chloride, or combinations thereof.

4. The ophthalmic formulation of any one of claims 1 to 3, wherein the formulation comprises from about 0.1% to about 99% pooled plasma proteins.

5. 4. The ophthalmic formulation of any one of claims 1 to 3, wherein the formulation comprises from about 0.01 mg / mL to about 1 g / mL of pooled IgG by weight.

6. 4. The ophthalmic formulation of any one of claims 1 to 3, wherein the amount of pooled IgG present in the ophthalmic formulation ranges from about 0.4% to about 4%.

7. 7. The ophthalmic formulation of claim 6, wherein the ophthalmic formulation comprises about 1% pooled IgG.

8. 8. An ophthalmic composition for use in the treatment or prevention of a clinical condition associated with the action of autoantibodies on or within the ocular surface of a patient in need of such treatment or prevention, the ophthalmic composition comprising the ophthalmic formulation of any one of claims 1 to 7, wherein the clinical condition is an inflammatory ocular surface or intraocular ocular disease, and / or an immunological ocular surface or intraocular ocular disease.

9. 8. An ophthalmic composition for use in reducing, alleviating or preventing ocular discomfort caused by the action of autoantibodies on or within the ocular surface of a patient suffering from a clinical condition, the ophthalmic composition comprising the ophthalmic formulation of any one of claims 1 to 7, wherein the clinical condition is an inflammatory ocular surface or intraocular ocular disease, and / or an immunological ocular surface or intraocular ocular disease.

10. 10. The ophthalmic composition for use according to claim 9, wherein the ocular discomfort comprises one or more of foreign body sensation, pain, photosensitivity, stinging, irritation, soreness, dryness, burning sensation, redness, itching or tingling.

11. The ophthalmic composition for use according to any one of claims 8 to 10, wherein the patient has autoantibodies present in a biological sample, the autoantibodies being anti-citrullinated protein antibodies.

12. The ophthalmic composition for use according to claim 11 , wherein the biological sample is an ocular fluid.

13. The clinical conditions include ocular graft versus host disease (oGVHD), Stevens-Johnson syndrome, ocular cicatricial pemphigoid (OCP), mild, moderate and severe aqueous deficient dry eye disease (DED), Meybomian gland disease, styes, ocular rosacea, blepharitis, superior limbal keratoconjunctivitis (SLK), aqueous sufficient DED, floppy eyelid syndrome, neurotrophic eye disease, symptom-sign disconnection (discordant DED), neuropathic pain, thyroid eye disease (Graves' ophthalmopathy), rheumatoid arthritis associated eye disease, lupus associated eye disease, Sjogren's syndrome, secondary Sjogren's syndrome, ocular rosacea, allergic keratoconjunctivitis (vernal 13. The ophthalmic composition according to any one of claims 8 to 12, wherein the eye disease is an inflammatory or immunological eye disease, including eye conditions associated with ocular inflammation, such as glaucoma, cataract surgery, refractive surgery (LASIK, LASEK, or PRK), keratoprosthesis surgery, radiation or chemical (alkaline or acidic) or traumatic injury, peripheral ulcerative keratitis, keratitis, episcleritis, scleritis, uveitis and glaucoma.

14. 14. The ophthalmic composition for use according to any one of claims 8 to 13, wherein the ophthalmic formulation is administered as an eye drop formulation, a topical solution formulation, a gel formulation, an emulsion formulation, a suspension formulation, an ointment formulation or an injectable formulation, and the eye drop formulation allows for corneal and intraocular penetration.

15. 14. The ophthalmic composition for use according to any one of claims 8 to 13, wherein the ophthalmic formulation is administered to the patient at least once a day, or the ophthalmic formulation, medicament or ophthalmic composition is administered to the subject at least once every 1 to 3 weeks.

16. The ophthalmic composition for use according to any one of claims 8 to 15, wherein the ophthalmic formulation is administered intraocularly as an intraocular injection.

17. 8. Use of an ophthalmic composition for the preparation of a medicament for the treatment or prevention of a clinical condition caused by the action of autoantibodies on or within the ocular surface of a patient in need of such treatment or prevention, said ophthalmic composition comprising the ophthalmic formulation of any one of claims 1 to 7, wherein said clinical condition is an inflammatory ocular surface or intraocular ocular disease, and / or an immunological ocular surface or intraocular ocular disease.

18. Use of an ophthalmic composition for the preparation of a medicament for the relief, alleviation or prevention of ocular discomfort caused by the action of autoantibodies on or within the ocular surface of a patient suffering from a clinical condition, said ophthalmic composition comprising the ophthalmic formulation of any one of claims 1 to 7, wherein said clinical condition is an inflammatory ocular surface or intraocular ocular disease, and / or an immunological ocular surface or intraocular ocular disease.

19. 19. The use of claim 18, wherein the ocular discomfort comprises one or more of foreign body sensation, pain, photosensitivity, stinging, irritation, soreness, dryness, burning, redness, itching or tingling.

20. The use according to any one of claims 17 to 19, wherein the patient has autoantibodies present in a biological sample.

21. The use according to claim 20, wherein the autoantibody present in the biological sample is an anti-citrullinated protein antibody.

22. 22. The use according to claim 20 or 21, wherein the biological sample is an ocular fluid.

23. The clinical conditions include ocular graft versus host disease (oGVHD), Stevens-Johnson syndrome, ocular cicatricial pemphigoid (OCP), mild, moderate and severe aqueous deficient dry eye disease (DED), Meybomian gland disease, styes, ocular rosacea, blepharitis, superior limbal keratoconjunctivitis (SLK), aqueous sufficient DED, floppy eyelid syndrome, neurotrophic eye disease, symptom-sign disconnection (discordant DED), neuropathic pain, thyroid eye disease (Graves' ophthalmopathy), rheumatoid arthritis associated eye disease, lupus associated eye disease, Sjogren's syndrome, secondary Sjogren's syndrome, ocular rosacea, allergic keratoconjunctivitis, ...

23. The use according to any one of claims 17 to 22, which is an inflammatory or immunological eye disease, including ocular conditions associated with ocular inflammation such as uveal keratitis (vernal), Thygeson's keratitis, retinal gliosis, aniridia, keratitis or post-operative / post-traumatic eye conditions, ocular surface reconstruction surgery, application of metabolites to the ocular surface, pterygium surgery, glaucoma surgery, cataract surgery, refractive surgery (LASIK, LASEK or PRK), keratoprosthesis surgery or ocular conditions associated with radiation or chemicals (alkaline or acidic) or traumatic injury, peripheral ulcerative keratitis, keratitis, episcleritis, scleritis, uveitis and glaucoma.

24. The use according to any one of claims 17 to 23, wherein the ophthalmic formulation is administered as an eye drop formulation, a topical liquid formulation, a gel formulation, an emulsion formulation, a suspension formulation, an ointment formulation or an injectable formulation allowing corneal and intraocular penetration.

25. 25. The use of any one of claims 17 to 24, wherein the ophthalmic formulation is administered to the patient at least once daily, or the ophthalmic formulation, medicament or ophthalmic composition is administered to the subject at least once every 1 to 3 weeks.

26. The use according to any one of claims 17 to 25, wherein the ophthalmic formulation is administered intraocularly as an intraocular injection.

27. A kit for use in reducing, alleviating or preventing ocular discomfort resulting from the action of autoantibodies on or within the eye of a patient suffering from a clinical condition, comprising: (i) an ophthalmic formulation according to any one of claims 1 to 7, and (ii) instructions for using the kit and for carrying out the administration of the ophthalmic formulation; Including the kit.

28. A diagnostic kit comprising: A test device for determining the concentration of anti-citrullinated protein autoantibodies in ocular fluids; and 10. A diagnostic kit comprising a dosing device for indicating a therapeutic treatment dose and regimen for administering the ophthalmic formulation of any one of claims 1-7 sufficient to treat and / or reduce the damaging effects of the anti-citrullinated protein autoantibody concentration in the ocular fluid.

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