Treatment and diagnosis of ocular diseases mediated by autoantibodies

Abstract

The present disclosure provides an ophthalmic formulation comprising: one or more pharmaceutically acceptable excipients; a pharmaceutically active compound capable of reducing the amount or deleterious effects of autoantibodies on the ocular surface, such as IgG; in particular, the present disclosure provides an ophthalmic formulation wherein the pharmaceutically active compound is 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, formation of symblepharon, shortening of the anterior chamber depth, lid margin / conjunctival keratinization, subconjunctival fibrosis, retinal gliosis and glaucoma.

Description

[0001] Cross-references to related applications

[0002] 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 incorporated herein by reference.

[0003] Statement on Federally Funded Research

[0004] This disclosure is made with government support under license number R01 EY024966 granted by the National Eye Institute (NEI) / National Institutes of Health (NIH). The government holds certain rights in this disclosure. Technical Field

[0005] This disclosure relates to an ophthalmic preparation capable of reducing the concentration or harmful activity of autoantibodies on the ocular surface and / or within the eye to prevent or treat inflammatory, immune, allergic, infectious, and traumatic ocular surface and / or intraocular diseases. Background Technology

[0006] Inflammatory, immune, allergic, infectious, and traumatic ocular surface and intraocular diseases can cause a variety of signs and symptoms, including but not limited to: eye discomfort, red eyes, dry eye syndrome, conjunctivitis, keratitis, symblepheron formation, fornix foreshortening, eyelid / conjunctival keratosis, and subconjunctival fibrosis. More specific types of ocular surface diseases include: ocular graft-versus-host disease (oGVHD); Steven Johnson syndrome; ocular cicatricial pemphigoid (OCP); mild, moderate, and severe delaminic dry eye (DED); meibomian gland disorders; ocular rosacea; styes; blepharitis; superior lip keratoconjunctivitis (SLK); adequate tear production in DED; blepharochalasis syndrome; neurotrophic eye diseases; symptom-sign disconnect (inconsistent DED); neuropathic pain; thyroid eye diseases (Graves' ophthalmopathy); rheumatoid arthritis-related eye diseases; lupus-related eye diseases; Sjogren's syndrome; and secondary Sjogren's syndrome. Syndrome; ocular rosacea; allergic keratoconjunctivitis (spring); viral keratoconjunctivitis (adenovirus EKC, herpesvirus); Thygeson's keratitis; aniridia; keratitis or postoperative / post-traumatic ocular symptoms; peripheral ulcerative keratitis; keratitis; episcleritis; scleritis; retinal gliosis; uveitis (anterior or posterior); and glaucoma (open-angle, closed-angle, or normal-tension).

[0007] Conventional treatments for some of these ocular surface diseases (particularly dry eye syndrome) include: (i) instillation of artificial tears to replenish and stimulate tear production; and (ii) use of anti-inflammatory drugs to reduce ocular surface inflammation. Typically, current treatments for dry eye include: topical application of artificial tear products / lubricants; tear maintenance management; stimulation of tear secretion; topical antibiotics (e.g., erythromycin or bacitracin ointment); oral administration of tetracyclines (e.g., tetracycline, doxycycline, or minocycline); and the use of anti-inflammatory compounds and corticosteroids. Conventional treatments for certain intraocular diseases include: (i) treatments to lower intraocular pressure; and (ii) intraocular steroid injections. These treatments are often time-consuming, unsatisfactory, and often ineffective or only occasionally effective. Furthermore, while conventional treatments may be effective to some extent in relieving the symptoms of dry eye syndrome, they have many undesirable side effects, such as burning and stinging sensations. Reducing local side effects and improving patient comfort and response to treatment is one of the objectives of this disclosure.

[0008] Another drawback of conventional treatments for ocular surface diseases is the inconsistent extent to which active pharmaceutical ingredients are absorbed into corneal cells and their residence time in the cornea, making it impossible to effectively treat ocular surface disease syndromes with intermittent application. Although ointments or creams may allow for longer residence times, these formulations may not damage the stratum corneum (the superficial keratinized layer of the skin) and may not reach the blood vessels and nerves located deeper within the eyelid tissue.

[0009] Another problem associated with conventional treatments is that they don't directly address the underlying cause. Identifying the cause of ocular surface disease allows for more effective treatment. Most conventional treatments only address the symptoms of ocular surface disease.

[0010] Therefore, there is a ongoing need for diagnostic kits, compositions, and methods for the effective treatment of clinical symptoms associated with ocular surface and intraocular diseases. Furthermore, there is a need to address the underlying causes of ocular surface and intraocular diseases. Summary of the Invention

[0011] Some aspects of this disclosure provide an ophthalmic preparation comprising: (a) one or more pharmaceutically acceptable ophthalmic excipients; and (b) immunoglobulin G (IgG) or a fragment thereof.

[0012] Some aspects of this disclosure provide an ophthalmic formulation comprising: (a) one or more pharmaceutically acceptable ophthalmic excipients; and (b) a plasma-derived human immunoglobulin G (IgG) conjugated to the formulation.

[0013] Additionally, this disclosure provides an ophthalmic preparation comprising: (a) one or more pharmaceutically acceptable ophthalmic excipients; and (b) an ophthalmic pharmaceutically active compound for treating a clinical condition selected from the group consisting of inflammatory, infectious, immune, allergic, and / or traumatic ocular surface or intraocular diseases, wherein the pharmaceutically active compound comprises IgG.

[0014] In various aspects, this disclosure provides an ophthalmic formulation comprising a pharmaceutically active compound capable of reducing the amount or harmful biological effects of autoantibodies on or within the ocular surface. These autoantibodies are generated in response to citrullinated proteins (referred herein to as "ACPA autoantibodies," which are generated in response to citrullination of proteins), or in response to homocitrullinated proteins (referred herein to as "anti-CarP antibodies," which are generated in response to carbamylation of proteins), or are natural autoantibodies generated in response to native proteins. Citrullination and carbamylation are two types of post-translational modifications (PTMs) of proteins. In related aspects, the pharmaceutically active compound comprises immunoglobulin G (IgG) or fragments thereof. These ophthalmic formulations may also comprise one or more pharmaceutically acceptable ophthalmic excipients.

[0015] In all respects, the disclosed ophthalmic formulations comprise one or more pharmaceutically acceptable ophthalmic excipients. For example, the pharmaceutically acceptable ophthalmic excipients are selected from: cyclodextrins; carbopol or carbomer or acrylic polymers; poloxamer; xylo-glucan; methylcellulose; hydroxypropyl methylcellulose; ethyl (hydroxyethyl) cellulose; pseudo-latex; cellulose acetate phthalate; gellan gum; alginate; carrageenan; hyaluronic acid; sodium acetate; disodium edetate; hydroxypropyl methylcellulose; acetic acid; ethanol; alginic acid; amerchol-cab; antipyrine; benzalkonium chloride; benzalkonium bromide. Boronide; Boric acid; Caffeine; Calcium chloride; Carbomer 1342; Carbomer 934P; Carbomer 940; Carbomer homopolymer type B (pentaerythritol allyl ether crosslinking); Sodium carboxymethyl cellulose; Castor oil; Cetyl alcohol; Chlorobutanol; Citric acid; Citric acid monohydrate; Creatine anhydride; Divinylbenzene styrene copolymer copolymer); ethylene vinyl acetate copolymer; gellan gum (low acyl); glycerin; glyceryl stearate; hydroxypropyl methylcellulose; lanolin; lorazine; lauroyl sarcosine; magnesium chloride; methylparaben; mineral oil; nonylbenzene alcohol ether-9; octyl styrene polyol-40; petrolatum; phenethyl alcohol; phenylmercuric acetate; phenylmercuric nitrate; poloxamer; poloxamer 188 or 407; polycarboflavone; polyethylene glycol 400 or 8000; polyethylene glycol 35 castor oil; polyethylene glycol 40 hydrogenated castor oil Polyethylene glycol 40 stearate; polypropylene glycol; polysorbate 20; polyvinyl alcohol; potassium chloride; potassium sorbate; povidone K29 / 32; povidone K30; povidone K90; povidones; propylene glycol; propylparaben; soda ash; sodium acetate; sodium bisulfate; sodium borate; sodium borate decahydrate; sodium carbonate; sodium chloride; sodium citrate; sodium metabisulfite; sodium nitrate; sodium sulfate; sodium sulfite; sodium thiosulfate; sorbic acid; sorbitol; stabilized oxychlorocomplex; sulfuric acid; thimerosal; titanium dioxide; tococelen; trisodium citrate dehydrate; tromethamine; tyloxapine; xanthan gum; zinc chloride; or combinations thereof.

[0016] Another aspect of this disclosure provides an ophthalmic preparation comprising: (a) a pharmaceutically acceptable ophthalmic excipient; (b) a therapeutically effective amount of a pharmaceutically active compound comprising human immunoglobulin G (IgG); and / or (c) a therapeutically effective amount of a combination of human plasma proteins, human lipids, or combinations thereof. As used herein, the term "therapeuticly effective" includes the ability to have immunomodulatory effects or to counteract (neutralize) autoantibodies.

[0017] In any disclosed ophthalmic formulation, the pharmaceutically active compound may comprise: 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 and fragments thereof; or combinations thereof.

[0018] In all respects, any disclosed ophthalmic preparation may further comprise a second pharmaceutically active compound selected from: steroids; anti-inflammatory agents, such as methylprednisolone, prednisolone, dexamethasone, cyclosporine, etc. Nonsteroidal anti-inflammatory drugs (NSAIDs); mucolytics, such as N-acetylcysteine, Nacystelyn, N-acetylglucosamine; PAD enzyme inhibitors, such as paclitaxel, glucocorticoids, or Cl-amidine; or NETs dissolving agents (DNase or heparin); Fab-targeting antibody fragments; Fc receptor blocking peptides; Fc receptor blocking antibodies; recombinant peptides containing pathogenic epitopes; conventionally synthesized DMARDs (methotrexate, leflunomide / teriflunomide, sulfasalazine, chloroquine / hydroxychloroquine); TNF-α targeted therapies (infliximab, adalimumab, etanercept, golimumab, sertozumab); B-cell targeted therapies (rituximab, oxaliplatin ... Famumab, belimumab, taberucizumab; T-cell targeted therapies (abatacept, beracip); interleukin targeted therapies (tocilizumab, anakinin, canananurumab, linnacicept, secukinumab); growth and differentiation factors (denomab, mavrilimumab); JAK pathway inhibitors (tofacitinib, baricitinib, filgotinib); and combinations thereof.

[0019] In some embodiments, the concentration or amount of IgG present in the ophthalmic preparation ranges from about 0.01 mg / mL to about 1 g / mL (1000 mg / mL) by weight, for example, from about 0.05 mg / mL to about 1 g / mL by weight, or from about 0.1 mg / mL to about 1 g / mL by weight, or from about 0.1 mg / mL to about 0.5 mg / mL by weight, or from 0.05 mg / mL to about 0.5 mg / mL by weight, or from 0.01 mg / mL to about 0.1 mg / mL by weight. In some 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 some 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.

[0020] 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.

[0021] In various exemplary embodiments, the amount of IgG is about 10 mg / mL or less. In some embodiments, the ophthalmic formulation may contain IgG at a concentration or amount of 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.

[0022] The term "combined plasma-derived combined human immunoglobulin G" refers to unseparated plasma from several donors, subsequently separated to isolate immunoglobulin G. This disclosure provides ophthalmic compositions comprising plasma from several donors, said plasma being dispensed unseparated at various dilutions based on IgG concentrations (0.01% to 10%) in single-use droppers or multi-dose vials. The consolidation of plasma can also be performed prior to the processing step. This combined plasma and immunoglobulin (PPIG) formulation differs from conventional plasma products (e.g., PRP) in that conventional plasma products are prepared from the blood of a single subject, while PPIG formulations are prepared from the combined plasma of hundreds to thousands of healthy subjects. The final product contains combined IgG and plasma proteins, with or without platelets and platelet-activating products. The combined plasma-derived IgG may also contain blood lipids.

[0023] In another iteration, a combined plasma-derived human immunoglobulin G (at a concentration of 0.01% to 10%) from hundreds to thousands of healthy subjects was formulated in plasma or serum (allogeneic or autologous) with one or more ophthalmic excipients. This ophthalmic formulation will provide a combination of: combined IgG to counteract autoantibodies; and proteins, cytokines, and serum / plasma growth factors that promote ocular surface regeneration.

[0024] In all respects, IgG present in any disclosed ophthalmic preparation includes: serum / plasma-derived combined immunoglobulin G (OSIG: ocular surface immunoglobulin), multimerized IgG1 Fc molecules (IgG1 Fc hexamer), IgG2a Fc multimers (Stadurmers), multivalent Fc structures, or combinations thereof.

[0025] In all respects, IgG present in any disclosed ophthalmic preparation is: an antigen-binding fragment wherein one or more antibody domains are truncated or absent; a genetically engineered antibody or its protein-binding fragment; a single-chain antibody, or an antibody that can bind to more than one epitope, or an antibody that can bind to one or more different antigens. For example, fragments of IgG are antigen-binding fragments, single-chain antibodies, Fv fragments, Fab fragments, Fab' fragments, or F(ab)2 fragments.

[0026] In all respects, IgG present in any disclosed ophthalmic preparation includes IgG1, IgG2, IgG3, and IgG4, or combinations thereof. Additionally or alternatively, IgG may include or be derived from any suitable source, such as: stem cell preparations containing IgG, manufactured IgG or IgG stem cells; glycoprotein-engineered sialylated IgG, IgG-Fc glycosylation, etc.; or combinations thereof.

[0027] In all respects, the IgG present in any disclosed ophthalmic preparation comprises a preselected concentration of autologous IgG purified from autologous plasma / serum using an isolation process (such as affinity chromatography using protein A beads, or another IgG isolation process).

[0028] In all respects, IgG present in any disclosed ophthalmic preparation counteracts autoantibodies or antibodies that specifically bind to citrullinated proteins.

[0029] In all respects, the pH of any disclosed ophthalmic preparation is from about pH 6 to about pH 8, or from about pH 6.2 to about pH 7.2, from about pH 6.4 to about pH 7.4, or from about pH 6.5 to about pH 7.5, from about pH 6.6 to about pH 7.6, or from 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.

[0030] In all respects, any disclosed ophthalmic preparation contains one or more pharmaceutically acceptable excipients, including: polyvinyl alcohol, povidone, hydroxypropyl methylcellulose, poloxamer, polyols, carbopol, praline, carbomer, carboxymethylcellulose, hydroxyethylcellulose, cyclodextrins, phosphate buffer, citrate buffer, Tris buffer, sodium chloride, potassium chloride, polysorbate 80, vegetable oil, preservatives, or combinations thereof.

[0031] Any disclosed ophthalmic preparation may be used to treat inflammatory, infectious, or immune diseases at the mucous membranes, or may exhibit immunomodulatory effects at the mucous membranes. The ophthalmic preparation may treat inflammatory, infectious, or immune diseases, or may have immunomodulatory effects in the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canal and ear cavity, synovial (joint) cavity, vaginal cavity, or at the mucous membranes of the skin.

[0032] In all respects, the disclosed ophthalmic preparations include multi-dose vials with preservatives or single-dose sterile containers without preservatives.

[0033] In various aspects, this disclosure provides a method for treating a clinical condition in a patient in need, the method comprising administering to the patient an ophthalmic preparation disclosed herein, wherein the clinical condition is an inflammatory ocular surface disease or intraocular disease, an infectious ocular surface disease or intraocular disease, and / or an immune ocular surface disease or intraocular disease. In various aspects, this disclosure provides a method for treating an inflammatory, infectious, and / or immune ocular disease in a patient in need, the method comprising administering to the patient an ophthalmic preparation disclosed herein.

[0034] In various aspects, this disclosure provides a method for reducing, alleviating, or preventing ocular discomfort in a patient with clinical symptoms, the method comprising administering to the patient an ophthalmic preparation disclosed herein, wherein the clinical symptoms are an inflammatory ocular surface disease or intraocular disease, an infectious ocular surface disease or intraocular disease, and / or an immune ocular surface disease or intraocular disease. In any of these methods, the ocular discomfort includes one or more of the following: foreign body sensation, photosensitivity, stinging, irritation, soreness, dryness, burning, redness, itching, or tingling.

[0035] This disclosure provides a method for treating clinical symptoms in patients in need, the method comprising administering the ophthalmic preparation disclosed herein to the patient, wherein the clinical symptoms are inflammatory, infectious, or immune diseases in the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canal and ear cavity, synovial (joint) cavity, vaginal cavity, or mucous membranes on the skin.

[0036] This disclosure also provides a method for inducing immunomodulatory effects on mucous membranes in patients in need, the method comprising administering the ophthalmic preparation disclosed herein to the patient, wherein the mucous membrane is in the oral cavity, nasal cavity, bladder, tracheobronchial passage, ear canal and ear cavity, synovial (joint) cavity, vaginal cavity, or on the skin.

[0037] This disclosure also provides for the use of any of the ophthalmic preparations in the preparation of a medicament for treating a clinical condition in a patient in need, wherein the clinical condition is an inflammatory ocular surface disease or intraocular disease, an infectious ocular surface disease or intraocular disease, and / or an immune ocular surface disease or intraocular disease. In various aspects, this disclosure provides for the use of any of the ophthalmic preparations in 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 disease or intraocular disease, an infectious ocular surface disease or intraocular disease, and / or an immune ocular surface disease or intraocular disease. In any of these uses, the ocular discomfort includes one or more of the following: foreign body sensation, pain, photosensitivity, stinging, irritation, soreness, dryness, burning, redness, itching, or tingling.

[0038] This disclosure also provides the use of the ophthalmic preparations disclosed herein in the preparation of medicaments for treating clinical symptoms in patients in need, wherein the clinical symptoms are inflammatory, infectious, and / or immune diseases in the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and ear cavities, synovial (joint) cavities, vaginal cavities, or on the mucous membranes of the skin. Additionally, this disclosure provides the use of the ophthalmic preparations disclosed herein in the preparation of medicaments for inducing immunomodulatory effects at the mucous membranes of patients in need, wherein the mucous membranes are in the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and ear cavities, synovial (joint) cavities, vaginal cavities, or on the skin.

[0039] This disclosure also provides compositions for treating clinical symptoms comprising any of the ophthalmic preparations described herein. In various aspects, the compositions can be used to treat inflammatory, infectious, and / or immune-mediated eye diseases in patients in need. In various aspects, compositions comprising any of the ophthalmic preparations described herein can be used to reduce, alleviate, or prevent ocular discomfort in patients suffering from clinical symptoms, wherein the clinical symptoms are inflammatory ocular surface diseases or intraocular diseases, infectious ocular surface diseases or intraocular diseases, and / or immune-mediated ocular surface diseases or intraocular diseases. In related aspects, the ocular discomfort includes one or more of the following: foreign body sensation, pain, photosensitivity, stinging, irritation, aching, burning, dryness, redness, itching, or tingling.

[0040] This disclosure provides compositions for treating clinical symptoms in patients in need, wherein the compositions comprise the ophthalmic preparations disclosed herein, wherein the clinical symptoms are inflammatory, infectious, or autoimmune diseases in the mucous membranes of the oral cavity, nasal cavity, bladder, tracheobronchial passage, ear canal and ear cavity, synovial (joint) cavity, vaginal cavity, or on the skin. Additionally, this disclosure provides compositions for inducing immunomodulatory effects at the mucous membranes of patients in need, wherein the compositions comprise the ophthalmic preparations disclosed herein, wherein the mucous membranes are in the oral cavity, nasal cavity, bladder, tracheobronchial passage, ear canal and ear cavity, synovial (joint) cavity, vaginal cavity, or on the skin.

[0041] Exemplary clinical conditions that can be treated according to the exemplary methods, uses, and compositions described herein include, but are not limited to, immune eye diseases, metabolic eye diseases, allergic eye diseases, traumatic eye diseases, infectious eye diseases, and hereditary eye diseases, such as, but not limited to; ocular graft-versus-host disease (oGVHD); Steven Johnson syndrome; ocular cicatricial pemphigoid (OCP); mild, moderate, and severe delaminic dry eye (DED); meibomian gland disease; stye; ocular rosacea; blepharitis; superior lip keratoconjunctivitis (SLK); delaminic DED; blepharospasm syndrome; neurotrophic eye diseases; symptom-sign disconnect (inconsistent DED); neuropathic pain; thyroid eye disease (Graves' ophthalmopathy); rheumatoid arthritis-related eye diseases; lupus-related eye diseases; Sjogren's syndrome; secondary Sjogren's syndrome. Sjogren's syndrome; ocular rosacea; allergic keratoconjunctivitis (spring); viral keratoconjunctivitis (adenovirus EKC, herpesvirus); Thygeson's keratitis; retinal gliosis; viral keratoconjunctivitis (adenovirus EKC); Thygeson's keratitis; aniridia; keratitis or postoperative / post-traumatic ocular symptoms; peripheral ulcerative keratitis; keratitis; episcleritis; scleritis; uveitis (anterior or posterior); retinitis; and glaucoma (open-angle, narrow-angle, or normal-tension). For example, the exemplary methods described herein can be used to treat keratitis caused by a variety of conditions, such as keratitis due to aseptic inflammation (e.g., PUK), or keratitis caused by viral infections (e.g., adenovirus subepithelial infiltration, herpesvirus dendritic epithelial ulceration), bacterial infections (e.g., Staphylococcus, Pseudomonas), or fungal infections (e.g., Candida, Acanthamoeba). Additionally, the exemplary methods described herein can be used to treat postoperative / post-traumatic ocular conditions or ocular conditions related to: posterior ocular surface reconstruction surgery; application of antimetabolites to the ocular surface; pterygium surgery; glaucoma surgery; cataract surgery; refractive surgery (LASIK, LASEK, or PRK); artificial cornea surgery; vitreoretinal surgery; or radiation or chemical (alkaline or acidic) or traumatic injury.

[0042] In various aspects, the patient possesses autoantibodies present in the biological sample. In related aspects, the autoantibodies present in the biological sample are anti-citrullinated protein antibodies. In some aspects, the biological sample is ocular fluid. In various aspects, the ocular fluid is tear fluid, eye wash, aqueous humor, or posterior vitreous fluid.

[0043] This disclosure provides a method for treating an eye disease, the method comprising the step of administering a therapeutically effective amount of an allogeneic or autologous IgG ophthalmic preparation to a patient requiring such treatment. This disclosure also provides the use of the therapeutically effective amount of the allogeneic or autologous IgG ophthalmic preparation in the preparation of a medicament for treating an eye disease in a patient requiring such treatment. Additionally, this disclosure provides a composition for treating an eye disease in a patient requiring such treatment, the composition comprising a therapeutically effective amount of an allogeneic or autologous IgG ophthalmic preparation.

[0044] In any of the disclosed methods, uses, or compositions, the ophthalmic preparation is administered as an eye drop, topical liquid, gel, emulsion, suspension, ointment, or injectable formulation. The ophthalmic preparation, agent, or composition may be administered to the subject at least once daily. In various embodiments, the IgG ophthalmic preparation is administered to the subject once, twice, three times, three times, four times, five times, six times, seven times, eight times, nine times, or ten times daily. In embodiments, the IgG ophthalmic preparation is administered to the subject at least once every week to three weeks. In various embodiments, one or more doses of the IgG ophthalmic preparation are administered weekly, every two weeks, monthly, or every two months. In all aspects, the concentration of IgG is defined as a 5%, 10%, or 20% ocular surface immunoglobulin (OSIG) solution. In various embodiments, the administered IgG is in the form of an OSIG solution at concentrations of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In yet another embodiment, the IgG ophthalmic preparation is administered intraocularly as an intraocular injection. In embodiments, the IgG ophthalmic preparation is administered to the ocular surface as eye drops or an ointment.

[0045] In some implementations, a conjugated IgG ophthalmic formulation (modified or unmodified) is manufactured using blood from a human subject and then administered at a specific concentration to the ocular surface or intraocular region of the same subject. To enable OSIG transtissue delivery, administration can be subconjunctival or subtendonal injection, or the OSIG formulation can be modified (using nanotechnology, such as polymers / biospheres, particles, or membranes (with or without surface modification)). Other methods to enhance OSIG penetration include: using penetrants, such as benzyl ammonium; charge-based strategies, such as iontophoresis; or increasing the residence time on the ocular surface, such as by increasing viscosity or adsorption in contact lenses or punctal plugs. OSIG eye drops can be formulated as microemulsions. Increasing viscosity by using high-molecular-weight hydrophilic polymers that do not diffuse through biological membranes and form a three-dimensional network in water can increase the time of contact with the cornea and the bioavailability of OSIG eye drops. Examples of such polymers include polyvinyl alcohol, poloxamer, hyaluronic acid, carbomer, carbopol, and polysaccharides, namely cellulose derivatives, gellan gum, and xanthan gum. The corneal and intraocular permeability of OSIG eye drops can be improved by using chelating agents, preservatives (such as benzalkonium chloride), surfactants, and bile salts to alter the continuity of the corneal epithelial structure, or by using cyclodextrin complexes. For intraocular delivery or delivery to the cornea, subconjunctiva, or lacrimal gland, OSIG's polymeric, solid, multi-compartment drug delivery system is used.

[0046] Nanoparticles are polymer carriers constructed from biodegradable, biocompatible, natural, or synthetic polymers that often possess mucosal adhesion properties. For ocular application, components used in their development include: poly(alkyl cyanoacrylate), polylactic acid, poly(ε-caprolactone), poly(lactic-co-glycolic acid), chitosan, gelatin, sodium alginate, and albumin. These forms can be categorized as: nanospheres; solid, monolithic spheres (with dispersed OSIG) constructed from a dense polymer matrix; and nanocapsules forming reservoirs, constructed from polymer membranes in solid or liquid form surrounding OSIG. Liposomes are also used for OSIG delivery across ocular tissues. Liposomes are phospholipid drug carriers, typically constructed from phosphatidylcholine, stearamine, and various amounts of cholesterol or lecithin and α-L-dispalmitoyl-phosphatidylcholine. Niosomes, discosomes, and dendrimers are also used for OSIG delivery across ocular tissues.

[0047] In another embodiment, this disclosure provides a method for detecting the presence of autoantibodies in a subject, the method comprising detecting the level of autoantibodies in an ocular fluid sample obtained from the subject, wherein the subject has an ocular surface disease or is at risk of developing an ocular surface disease. In a related aspect, the method further comprises the step of comparing the level of the autoantibodies in the sample obtained from the subject with the level of a control autoantibody. For example, the ocular fluid is tear fluid, eye wash, aqueous humor, or vitreous humor.

[0048] This disclosure also provides a method for diagnosing, determining the risk of development, or monitoring ocular surface diseases in subjects, the method comprising: detecting the level of an autoantibody in a sample obtained from the subject; and comparing the level of the antibody in the sample obtained from the subject with a control level of the autoantibody, wherein the control level of the autoantibody is used to diagnose, determine the risk of development, or monitor ocular surface diseases in the subject. In related aspects, the autoantibody is a natural autoantibody, an anti-CarP autoantibody, or an ACPA antibody. In various aspects, the autoantibody includes a variety of autoantibodies. In related aspects, the antigen for detecting the autoantibody includes citrullinated proteins, citrullinated peptide sequences, or combinations thereof. In related aspects, the antigen for detecting the autoantibody includes carbamoylated proteins, carbamoylated peptide sequences, or combinations thereof.

[0049] In any of the disclosed methods, the sample is an eye fluid, wherein the eye fluid is tears, eye wash, aqueous humor from the anterior chamber, or vitreous humor from the posterior chamber.

[0050] In any disclosed method, the level of autoantibodies is detected by enzymatic, spectroscopic, chromatographic, immunological, or combinations thereof. In an exemplary embodiment, a method may include steps of determining the progression of ocular surface disease or determining the efficacy of treatment in a subject who has, is suspected of having, or is susceptible to ocular surface disease. In any disclosed method, the control levels of the autoantibodies include: the level of the autoantibodies in the subject prior to the initiation of treatment; the level of the autoantibodies in the subject at an earlier stage of treatment; or combinations thereof.

[0051] This disclosure also provides a diagnostic kit comprising an array of antigen plates for detecting autoantibodies, said autoantibodies comprising citrullinated proteins, citrullinated peptide sequences, or combinations thereof. The diagnostic kit can be used to perform any of the methods described herein.

[0052] In an exemplary embodiment, the Fc receptor blocking composition can be formulated for ocular applications. For example, the Fc receptor blocking composition may be further defined by an Fc receptor blocking ocular peptide concentration and optionally a suitable carrier. In another embodiment, the Fc receptor blocking composition may be further defined by an Fc receptor blocking ocular antibody concentration and optionally a suitable carrier. Additionally, the Fc blocking composition can be used to treat inflammatory, infectious, or immune diseases at the mucous membrane, or exhibit immunomodulatory effects at the mucous membrane. The Fc blocking composition can treat inflammatory, infectious, or immune diseases, or have immunomodulatory effects in the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canal and ear cavity, synovial (joint) cavity, vaginal cavity, or at the mucous membrane of the skin.

[0053] This disclosure also provides a concentrated ocular or mucosal IgG composition. Based on the principles herein, a concentrated ocular IgG composition is proposed. The composition may further comprise an anti-inflammatory agent.

[0054] This disclosure also provides a therapeutic dose for treating ocular immune diseases, wherein the ocular immune disease is indicated by the level of anti-citrullinated protein autoantibodies determined from eye fluid removed from the treatment site, and the therapeutic dose can be formulated using a concentrated ocular IgG composition. In some embodiments, the therapeutic dose may be determined based on tear fluid removed from a subject suffering from an immune disease, such as dry eye. In some embodiments, a therapeutic dose may comprise a composition further defined by a concentrated ocular IgG composition, the concentration of which reduces the concentration of ACPA antibodies and / or the effect produced by application at the treatment site. For example, the concentrated ocular IgG composition that the therapeutic dose may comprise ranges from about 0.01 mg / mL to about 1 g / mL by weight.

[0055] In some embodiments, a diagnostic kit includes: a testing device for determining the concentration of anti-citrullinated protein autoantibodies in ocular fluid (such as tears); and a dosing device for indicating a therapeutic dose and regimen sufficient to treat and / or reduce the damaging effect of the concentration of the anti-citrullinated protein autoantibodies in the ocular fluid. In related aspects, the ocular fluid is tears, eye wash, aqueous humor, or posterior vitreous fluid.

[0056] In an exemplary embodiment, a method for preparing a therapeutic dose for detecting immune damage in eye drops removed from a treatment site includes: providing a concentration of IgG configured to reduce the level and / or action of anti-citrullinated protein autoantibodies when administered to the treatment site; and adding a carrier to the concentration of IgG to form a therapeutic dose configured to deliver the IgG to the treatment site.

[0057] In another exemplary embodiment, a method of preparing a therapeutic dose may include providing a concentration of IgG in an ocular surface immunoglobulin (OSIG) solution ranging from about 0.01% to about 20%, or an OSIG solution ranging from about 0.1% to about 0.5%, or an OSIG solution ranging from about 0.4% to about 1%. For example, a concentration of IgG in an OSIG solution 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%. In one embodiment, OSIG Flebogamma 5% DIF is used to form a formulation to test its toxicity to human cells. Figure 19 Flebogamma 5% DIF at a concentration of 4 mg / mL is non-toxic to human epithelial cells and is therefore a suitable exemplary embodiment. OSIG solutions can be prepared using any commercially available IgG or conjugated plasma IgG. Other embodiments using commercially available formulations to prepare OSIG solutions at non-toxic concentrations are also suitable examples, based on the principles herein. In a related aspect, the method further includes the step of periodically delivering the therapeutic dose to the treatment site for a treatment period to achieve improvement in immune or inflammatory symptoms or until the concentration indicated by the testing device is within the normal range, the treatment period being defined by a response detected using the diagnostic kit at at least one of twice daily and twice monthly, or any other suitable time period.

[0058] In embodiments, the B-cell-targeting ocular composition may include a suitable B-cell composition (such as rituximab) formulated as eye drops or other suitable delivery methods for treating eye diseases or other mucosal diseases. For example, the B-cell-targeting ocular composition has immunomodulatory effects in mucous membranes such as the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canal and ear cavity, synovial (joint) cavity, vaginal cavity, or on the skin.

[0059] In addition to applying the compositions described herein to the eyes, these IgG compositions can also be applied to mucous membranes in other sites, such as the oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and ear cavities, synovial (joint) cavities, vaginal cavity, or 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 in various sites (such as the eyes, oral cavity, nasal cavity, bladder, tracheobronchial passages, ear canals and ear cavities, synovial (joint) cavities, vaginal cavity) or on the skin to treat inflammatory and / or immune diseases at these mucosal sites.

[0060] In yet another exemplary embodiment, B-cell-targeting therapeutic agents (rituximab, ofamumumab, belimumab, asceticip, taberucizumab) can form compositions for treating immune eye diseases, metabolic eye diseases, allergic eye diseases, traumatic eye diseases, infectious eye diseases, and hereditary eye diseases, such as, but not limited to, ocular graft-versus-host disease (oGVHD); Steven Johnson syndrome; ocular cicatricial pemphigoid (OCP); mild, moderate, and severe dry eye disease with acridity (DED); meibomian gland disease; superior limbal keratoconjunctivitis (SLK); DED with adequate tear production; blepharospasm syndrome; neurotrophic eye diseases; symptom-sign disconnect (inconsistent DED); neuropathic pain; thyroid eye disease (Graves' ophthalmopathy); rheumatoid arthritis-related eye diseases; lupus-related eye diseases; Sjogren's syndrome. Rituximab can be used to treat various eye conditions, including: secondary Sjogren's syndrome; ocular rosacea; allergic keratoconjunctivitis (vernal); viral keratoconjunctivitis (adenovirus EKC); Thygeson's keratitis; retinal gliosis; aniridia; keratitis or postoperative / post-traumatic eye conditions; peripheral ulcerative keratitis; keratitis; episcleritis; scleritis; uveitis; retinitis; and glaucoma. For example, rituximab can be formulated as eye drops or other suitable delivery methods to treat eye diseases. Attached Figure Description

[0061] Figure 1 This is a box plot showing the presence of ACPA in ocular cleansing agents in healthy subjects.

[0062] Figure 2This is a box plot showing the presence of ACPA autoantibodies in ocular cleansers from patients with several ocular surface diseases.

[0063] Figure 3 This is a graph showing the percentage of patients with specific ocular surface diseases who are positive for all ACPA in ocular surface cleansers.

[0064] Figure 4 This is a box plot showing ACPA levels in the tears of patients with Sjögren's syndrome before and after treatment.

[0065] Figure 5 This is a box plot showing ACPA levels in the tear fluid of patients with GVHD before and after treatment.

[0066] Figure 6 This is an immunofluorescence staining photograph of an imprint cytology specimen from the ocular surface, showing the presence of citrullinated protein in non-epithelial cells of patients with Sjögren's syndrome. Sjögren's syndrome – cit / k14 or cit / NE → N = 5; healthy – cit / k14 → N = 2.

[0067] Figure 7 This is an immunofluorescence staining photograph of an imprint cytology specimen from the ocular surface, showing the presence of citrullinated protein in neutrophils from patients with Sjögren's syndrome. cit / NE→N=2.

[0068] Figure 8 This is an immunofluorescence staining photograph of an imprint cytology specimen from the ocular surface, showing the presence of citrullinated proteins in the epithelial cells of patients with ocular GVHD (oGVHD). Def. oGVHD, cit / k14→N=4; no oGVHD, cit / k14→N=2.

[0069] Figure 9 This is an immunofluorescence staining image of the ocular surface mucous membrane, showing the presence of induced Fc-γ receptors on neutrophils. Isolated neutrophils N=1; Pt mucus N=2

[0070] Figure 10 These are photographs of immunofluorescence staining of impression cytology specimens from the ocular surface, showing the presence of Fc-γ receptors on non-epithelial cells (neutrophils and monocytes). FcR+NE:N = 5 different individuals, 4 different diseases.

[0071] Figure 11 These are photographs of dot blot analysis and graphs showing the reactivity of ACPA in tears to NET-citrullinated proteins.

[0072] Figure 12These are photographs of dot blot analysis and images showing the polyclonal nature of autoantibodies in ACPA-positive tears.

[0073] Figure 13 This is an immunofluorescence staining image of the ocular surface mucous membrane, showing the presence of citrullinated protein H4R3cit on the patient's mucous cell aggregates.

[0074] Figure 14 This is a photograph of a mouse cornea, showing ocular surface staining with fluorescein to reveal corneal epithelial disease. Data shows that ACPA antibodies can induce corneal epithelial disease, but not all ACPA antibodies can.

[0075] Figure 15 This is an image of an immunofluorescence staining of an imprint cytology specimen from the ocular surface of the mouse cornea, showing that ACPA antibody #1 induces NETosis and citrullination of the mouse corneal epithelium.

[0076] Figure 16 These are photographs of mouse corneas, showing ocular surface staining with fluorescein to reveal corneal epithelial disease. Data show that ACPA-induced pathological effects on the cornea are mediated via Fc receptors. Mouse IgG competes with ACPA antibodies for binding to Fc receptors, and Fc receptor blockers can block all Fc receptors. This competitive and peptide-blocking effect may eliminate the pathological effects of ACPA on the cornea, suggesting that methods preventing ACPA antibodies from interacting with Fc receptors may be a potential therapeutic strategy for citrullination-related eye diseases.

[0077] Figure 17 This is an image of immunofluorescence staining of neutrophils, showing ACPA-H4R3cit inducing NETosis in vitro.

[0078] Figure 18 This data demonstrates 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 elevation in LDH levels.

[0079] Figure 19 Exemplary images of eyes with high levels of autoantibodies (ACPA) in the tear film and in severe ocular surface disease are provided.

[0080] Figure 20 A and 20B provide exemplary images of an eye with high levels of ACPA in the tear film but without systemic autoimmune disease and with negative ACPA autoantibodies and rheumatoid arthritis seroconversion.

[0081] Figure 21A and 21B provide exemplary images of eyes with asymmetrical ocular disease; the eyes with more severe ocular surface disease have higher levels of autoantibodies (ACPA) in their tears.

[0082] Figure 22 Images A and 22B provide images from a 56-year-old woman suffering from severe tear deficiency and severe ocular surface disease due to ocular graft-versus-host disease (Case Study 1). These images demonstrate that OSIG treatment alleviated the signs and symptoms of severe dry eye disease and reduced the levels of inflammatory biomarkers in the tears.

[0083] Figure 23 Images were provided from a 31-year-old woman with graft-versus-host disease of the eye, suffering from severe tear deficiency and severe ocular surface disease (Case No. 2). These images demonstrate that OSIG treatment alleviated the symptoms of neurotrophic keratitis.

[0084] Figure 24 Images were provided from a 74-year-old male with neurotrophic keratitis due to a history of LASIK ophthalmic surgery in his left eye (Case Study 3). These images demonstrate that OSIG treatment alleviated the signs and symptoms of severe dry eye and reduced the levels of inflammatory biomarkers in the tear film.

[0085] Figure 25 Images were provided from a 29-year-old male suffering from tear deficiency and severe ocular surface disease due to Steven Johnson syndrome (Case Study 6). These images demonstrate that OSIG treatment alleviated the signs and symptoms of severe dry eye caused by Steven Johnson syndrome. Detailed Implementation

[0086] Based on the principles of this study, autoantibodies (such as anti-citrullinated protein antibodies (ACPA)) were found in the tears of patients with the following conditions: eye diseases such as dry eye disease (DED), Sjogren's syndrome, meibomian gland disease, superior limbal keratoconjunctivitis (SLK), ocular cicatricial pemphigoid, Steven Johnson syndrome, ocular graft-versus-host disease, peripheral ulcerative keratitis, episcleritis, scleritis; and these autoimmune diseases. Surprisingly, ACPA autoantibodies were present in the tears of patients even when ACPA and rheumatoid factor were not detected in serum, indicating that tear autoantibodies are produced within / around the ocular tissue. This result is unexpected because the presence of ACPA autoantibodies in the tears of patients with eye diseases was not anticipated, as they did not have ACPA or rheumatoid factor in their serum and had no evidence of systemic autoimmune disease. Patients with severe unilateral disease often exhibit elevated ACPA levels in their tears. Furthermore, eyes with very high ACPA levels in tears frequently indicate severe disease (such as corneal melting and scarring) and are poorly controlled despite aggressive treatment. In the study described in this article, the presence of citrullinated proteins and Fc receptors on the ocular surface of these patients was identified. Laboratory experiments confirmed that exposure of mouse corneas to ACPA induces ocular surface disease, and that targeting the interaction between ACPA and ocular tissues using IgG or Fc receptor blockers reduced the detrimental effects of ACPA on the ocular surface. In conclusion, combined IgG and / or synthetic variants thereof are suitable forms of therapeutic agents for ocular diseases caused by or affected by autoantibodies. Additionally, the polyclonal nature of autoantibodies identified in tears (e.g., the presence of cit histones, cit α-enolase, cit fibrinogen, etc.) makes combined IgG an attractive therapeutic agent, as targeting individual antibodies is unattractive and laborious.

[0087] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “a”, “an”, and “the” include plural indicators.

[0088] In an exemplary embodiment, an ophthalmic formulation comprises: (a) a pharmaceutically active compound capable of reducing the amount of autoantibodies on or within the ocular surface, such autoantibodies being generated in response to citrullinated proteins (ACPA autoantibodies generated due to citrullination of proteins), or in response to homocitrullinated proteins (anti-CarP antibodies generated due to carbamylation of proteins), or natural autoantibodies generated in response to natural proteins; (b) an optional second pharmaceutically active compound selected from the group consisting of PAD enzyme inhibitors, NETs dissantling agents, and Fc receptor blockers, and combinations thereof; and (c) a pharmaceutically acceptable ophthalmic excipient. Unless the context otherwise requires, the term "pharmaceutically active compound" means a compound that is pharmaceutically active when applied topically to the ocular surface or injected into the ocular cavity. Thus, for example, the term "pharmaceuticalally active NSAID" means an NSAID that is pharmaceutically active when applied topically to the ocular surface or injected into the ocular cavity.

[0089] Another aspect of this disclosure provides an ophthalmic preparation comprising: (a) a pharmaceutically acceptable ophthalmic excipient; (b) a therapeutically effective amount of a pharmaceutically active compound comprising immunoglobulin G (IgG); and (c) optionally a second pharmaceutically active compound selected from the group consisting of steroids, anti-inflammatory agents, mucolytics, PAD enzyme inhibitors, NETs dissolving agents, and Fc receptor blockers, as well as combinations thereof. The pharmaceutically active compound comprising immunoglobulin G (IgG) is capable of treating clinical symptoms selected from the group consisting of inflammatory and immune ocular surface diseases that can cause symptoms such as: eye discomfort, dry eye syndrome, keratitis, symblepheronformation, fornix foreshortening, palpebral / conjunctival keratosis, and subconjunctival fibrosis. "Therapeutically effective amount" means the amount of compound sufficient to achieve such treatment of a disease or clinical symptom when administered to a mammal to treat the disease or clinical symptom. The "therapeutic effective dose" will vary depending on the compound, the disease or clinical symptom and its severity, as well as the age, weight, etc. of the mammal being treated. "Treatment" or "treatment" for a clinical symptom or disease includes: (1) preventing the development of a clinical symptom or disease, i.e., preventing the development of clinical symptoms of the symptom or disease in mammals that may be susceptible to the symptom or disease but have not yet experienced or exhibited symptoms of the symptom or disease; (2) inhibiting a clinical symptom or disease, i.e., preventing or reducing the development of a clinical symptom or disease or its symptoms; or (3) alleviating a clinical symptom or disease, i.e., causing the clinical symptom or disease or its symptoms to subside.

[0090] Another aspect of this disclosure provides an ophthalmic formulation comprising: (a) a pharmaceutically acceptable ophthalmic excipient; (b) a therapeutically effective amount of a pharmaceutically active compound comprising combined human immunoglobulin G (IgG); and / or (c) a therapeutically effective amount of combined human plasma proteins (dilution ranging from 0.1% to 99% of human plasma proteins) and combined human lipids (dilution ranging from 0.1% to 99%).

[0091] The ophthalmic preparations disclosed herein can be used to treat a variety of clinical conditions associated with inflammatory and immune ocular surface diseases. Exemplary clinical conditions that can be treated with the ophthalmic preparations disclosed herein include, but are not limited to, ocular surface diseases that can cause symptoms such as: eye discomfort, dry eye syndrome, mucocellular aggregates / debris in the tear film, symblepheron formation, fornix foreshortening, eyelid / conjunctival keratosis, and subconjunctival fibrosis. More specific types of ocular surface diseases include: ocular graft-versus-host disease (oGVHD); Steven Johnson syndrome; ocular cicatricial pemphigoid (OCP); mild, moderate, and severe delaminic dry eye (DED); meibomian gland disorders; superior limbal keratoconjunctivitis (SLK); adequate tear production DED; blepharospasm syndrome; neurotrophic eye diseases; symptom-sign disconnect (inconsistent DED); neuropathic pain; thyroid eye disease (Grave's ophthalmopathy); rheumatoid arthritis-related eye diseases; lupus-related eye diseases; Sjogren's syndrome; and secondary Sjogren's syndrome. Syndrome; ocular rosacea; allergic keratoconjunctivitis (spring); aniridia; keratitis due to aseptic inflammation or viral, bacterial or fungal infection; postoperative / post-traumatic ocular conditions; peripheral ulcerative keratitis; episcleritis; scleritis; uveitis; and glaucoma. Exemplary postoperative / post-traumatic ocular conditions that can be treated with the ophthalmic preparations disclosed herein include, but are not limited to, ocular conditions related to: posterior ocular surface reconstruction surgery; application of antimetabolites to the ocular surface; pterygium surgery; glaucoma surgery; cataract surgery; laser vision correction surgery (LASIK, LASEK, EPI-LASIK); artificial corneal surgery; and radiation damage.

[0092] Other ophthalmic clinical conditions that can be treated with the ophthalmic preparations disclosed herein include, but are not limited to: dry eye syndrome (keratoconjunctivitis sicca); Sjogren's syndrome; congenital acritonia; dry eye disease (dry eye due to vitamin A deficiency); keratomalacia; thyroid eye disease; ocular rosacea; eyelid diseases; meibomian gland diseases; meibomian gland dysfunction; ectropion; blepharitis; eyelid skin laxity; sarcoidosis; stye; meibomianitis; meibomian gland cyst; ptosis; pterygium; eyelid edema; eyelid dermatitis; trichiasis; eyelash loss; dacryoadenitis; Stevens-Johnson syndrome; ocular graft-versus-host disease; dacryoadenitis; conjunctivitis; keratoconjunctivitis; palpebral conjunctivitis; blepharokeratoconjunctivitis; allergic conjunctivitis; vernal conjunctivitis; conjunctival hyperemia. suffusion; conjunctivochalasis; subconjunctival hemorrhage; pterygium; conjunctival macular degeneration; conjunctival edema; iritis; iridocyclitis; anterior uveitis; glaucoma; conjunctivitis; keratitis; scleritis; episcleritis; peripheral ulcerative keratitis; neurotrophic keratitis; neurotrophic eye disease; corneal ulcer; ulcerative keratitis; corneal abrasion; photokeratitis; ultraviolet keratitis; exposure keratitis; superficial punctuate keratitis; Thygeson's superficial punctuate keratitis Herpes simplex keratitis; herpes zoster keratitis; rosacea; postoperative inflammation following ocular surgery (i.e., eyelid surgery, cataract surgery, corneal surgery, refractive surgery (including optical refractive corneal ablation), glaucoma surgery, lacrimal gland surgery, conjunctival surgery, ocular muscle surgery); ocular surface conditions caused by chemical burns, thermal burns, or physical trauma.Ocular symptoms caused by the following autoimmune or vascular diseases: rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, Reiter's syndrome, enteropathic arthritis, psoriatic arthritis, discoid and systemic lupus erythematosus, multiple sclerosis, Graves' disease, antiphospholipid syndrome, sarcoidosis, Wegner's granulomatosis, Behcet's syndrome, polyarteritis nodosa, Takayasu's arteritis, dermatomyositis, psoriasis, relapsing polychondritis, vasculitis, and sickle cell anemia.

[0093] In some embodiments, the ophthalmic preparations of this disclosure are used to treat dry eye syndrome. Dry eye syndrome is primarily classified into two categories: (i) tear deficiency dry eye syndrome (ADDE) and (ii) evaporative dry eye syndrome (EDE). There are also cases of dry eye syndrome with mixed mechanisms (i.e., both ADDE and EDE). ADDE is primarily caused by failure of tear secretion. ADDE can be further subdivided into Sjogren's syndrome dry eye disease (where the lacrimal and salivary glands are targeted by an autoimmune process (e.g., rheumatoid arthritis)) and non-Sjogren's syndrome dry eye disease (tear dysfunction, but excluding the systemic autoimmune features of Sjogren's syndrome, such as age-related dry eye). Conversely, EDE is primarily caused by excessive loss of moisture due to exposure of the ocular surface in the presence of normal tear secretion function. The causes can be external (e.g., ocular surface conditions due to certain external exposures, contact lens wear, or vitamin A deficiency) or internal (e.g., meibomian gland dysfunction and eyelid opening disorders). The meibomian glands secrete a mixture of lipids and other components that form the outer layer of the tear film in front of the eyes. This lipid layer helps reduce tear film evaporation. Meibomian gland dysfunction (MGD) leads to evaporative dry eye. One of the most well-known clinical findings in MGD is the presence of numerous telangiectatic vessels at the eyelid margins. MGD can also be associated with tear-deficient dry eye, as seen in ocular graft-versus-host disease (oGVHD).Other specific dry eye syndromes that can be treated with the compositions of this 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 syndrome; dry eye caused by conjunctivochalasis; dry eye caused by palpebral conjunctivitis; dry eye caused by blepharokeratoconjunctivitis; dry eye caused by superficial punctuate keratitis; dry eye caused by thygeson's superficial punctuate keratopathy; dry eye caused by oGVHD; and Sjogren's dry eye syndrome. Dry eye syndrome; Stevens-Johnson syndrome; MGD; dry eye syndrome caused by meibomian gland disease; dry eye syndrome caused by vitamin A deficiency; dry eye syndrome caused by pharmacological effects (i.e., hormone replacement therapy, blood pressure medication, antihistamines, antidepressants, anticholinergics, glaucoma medications, antihypertensive drugs, diuretics, sedatives, isotretinoin, nasal decongestants, oral contraceptives, beta-blockers, phenothiazines, atropine, analgesic opioids); dry eye syndrome caused by pregnancy; dry eye syndrome caused by LASIK surgery or refractive surgery; dry eye syndrome caused by collagen vascular diseases (i.e., systemic lupus erythematosus, Wegener's granulomatosis, rheumatoid arthritis, relapsing polychondritis). Dry eye caused by: polychondritis; lacrimal gland infiltration caused by tumors or sarcoidosis; lacrimal gland radiation fibrosis; ablation of lacrimal gland, meibomian gland, or goblet cells; sensory denervation; thermal or chemical burns; underlying diabetic symptoms; viral, fungal, or bacterial infections; prolonged contact lens wear; eyelid diseases or injuries (i.e., ptosis); corneal dystrophy; autoimmune diseases; age-related dry eye; and combinations thereof.

[0094] In some specific embodiments, the ophthalmic preparations of this disclosure are used to treat meibomian gland dysfunction (MGD). In other embodiments, the ophthalmic preparations of this disclosure are used to treat tear-deficiency dry eye syndrome (ADDE). In some cases, methods for treating ADDE include treating patients requiring treatment for Sjögren's dry eye syndrome, ocular graft-versus-host disease (oGVHD), or non-Sjögren's dry eye syndrome. In other embodiments, methods for treating dry eye syndrome include treating patients requiring treatment for evaporative dry eye syndrome (EDE). Still in other embodiments, methods of this disclosure include treating patients requiring treatment for dry eye syndrome with a mixed mechanism consisting of ADDE and EDE. Still in other embodiments, methods of this disclosure include treating patients with dry eye syndrome due to complications of refractive surgery or due to one or more of the following causes: vitamin A deficiency, ocular surface disease, allergy, aging, contact lens wear, medication use, or eyelid disease.

[0095] Two forms of post-translational modification (PTM) – citrullination and carbamylation – produce two chemically highly correlated non-standard amino acids in peptides – citrulline and homocitrulline, respectively. Increasing evidence suggests that protein citrullination and carbamylation have pathophysiological roles, such as the role of autoantibodies against these proteins in rheumatoid arthritis. However, the roles of citrullination and carbamylation, and ACPA, in ocular surface diseases have not been previously described. The study presented in this article identifies the presence of ACPA in ocular surface fluid.

[0096] In some embodiments, the ophthalmic formulation comprises immunoglobulin G (IgG) as a pharmaceutically active ingredient. Types of IgG that may be used in the ophthalmic formulations of this disclosure include, but are not limited to: (i) concomitant immunoglobulin G (OSIG) derived from serum / plasma; (ii) autologous IgG purified from autologous plasma / serum; (iii) polymerized IgG1 Fc molecules (IgG1Fc hexamer); (iv) IgG2a Fc polymers (Stadhumers); (v) multivalent Fc structures; (vi) glycoengineered sialylated IgG; and (vii) IgG-Fc glycosylation or combinations thereof. In some embodiments, a commercially available OSIG formulation is formulated together with ophthalmic excipients (diluted or concentrated as needed) to achieve an IgG concentration three to six times that normally exists at the application site (physiological or pathological), and then dispensed as eye drops, injections, or other suitable delivery methods. Examples of commercially available IgG solutions include “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 mixed with ophthalmic excipients to the desired IgG concentration (e.g., 4 mg / ml) and the desired pH of at least 6.0 to prepare the disclosed OSIG compositions.

[0097] In some embodiments, ophthalmic formulations comprise functional equivalents of intact IgG antibodies, such as antigen-binding fragments in which one or more antibody domains are truncated or absent (e.g., Fv, Fab, Fab', or F(ab)2 fragments), and genetically engineered antibodies or their protein-binding fragments, including single-chain antibodies or antibodies that can bind to more than one epitope (e.g., bispecific antibodies) or antibodies that can bind to one or more different antigens (e.g., bispecific or multispecific antibodies), may also be used in this disclosure.

[0098] Immunoglobulins are a group of closely related glycoproteins, composed of 82%–96% protein and 4%–18% carbohydrates. These approximately 150 kDa glycoproteins are present in plasma at an average concentration of 7 to 12 g / L, depending on individual variability and environmental levels of exposure to antigens. Immunoglobulin G (IgG) is the major effector molecule in the human humoral immune response, accounting for approximately 75% of total Ig in the plasma of healthy individuals. The basic Ig molecule has a four-chain structure, comprising two identical heavy (H) chains and two identical light (L) chains linked together by interchain disulfide bonds. IgG has a dual function, characterized by its ability to recognize and specifically react with antigens while performing a series of non-specific effector functions, thereby rendering the antigen harmless and ultimately eliminating it. This functional dichotomy of IgG is reflected in the molecular structure, which includes two variable regions (Fab) responsible for antigen binding and constant regions (Fc or crystallizable fragments) mediating specific effector functions. The presence of complex oligosaccharide structures modulates IgG function, particularly complement activation and binding to FcγR. In some embodiments, the ophthalmic formulation of this disclosure will consist of immunoglobulin G (IgG) with enhanced sialylation (sialylated IgG), which is achieved using standard chemical methods.

[0099] In some embodiments, the concentration of IgG in the ophthalmic formulations of this disclosure is defined by a 10% ocular surface immunoglobulin (OSIG) solution or any other suitable concentration. OSIG is a therapeutic formulation of pooled normal multispecific human IgG obtained from the serum / plasma of a large number of healthy donors. This formulation contains antibodies against microbial antigens, autoantigens (natural autoantibodies), and anti-idiotype antibodies that recognize other antibodies. The plasma used in OSIG production comes from two sources: approximately 20% from blood donors and another 80% from plasma donors. Individual plasmas are pooled; the pool size is at least 1,000 donors but can be up to 100,000 donors. Thousands of donors contribute a typical plasma pool for isolating immunoglobulins, representing a large amount of antibody specificity against infectious agents (such as bacteria and viruses) and a large amount of autoantigens reflecting the cumulative environmental exposure of the donor population. OSIG formulations consist of intact IgG molecules, with the distribution of IgG subclasses (IgG1, IgG2, IgG3, and IgG4) corresponding to that of normal serum. The main purification processes used for the production of OSIG may include: (i) separation, such as using polyethylene glycol (PEG), a synthetic polymer, which can also be used to separate proteins from natural mixtures (such as plasma) by fractional precipitation via exclusion mechanisms; and (ii) chromatography, such as anion exchange chromatography, hydrophobic charge-induced chromatography, or size exclusion chromatography. Biological effects of OSIG that may contribute to the efficacy of the ophthalmic formulations disclosed herein include: (i) functional blockade of Fc receptors. Due to Fc-mediated biological effects, OSIG saturates Fc receptors, leading to reduced cell destruction; (ii) autoantibody cancellation and inhibition of autoantibody production. OSIG formulations contain anti-idiotype antibodies, which are antibodies capable of specifically interacting with the variable region (antigen recognition site) of an autoantibody. This interaction has the potential to cancel out autoantibodies and inhibit their production via binding to self-reactive B lymphocytes; and (iii) complement inhibition. The Fc portion of OSIG can bind to complement C3b and C4b fragments, thereby inhibiting their tissue deposition and the production of C5 convertase, (iv) the modulation of cytokine (including IL-1, -2, -3, -4, -5, -10, TNF-α, and GM-CSF) and cytokine antagonist production (IL-1 receptor antagonists); and (v) signaling via the inhibitory Fc receptor FcγRIIB.

[0100] In the ophthalmic formulations of this disclosure, the amount of IgG can range from about 0.01 mg / mL to about 1 g / mL, typically from about 1 mg / mL to about 10 mg / mL. In some embodiments, the concentration or amount of IgG can be about 0.01 mg / mL, and in other embodiments, the concentration or amount of IgG can be about 0.05 mg / mL. In some embodiments, the concentration or amount of IgG can be about 1 mg / mL, and in other embodiments, the concentration or amount of IgG can 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.

[0101] This disclosure covers the presence of autoantibodies on the ocular surface (tears) or inside the eye (water and vitreous) in various eye diseases, with anti-citrullinated protein antibody (ACPA) being a prototype example. The autoantibodies that may be present and covered by this disclosure include antibodies against the following proteins: β2-glycoprotein, C1q, CENP-B (centromerein B), CENP-A (centromerein A), Jo-1, Ku, Mi-2, myeloperoxidase (MPO), PCNA (proliferating cell nuclear antigen A), PL-12 (alanyl-tRNA synthetase), PM / Scl 100, protease 3, RNP (ribonucleoprotein), RNP / Smith (RNP / Sm), ribosomal P, Scl-70, Sm, SSB / La (Sjögren's syndrome-associated antigen B / La), SSA / Ro60 (Sjögren's syndrome-associated antigen A / Ro60 kDa), and SSA / Ro52 (Sjögren's syndrome-associated antigen A / Ro52 kDa). Although not designed to be exhaustive, the autoantibodies (ACPA or non-ACPA) covered by this disclosure may be derived from the proteins or fragments thereof (citrullinated or native) listed in Table 1.

[0102]

[0103] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable ophthalmic excipient" means an excipient that can be used to prepare the pharmaceutical compositions of this disclosure. Such excipients are generally considered safe, non-toxic, and neither biologically active nor undesirable by those skilled in the art, and include veterinary and human pharmaceutically acceptable excipients. As used in the specification and claims, "pharmaceuticalally acceptable excipient" includes one or more such excipients. Exemplary pharmaceutically acceptable excipients include: salts (such as sodium chloride) or tensile agents, gums, resins, solvents (such as water), non-aqueous solvents (such as ethanol, oils, pH-maintaining buffers, pH adjusters (e.g., bases, such as sodium hydroxide; and acids, such as hydrochloric acid), emulsifiers, thickeners, microemulsion or nanoemulsion forming agents, preservatives, surfactants, etc. Exemplary pharmaceutically acceptable excipients that can be used in ophthalmic formulations of this disclosure include, but are not limited to: water, benzyl alcohol, sodium hydroxide, hydrochloric acid, Castrol oil, citrate buffers, Tris buffers, phosphate buffers, and other excipients known to those skilled in the art.

[0104] In some embodiments, the ophthalmic preparations of this disclosure may also contain salts, such as sodium chloride. In other embodiments, the ophthalmic preparations of this invention may also contain non-aqueous solvents, such as benzyl alcohol, ethanol, or other non-aqueous solvents known to those skilled in the art.

[0105] In other embodiments, the ophthalmic formulation of this disclosure is adjusted from a pH of about 5.0 to a pH of about 8.5, typically from a pH of about 5.0 to a pH of about 8.0, and typically from a pH of about 5.0 to a pH of about 7.5. Other 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, or 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 preparations of this disclosure can be adjusted as needed using, for example, sodium hydroxide and / or hydrochloric acid, to achieve a desired pH level.

[0106] Ophthalmic preparations can be formulated as eye drops, topical liquids, ointments, lotions, suspensions, or gels (e.g., sodium IgG in a hydrogel). Ophthalmic preparations can also be formulated as oil or suspension nanoemulsions. Additionally, ophthalmic preparations can be formulated as injectable formulations.

[0107] In some embodiments, the ophthalmic formulations of this disclosure are preservative-free and are formulated for single use or in multi-dose vials. If preservatives are used, suitable preservatives include, but are not limited to: benzyl ammonium, purite, chlorobutanol, sodium perborate, stabilized oxychloro complex (SOC), polyquaternium-1 (PQ-1), thimerosal, benzyl alcohol, sorbic acid, methylparaben / propylparaben, chlorhexidine, disodium EDTA, sofZia, and other preservatives known to those skilled in the art of ophthalmic or ophthalmic formulation chemistry.

[0108] When present, the second pharmaceutically active compound is selected from the group consisting of steroids, anti-inflammatory agents, mucolytics, and combinations thereof. Exemplary steroids suitable for ophthalmic formulations of this disclosure include, but are not limited to, methylprednisolone, prednisolone, dexamethasone, loreprednol etabonate, fluocinolone acetonide, difluprednisolone, fluorometholone, methylhydroxyzine, fluocinolone acetonide, limexoloxetine, and combinations thereof. When steroids are used in ophthalmic formulations of this disclosure, the amount of steroid 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 typically from about 0.1% w / w to about 0.3% w / w. It should be understood that the scope of this disclosure is not limited to these specific ranges of steroid amounts. In particular, the amount of steroid present in ophthalmic formulations of this disclosure generally depends on the steroid used. For example, the amount of steroid present can vary depending on the activity of the specific steroid used, the molecular weight of the steroid, and the purpose of using the steroid in the ophthalmic formulation disclosed herein.

[0109] Exemplary anti-inflammatory agents suitable for ophthalmic formulations of this disclosure include, but are not limited to: cyclosporine; Tacrolimus; interleukin-1 receptor antagonists (analexin); other NSAIDs, such as ketoroxyprofen, diclofenac, flurbiprofen, bromfenac, napafenac; and combinations thereof. When an anti-inflammatory agent is used in the ophthalmic formulations of this disclosure, the amount of the anti-inflammatory 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 typically from about 0.1% w / w to about 0.3% w / w. It should be understood that the scope of this disclosure is not limited to these specific ranges of the amount of the anti-inflammatory agent. In particular, the amount of anti-inflammatory agent present in the ophthalmic formulations of this disclosure generally depends on the specific anti-inflammatory agent used, such as the activity of the specific anti-inflammatory agent used, the molecular weight of the anti-inflammatory agent, etc.

[0110] Exemplary mucolytic agents that can be used in ophthalmic formulations of this disclosure include, but are not limited to: N-acetylcysteine; Nacystelyn; dextran; DNase I (Dairycium α); coagulants; thymosin β4; 14- and 15-membered macrolide antibiotics (e.g., erythromycin, non-antimicrobial derivatives of erythromycin (e.g., EM703 and EM900), clarithromycin, roxithromycin, fedamycin, telithromycin, and azithromycin); and combinations thereof. It should be understood that antibiotics are used primarily because of their mucolytic activity. When a mucolytic agent is used in an ophthalmic formulation of this 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 typically from about 0.1% w / w to about 0.3% w / w. It should be understood that the scope of this disclosure is not limited to these specific ranges of the amount of mucolytic agent. In particular, the amount of mucolytic agent present in the ophthalmic formulations of this disclosure generally depends on the specific mucolytic agent used, such as the activity of the mucolytic agent, the molecular weight of the mucolytic agent, etc.

[0111] The ophthalmic formulations disclosed herein may be homogeneous or heterogeneous. In some embodiments, the ophthalmic formulations of this disclosure comprise oils or fatty acid esters. Fatty acid esters have the meaning commonly understood in the art as esters formed between alcohols and fatty acids. Exemplary fatty acid esters that may be used in formulations of this disclosure include, but are not limited to: triglycerides commonly referred to as vegetable oils; monoglycerides and diglycerides of fatty acids; methyl esters of fatty acids; and other fatty acid esters known to those skilled in the art. It should be understood that fatty acid esters may be mixtures of several compounds or substantially pure compounds. Typically, fatty acid esters are vegetable oils. Specific examples of vegetable oils that may 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.

[0112] Various mediators can be used in the ophthalmic formulations of this disclosure. These mediators include, but are not limited to: purified water (aqueous), polyvinyl alcohol, povidone, hydroxypropyl methylcellulose, poloxamer, carboxymethyl cellulose, hydroxyethyl cellulose, polyols, sodium hyaluronate, praline, corbopol, cyclodextrin, and mixtures of two or more thereof. Excipients are used in the formulation in the desired amount to provide the concentration of the active compound disclosed herein. In one particular embodiment, the mediator comprises water.

[0113] In some embodiments of this disclosure, emulsion-stabilizing polymers are used. While not intended to limit the scope of this disclosure, emulsion-stabilizing polymers typically contain hydrophilic groups such as cellulose, sugars, ethylene oxide, hydroxides, carboxylic acids, or other polyelectrolytes. Without being bound by any theory, it is believed that these polymers help stabilize the emulsion by increasing the viscosity of the formulation and reducing interfacial tension. Surfactants (such as polysorbate 80 or other ophthalmologically acceptable surfactants) can be used to stabilize the emulsion. Some examples of emulsion-stabilizing polymers that can be used in this disclosure include, but are not limited to, carbomer, Sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, polyethylene glycol, and mixtures of two or more thereof.

[0114] The ophthalmic preparations disclosed herein can be packaged in various packaging forms known in the field of topical ophthalmology. In one particular embodiment, the ophthalmic preparation is packaged in a sterile, preservative-free, single-use package or vial or container (i.e., a unit-dose vial). Each vial (e.g., as small as 0.9 mL) can be made of low-density polyethylene to contain a small amount of the preparation, such as 0.4 mL for a single use. Thus, in the case of sterilizing the ophthalmic preparation and containing it in a single-use, single-dose container for topical application in the form of drops, multiple vials in the form of 30 vials, 60 vials, etc., can be packaged in a covered tray, for example, a polypropylene tray with a peelable aluminum cap. The entire contents of each tray can be dispensed as is, one vial or package per use, and discarded immediately after each use. For example, plastic ampoules or vials or containers can be manufactured using blow-fill-seal (BFS) technology. The BFS method can involve plastic extrusion, molding, aseptic filling, and hermetically sealed in a continuous operation, and those methods are known in the art. In another embodiment, the formulation is packaged in multi-dose vials, allowing for aseptic dispensing of the material each time using a dedicated container / barrier that maintains sterility. In yet another embodiment, the ophthalmic formulation is packaged as a sterile product in a conventional vial / container.

[0115] In some embodiments, the dosage form of this disclosure is an eye drop or an eye drop preparation of a heterogeneous aqueous solution.

[0116] In one particular implementation, an ophthalmic preparation containing IgG (OSIG) is formulated as eye drops and used to treat inflammatory and immune ocular surface diseases that can cause eye discomfort, mucosal cell aggregates / debris in the tear film, symblepheron formation, fornix foreshortening, eyelid / conjunctival keratosis, or subconjunctival fibrosis. Specific clinical conditions that can be treated with the ophthalmic preparations disclosed herein include conditions such as: ocular graft-versus-host disease (oGVHD); Steven Johnson syndrome; ocular cicatricial pemphigus (OCP); mild, moderate, and severe dry eye with acridity (DED) (secondary to Sjögren's syndrome, non-Sjögren's syndrome, idiopathic, and other causes); meibomian gland disorders (dysfunction or atrophy); superior limbal keratoconjunctivitis (SLK); DED with adequate tear production (consistent or inconsistent); blepharochalasis syndrome; neurotrophic eye diseases; aniridia; and postoperative / post-traumatic pathology, such as after ocular surface reconstruction surgery, application of antimetabolites (mitomycin C, 5-fluorouracil, etc.) to the ocular surface, or artificial corneal repair surgery or radiation injury.

[0117] Another aspect of this disclosure provides a method for treating an eye disease in a subject requiring such treatment. Such a method typically involves administering to the subject a therapeutically effective amount of a subtype of an anticoagulant below the anticoagulant dose to treat the eye disease. In some embodiments, the subject is administered an IgG ophthalmic preparation at least twice daily. Still in other embodiments, the subject is administered an IgG ophthalmic preparation at least twice monthly. The ophthalmic preparation may be an aqueous solution, an aqueous suspension, a gel, etc.

[0118] Another aspect of this disclosure provides a method for diagnosing or monitoring ocular surface diseases in a subject. The method includes comparing the level of autoantibodies in a sample obtained from the subject with a control level of said autoantibodies to diagnose or monitor ocular surface conditions in the subject. As used herein, the term "control level of autoantibodies" refers to a level that can be compared with the level of autoantibodies in a test sample, including: (i) autoantibodies in a subject without ocular surface disease, (ii) autoantibodies in a subject with ocular surface disease, and (iii) autoantibody levels from the same subject before treatment, at the beginning of treatment, or before the onset of any symptoms of ocular surface disease.

[0119] In some implementations, the control level can be a normal level, meaning the level in samples from healthy patients, i.e., subjects without ocular surface disease. This control level may be more specifically referred to as a "negative control." This allows for the determination of a control level based on autoantibodies, where the autoantibody levels in the sample being evaluated for ocular surface disease have a measurable difference or are substantially no difference compared to the control level.

[0120] In another embodiment, the control level may be the level of autoantibodies established in samples from a subject or a group of individuals believed to have an ocular surface disease. This may be more specifically referred to as the “positive control level.” As used herein, the term “positive control” refers to the level of autoantibody expression or biological activity established in a sample derived from a subject, another individual, or a group of individuals, where, based on data from that sample, the sample is believed to have a disease.

[0121] In other implementations, a control level can be established from previous samples from the subject, thereby enabling monitoring of disease progression or regression over time and / or assessment of the efficacy of treatment.

[0122] Unless otherwise stated or required by context, the term "monitoring" refers to determining the progression of ocular surface disease or the effectiveness of a particular treatment or medication. The term "diagnosis" refers to the process of determining the presence of ocular surface disease in a subject. It may also include determining what specific ocular surface disease is present in the subject.

[0123] Autoantibodies can be used to diagnose ocular surface diseases in order to initiate appropriate therapy or treatment, such as with IgG eye drops or another appropriate pharmaceutical product.

[0124] Changes in autoantibody levels can be used to assess response to treatment or the effectiveness of treatment. Treatment intensity can also be titrated to autoantibody levels.

[0125] This method can be used to provide rapid results, as an in-office test for the diagnosis and management of ocular surface diseases, and for initiating / titering IgG eye drops for such diseases, either as a single agent or in combination with other active pharmacological agents. The test can be designed to measure the composition of tears or other ocular fluids. (See also: [link to related information]). Figure 1 Concentrations below 5 have not been considered significant to date. However, levels above 5 can be shown to indicate treatable symptoms and are considered within the scope of this disclosure.

[0126] Because the IgG composition is stable over a long period, treatment can be administered periodically. Treatment can be designed to be administered at least twice daily or at least twice monthly, or according to any other suitable treatment regimen, provided that the treatment is sufficient to reduce the levels or the harmful biological effects caused by autoantibodies in the eye drops.

[0127] The methods of this disclosure utilizing autoantibodies can also be used to diagnose or monitor ocular surface diseases, such as oGVHD, Sjogren's syndrome, and other dry eye conditions. Such methods typically involve measuring the level of one or more autoantibodies present in a biological sample (e.g., tears, blood / serum, or ocular surface cells) obtained from the test subject and comparing it to a control level. The control level could be the subject's previous autoantibody level, the autoantibody level of a healthy subject, or the autoantibody level of a subject with an ocular surface disease.

[0128] Another aspect of this disclosure provides sensors, biosensors, multianalyte plates, arrays, assays, and kits for determining the levels of one or more autoantibodies obtained from a subject. Autoantibodies and methods of use thereof can be used to assist in the diagnosis and assessment of the onset and progression of ocular surface diseases. This disclosure also relates to the use of autoantibodies in clinical screening, prognostic assessment, treatment evaluation, drug screening, and drug development.

[0129] In a particular aspect, this disclosure provides a method for diagnosing or monitoring ocular surface diseases in a subject, the method comprising: obtaining a biological sample from the subject and comparing the levels of one or more autoantibodies in the biological sample with control levels.

[0130] The level of autoantibodies can be readily determined using any conventional method available to those skilled in the art. Exemplary methods include, but are not limited to, direct or indirect methods, such as coupled or uncoupled enzymatic methods; electrochemical methods; spectroscopic methods (e.g., spectrophotometry, fluorescence methods, photometry, polarimetry, etc., using a UV / VIS spectrometer); chromatographic methods (e.g., HPLC, gas chromatography, MPLC, LPLC, etc.); and immunological methods (e.g., ELISA, DOT-BLOT analysis, etc.).

[0131] Another aspect of this disclosure provides a method for diagnosing or monitoring ocular surface diseases or their predisposition. Such a method includes comparing the levels of one or more autoantibodies present in a biological sample obtained from a test subject to a control level. In one particular embodiment, the method of this disclosure is used to monitor the efficacy of a treatment (e.g., a therapeutic substance) in subjects who have, are suspected of having, or are susceptible to ocular surface diseases.

[0132] Another aspect of this disclosure provides a multianalyte plate or array capable of detecting one, two, three, four, or more (hundreds) of the autoantibodies of this disclosure. The multianalyte plate is capable of detecting a variety of different analytes. The array is capable of detecting a single analyte in multiple samples, or, as a multianalyte array, is capable of detecting many different analytes in a sample. The multianalyte set or multianalyte array according to the invention is capable of detecting one or more autoantibodies described herein, and is also capable of detecting autoantibodies other than those specifically described herein. Detection methods may include bead-based methods (e.g., the Luminex platform) or Phadia ImmunRCap.

[0133] Diagnostic or monitoring test kits suitable for performing the methods according to this disclosure are also provided, optionally together with instructions for use. The diagnostic or monitoring kit may include one or more biosensors according to this disclosure, and the kit may include a single sensor, a biosensor, or a combination of sensors and / or biosensors. The diagnostic or monitoring kit may include a plate or array according to this disclosure. The diagnostic or monitoring kit may include assays or combinations of assays according to the invention.

[0134] Furthermore, methods, sensors, biosensors, multianalyte plates, arrays, or kits according to this disclosure are provided to identify the use of substances capable of modulating or treating ocular surface diseases. Substances capable of modulating or treating ocular surface diseases may be anti-inflammatory, immunomodulatory, immunosuppressive, antibiotic substances, or palliative drugs (artificial tears, contact lenses, or punctal plugs) used to treat ocular surface diseases.

[0135] Throughout this specification and the appended claims, the term “treating” or “treatment” includes: (1) preventing disease, i.e., preventing the development of clinical symptoms of the disease in subjects who are susceptible to the disease but have not yet experienced or shown symptoms of the disease; (2) suppressing disease, i.e. preventing or reducing the development of the disease or its clinical symptoms; or (3) alleviating disease, i.e. causing the disease or its clinical symptoms to subside.

[0136] Higher levels of autoantibodies in test biological samples, relative to negative control levels (e.g., biomarker levels in subjects without ocular surface disease), indicate the presence of ocular surface disease, such as oGVHD, Sjögren's syndrome, and other dry eye conditions.

[0137] According to the monitoring methods of the present invention, the methods for determining developmental and diagnostic risks can be used to confirm the presence or susceptibility to ocular surface diseases; to monitor the development of ocular surface diseases by assessing onset and progression, or to evaluate improvement or regression of the disease. Monitoring and diagnostic methods can also be used to evaluate clinical screening, prognosis, treatment options, and methods for assessing treatment benefits, i.e., for drug screening and drug development. Effective diagnostic and monitoring methods provide a very powerful “patient solution” by establishing accurate diagnoses, with the potential to improve prognosis, and can quickly determine the most appropriate treatment (thus reducing unnecessary exposure to harmful drug side effects), thereby reducing “downtime” and relapse rates.

[0138] Methods for monitoring the efficacy of therapies can be used to monitor the effectiveness of existing and new therapies in human subjects and non-human animals (e.g., animal models). These monitoring methods can be incorporated into the screening of new drug substances and combinations. Regulation of protein biomarker levels can serve as an indicator of ocular surface disease or a predisposition to ocular surface disease. Elevated levels of autoantibodies over time indicate disease onset or progression, i.e., disease worsening, while decreased levels of protein biomarkers indicate disease improvement or remission. The identification of autoantibodies in ocular surface diseases allows for the integration of diagnostic procedures and treatment regimens.

[0139] Currently, there is no method available to determine effective treatments, and rapid assessment of drug response has not been possible to date. Traditionally, for a given treatment, many ocular surface disease therapies require treatment trials lasting weeks to months. The detection of autoantibodies disclosed in this disclosure can be used to screen subjects before they participate in clinical trials. Autoantibodies provide a means of indicating treatment response, response failure, adverse reactions, medication adherence, and achieving adequate serum drug levels. Autoantibodies can be used to warn of adverse drug reactions, a major problem encountered with all ocular surface disease medications. Autoantibodies can be used in the development of personalized ocular surface disease treatments because assessment of response can be used to fine-tune dosage, reduce the number of prescription drugs, reduce delays in obtaining effective treatment, and avoid adverse drug reactions. Therefore, by monitoring autoantibodies disclosed in this disclosure, patient care can be precisely tailored to match needs determined by the pharmacogenomics characteristics of the disease and the patient; thus, autoantibodies can be used to titrate optimal dosage, predict positive treatment responses, and identify patients at high risk of serious side effects.

[0140] Autoantibodies can be measured by direct or indirect detection methods. Biomarkers can be detected directly or indirectly by interaction with one or more ligands (such as enzymes, binding receptors, or transporters), antibodies, peptides, aptamers, or oligonucleotides, or any synthetic chemical receptor or compound capable of specifically binding to a biomarker. Ligands may have detectable labels, such as luminescent, fluorescent, or radioactive labels and / or affinity labels.

[0141] In one particular embodiment, the autoantibodies of this disclosure are detected and measured using: mass spectrometry-based techniques; chromatographic techniques; enzyme detection systems (by direct or indirect measurement); or sensors, such as sensor systems with amperometric, potentiometric, conductivity, impedance, magnetic, optical, acoustic, or thermal transducers. Sensors can be combined with physical, chemical, or biological detection systems. Examples of sensors include biosensors, i.e., sensors with, for example, nucleic acid-based biorecognition systems, such as oligonucleotide probes or aptamers, or proteins such as enzymes, binding proteins, receptor proteins, transporter proteins, or antibodies. Biosensors can be combined with immunological methods, electrical, thermal, magnetic, optical (e.g., holographic), or acoustic techniques for detecting biomarkers. Using such biosensors, target autoantibodies can be detected at expected concentrations found in biological samples. The methods of this disclosure are applicable to clinical screening, prognostic assessment, monitoring treatment outcomes, identifying patients most likely to respond to a specific treatment, drug screening and development, and assisting in the identification of new targets for drug therapy. The identification of key disease-specific autoantibodies is crucial for the integration of diagnostic procedures and treatment regimens.

[0142] Methods involving the detection and / or quantification of the autoantibodies disclosed herein can be performed using benchtop instruments or integrated into disposable, diagnostic, or monitoring platforms suitable for use in non-laboratory settings, such as in a physician's office or at a patient's bedside. Suitable sensors or biosensors for performing the methods of this disclosure include "credit" cards with optical or acoustic readers. The sensors or biosensors can be configured to allow the electronic transmission of collected data to a physician for interpretation, thus forming the basis of e-medicine.

[0143] For purposes of illustration and description, the foregoing discussion of this disclosure has been provided. The foregoing is not intended to limit this disclosure to one or more of the forms disclosed herein. While the description of this disclosure includes descriptions of one or more embodiments, as well as certain variations and modifications, other variations and modifications will, upon understanding this disclosure, be within the scope of this disclosure, for example, as may be possible within the skill and knowledge of those skilled in the art. The purpose is to obtain the right to include alternative embodiments to the permissible extent, including alternatives, interchanges, and / or equivalent structures, functions, scopes, or steps claimed, whether or not such alternatives, interchanges, and / or equivalent structures, functions, scopes, or steps are disclosed herein, and it is not intended to disclose any patentable subject matter.

[0144] In addition to the examples below, clinically significant results relevant to this disclosure are also provided in Kwon et al., Pathological consequences of anti-citrullinated protein antibodies in tears and the therapeutic potential of combined human immunoglobulin eye drops in dry eye disease, Ocul Surf., October 2019. (pii:S1542-0124(19)30377-5.doi:10.1016 / j.jtos.2019.10.004) https: / / doi.org / 10.1016 / j.jtos.2019.10.004 Kwon et al. have incorporated this paper in its entirety by reference.

[0145] Example

[0146] Example 1: Levels of autoantibodies (ACPA) in healthy subjects and ocular surface diseases

[0147] The presence of autoantibodies (ACPA) was demonstrated in the ocular surface irrigation fluid of healthy subjects. Figure 1 To establish a cutoff value for diagnosing positive ACPA autoantibodies in subjects, ocular surface irrigation was performed using 50 μl of artificial tears in healthy subjects (without tear deficiency dry eye (DED)), and the ACPA level of 10 μl of recovered irrigation fluid was analyzed by ELISA using a commercial plate (CCP3). All negative values ​​were considered as 0. The mean + 3SD was used to establish a threshold for positive ACPA values. The mean of ACPA, all negative values ​​= 0, was 0.57, and 3SD + 1.2 = 4.17. Therefore, the determined cutoff value was 5.0.

[0148] The presence of autoantibodies (ACPA) has also been demonstrated in the ocular surface rinsing solutions of patients with ocular surface diseases. Figure 2 and Figure 3 ).like Figure 2As shown, ACPA levels were determined in ocular surface irrigation fluid from patients with ocular surface diseases. These patients were receiving appropriate topical eye drop treatment (artificial tears, (cyclosporine), xiidra, or steroids). In several ocular autoimmune diseases, such as Sjogren's syndrome, tear-deficient dry eye disease, mixed-mechanism DED, ocular cicatricial pemphigoid (OCP), Stevens Johnson syndrome, and ocular graft-versus-host disease (oGVHD), tear ACPA levels were positive (i.e., ACPA > 5.0). Patients with evaporative DED (such as meibomian gland dysfunction) also had high ACPA levels. Superior limbal keratoconjunctivitis (SLK) and neurotrophic keratitis also had high ACPA levels. Surprisingly, patients with discontinuous symptoms and signs (discordant DED) also had high ACPA levels. Therefore, all these eye diseases, including other intraocular autoimmune / inflammatory ocular glaucoma, will benefit from OSIG eye drop treatment to reduce the contribution of autoantibodies to the signs and symptoms of eye diseases. Figure 3 The data showed the percentage of all ACPA-positive patients with specific eye diseases in ocular surface cleaning solutions.

[0149] Example 2: Conventional treatments have insufficient effect on ACPA levels.

[0150] Experiments were conducted to demonstrate the insufficient effect of conventional treatment on the ACPA content in ocular surface cleansing solutions. Figure 4 and 5 ). Figure 4 This study shows ACPA levels in the tear fluid of patients with Sjögren's syndrome before and after initiating routine treatment. The ACPA levels in untreated patients (Sjögren's syndrome, pre-Rx) were significantly higher than those in patients already receiving treatment. Levels were lower after initiation of treatment (Sjögren's syndrome, post-Rx) compared to healthy subjects, but still significantly higher. These data suggest that ACPA levels in the tear fluid remain high even when patients with Sjögren's syndrome are receiving treatment, thus highlighting the potential for targeted ACPA reduction (e.g., OSIG treatment) to decrease its contribution to ocular surface disease.

[0151] ACPA levels in oGVHD tears before and after treatment initiation. ACPA levels in untreated oGVHD patients (pre-oGVHD - Rx) were significantly higher than ACPA levels after treatment initiation (post-oGVHD - Rx). Figure 5One month after treatment began, ACP levels were significantly higher than in healthy subjects. This data suggests that even when oGVHD patients are receiving treatment, ACPA levels in the tear film remain high, thus highlighting the need for targeted ACPA treatment (e.g., OSIG therapy) to reduce ACPA's contribution to ocular surface disease.

[0152] Example 3: Citrullinated proteins are present on the ocular surface of patients with ocular surface diseases.

[0153] Next, the presence of citrullinated proteins in the ocular surface of patients with ocular surface diseases was confirmed. For example... Figure 6 As shown, citrulline is expressed in non-epithelial cells of patients with Sjögren's disease—imprint cytology. The aim of this experiment was to examine whether ocular surface cells in patients with Sjögren's disease are citrullinated. Research approval 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 of the study and its potential consequences were explained. The study was conducted in accordance with the requirements of the Health Insurance Portability and Responsibility Act (HIPAA) and the purposes of the Declaration of Helsinki.

[0154] Apply antibiotics to the patient's eyes. Apply filter paper (PALL, #60298) to the temporal conjunctiva and lower eyelid to remove the superficial layer of ocular surface epithelium. Fix the filter paper in a petri dish with 4% paraformaldehyde (PFA) for 20 minutes, then wash in 1X PBS for 5 minutes, and attach it to a microscope slide using an adhesive patch. Draw a waterproof layer around the sample with a Papanicolaou pen, then saturate with PBS-T (0.025% Triton in 1X PBS) for 1 hour. Gently shake the filter paper with 1X PBS for 5 minutes, then block with 2.5% donkey serum (secondary host bacteria) and 1% BSA in 1X PBS for at least 1 hour. Then incubate the filter paper with the primary antibody 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).

[0155] The next day, the filter paper was washed three times with 1X PBS, gently agitated for 5 minutes, and then incubated with secondary antibodies at room temperature for 1 hour. The secondary antibodies used were: Alexa Fluor 594 donkey anti-mouse IgG (1:1000; Jackson ImmunoResearchLab, #715-585-150) and Alexa Fluor 488 donkey anti-rabbit IgG (1:1000; Jackson ImmunoResearchLab, #711-546-152). After incubation, the filter paper was washed three times with 1X PBS, 5 minutes each time, gently agitated in the dark. The slides were dried and incubated with ProLong DAPI. TM Fixation was performed using Gold anti-fading fixative (Invitrogen, P36931). A coverslip was placed and sealed with nail polish. The slides were completely dry before imaging. Images were captured at 63x magnification using a Zeiss LSM 710 confocal microscope (Leica, UIC Ophthalmology Core device) and analyzed using Zeiss LSMImage Software. Imprint cytology of patients with Sjögren's syndrome revealed citrulline expression on non-epithelial cells (neutrophils). These non-epithelial cells are likely neutrophils, supported by NETs. These data suggest that non-epithelial cells in patients with Sjögren's syndrome are citrullinated.

[0156] Example 4: Citrullinated proteins are not present in healthy subjects.

[0157] In Sjögren's syndrome, citrullinated proteins appear to be predominantly neutrophils. Figure 6 and 7 The purpose of this experiment was to verify whether citrullinated non-epithelial cells were neutrophils. Figure 7 Perform an imprint cytology examination as described in Example 3 above.

[0158] The primary antibodies used were mouse monoclonal anti-citrulline antibody (1:1000; Millipore, MABN328) and rabbit monoclonal anti-neutrophil protease antibody (1:500; Abcam, ab131260). The next day, the filter paper was washed three times with 1X PBS, gently agitated for 5 minutes, and then incubated with secondary antibodies at room temperature for 1 hour. The secondary antibodies used were: Alexa Fluor 594 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 paper was washed three times with 1X PBS, 5 minutes each time, gently agitated in the dark. The slides were dried and incubated with ProLong DAPI. TM Fix the slide with Gold anti-fading fixative (Invitrogen, P36931). Place a coverslip and seal with nail polish. Allow the slide to dry completely before imaging. Capture images at 63x magnification using a Zeiss LSM 710 confocal microscope (Leica, UIC Ophthalmology Core instrument) and analyze them using Zeiss LSM image software.

[0159] Citrulline-positive cells are neutrophils with multilobed nuclei. This suggests that neutrophils are a major source of citrulline on the ocular surface. Citrullinated neutrophil proteins may be the source of the ACPA reaction. This was confirmed by dot blot assay showing the reaction of ACPA-positive tears with citrullinated neutrophil proteins. These data indicate that neutrophils are the source of citrullination, and that citrullinated neutrophil proteins may be the source of the ACPA reaction.

[0160] Example 5: In oGVHD, citrullinated proteins appear to be predominantly epithelial.

[0161] Next, we will demonstrate that citrulline is expressed on epithelial cells in confirmed oGVHD. The aim of this experiment is to compare citrulline expression between patients with xerosis and those with confirmed oGVHD. Figure 8Apply antibiotics to the patient's eyes and apply filter paper (PALL, #60298) to the temporal conjunctiva and lower eyelid to remove the superficial layer of ocular surface epithelium. Fix the filter paper in a petri dish with 4% paraformaldehyde (PFA) for 20 minutes. Then wash them in 1X PBS for 5 minutes and attach them to a microscope slide using an adhesive label. Draw a waterproof layer around the sample with a Papanicolaou pen and then saturate with PBS-T (0.025% Triton in 1X PBS) for 1 hour. Gently shake the filter paper with 1X PBS for 5 minutes and then block with 2.5% donkey serum (secondary host bacteria) and 1% BSA in 1X PBS for at least 1 hour. Then incubate the filter paper with the primary antibody 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, filter paper was washed three times with 1X PBS and gently shaken for 5 minutes, then incubated with secondary antibodies at room temperature for 1 hour. The secondary antibodies used were: Alexa Fluor 594 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).

[0162] After incubation, wash the filter paper three times with 1X PBS, 5 minutes each time, gently agitating in the dark. Dry the slide and use ProLong with DAPI. TM Fixation was performed using Gold anti-fading fixative (Invitrogen, P36931). Coverslips were placed and sealed with nail polish. Slides were completely dry before imaging. Images were captured at 63x magnification using a Zeiss LSM 710 confocal microscope (Leica, UICO phthalmology Core device) and analyzed using Zeiss LSM Image Software. Unlike Sjögren's syndrome where neutrophils are the primary source of citrulline, the entire epithelial cell line in definitive oGVHD patients was citrullinated, which was associated with high levels of citrulline and PAD4 enzymes. These data suggest that strong citrullination of epithelial cells supports high levels of citrulline and PAD4 enzymes. Co-localization of citrulline and K14 was not observed in two different Sjögren's syndrome patients tested, but it was observed in oGVHD patients. Furthermore, co-localization of citrulline with neutrophils was observed in two different Sjögren's syndrome patients.

[0163] Example 6: Inducible Fc receptor expressed on neutrophils

[0164] Patients without oGVHD lack citrullinated proteins. These findings suggest that one source of autoantibody production in DED patients is citrullinated neutrophils and / or ocular surface cells. The presence of Fc receptors (receptors for IgG) on the ocular surface was also demonstrated, and ACPA antibodies interact with these Fc receptors to generate surface disease. It is hypothesized that Fc receptors are not expressed in immature neutrophils but can be induced. Fc receptors are expressed in neutrophils within mucosal cell aggregates.

[0165] The aim of this experiment was to support the hypothesis of inducible Fc receptors on neutrophils. Peripheral blood was collected via venipuncture in BD vacuum sodium heparin tubes (BD Biosciences, #367878) and immediately transported to the laboratory for neutrophil isolation. Neutrophils were isolated using MACSxpress magnetic beads (MACSxpress Neutrophil Isolation Kit, Miltenyi Biotech, #130-104-434) by immunomagnetic consumption of non-target cells, according to the manufacturer's instructions. Residual red blood cells were removed using the MACSxpress Erythrocyte Removal Kit (Miltenyi Biotec, #130-094-183). The isolated neutrophils were resuspended in 3 mL of serum-free, phenol red-free RPMI-1640 medium (GIBCO, #11835-030). After cell count, 50,000 cells / 200 μL of neutrophils were loaded into an EZ single-cell funnel (Thermo Scientific, #A78710003). Mucus samples from patients were prepared using 4 U / mL DNase I and 1x Buffer in 100 μL of solution and incubated at 37°C for 30 minutes to achieve a single-cell suspension. The 10-fold diluted mucus samples were loaded into an EZ single-cell funnel (Thermo Scientific, #A78710003). The samples were centrifuged at 1000 rpm for 5 minutes using a Cytospin 4 (Thermo Scientific, Kalamazoo, MI) to achieve monolayer cell deposition within a defined area (Thermo Scientific, #5991056). After centrifugation, the slides were air-dried for 5 minutes and then fixed in 4% paraformaldehyde (PFA) solution (Electron Microscopy Sciences, Hatfield, PA, #15710) for 20 minutes. Draw a waterproof layer around the sample using a Papanicolaou pen, then infiltrate with PBS-T (0.025% Triton in 1X PBS) for 1 hour. Wash the sample gently with 1X PBS for 5 minutes, then block with 2.5% donkey serum (secondary host species) and 1% BSA in 1X PBS for at least 1 hour. Then incubate the sample 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 protease antibody (1:500; Abcam, ab131260), and rabbit monoclonal IgG antibody (0.03ug / mL; Abcam, ab172730).

[0166] The next day, the samples were gently washed three times with 1X PBS, agitated 5 minutes each time, and then incubated with secondary antibodies at room temperature for 1 hour. The secondary antibodies used were: Alexa Fluor 594 donkey anti-mouse IgG (1:1000; Jackson ImmunoResearchLab, #715-585-150) and Alexa Fluor 488 donkey anti-rabbit IgG (1:1000; Jackson ImmunoResearchLab, #711-546-152). After incubation, the samples were washed three times with 1X PBS, agitated 5 minutes each time, in the dark. The slides were dried and incubated with ProLong DAPI. TM Fix the slide with Gold anti-fading fixative (Invitrogen, P36931). Place a coverslip and seal with nail polish. Allow the slide to dry completely before imaging. Capture images at 63x magnification using a Zeiss LSM 710 confocal microscope (Leica, UIC Ophthalmology Core instrument) and analyze them using Zeiss LSMImage Software.

[0167] like Figure 9 As shown, the Fc receptor was expressed only on PMA-stimulated neutrophils and myxoid bodies of patients, indicating that neutrophils were activated. Positive staining was confirmed using isotype and negative controls. In summary, naive neutrophils do not express the Fc receptor, while activated neutrophils do, confirming that Fc receptor expression is inducible.

[0168] As a separate experiment, the presence of Fc receptor-positive neutrophils on the ocular surface was confirmed. Imprint 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 with 1X PBS and gently shaken for 5 minutes, then incubated with the secondary antibodies at room temperature for 1 hour. The secondary antibodies used were: Alexa Fluor 594 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, wash the filter paper three times with 1X PBS, 5 minutes each time, gently agitating in the dark. Dry the slide and use ProLong with DAPI.TM Fixation was performed using Gold anti-fading fixative (Invitrogen, P36931). A coverslip was placed and sealed with nail polish. The slides were completely dry before imaging. Images were captured at 63x magnification using a Zeiss LSM 710 confocal microscope (Leica, UICO phthalmology Core device) and analyzed using Zeiss LSM Image Software. After immunofluorescence staining, the same filter paper was stained with hematoxylin and eosin. The filter paper was stained with hematoxylin (FisherScientific, SH26-500D), rinsed with acid, immersed in an indigo solution, and stained with eosin (Thermo Scientific, Waltham, MA). The slides were examined using an upright Axioscope 100 microscope (Carl Zeiss Meditec GmbH, Hamburg, Germany), imaged using a Zeiss MRc color camera, and then analyzed using Zeiss Axiovision.

[0169] Figure 10 The study demonstrated that the Fc receptor was expressed on K14-negative cells (non-epithelial cells) mixed with epithelial cells. Some of these non-epithelial cells had multilobed nuclei (white boxes), reminiscent of neutrophils, suggesting neutrophils, which was further confirmed by H&E staining. Other cells were large monocytes, possibly T cells. Literature has confirmed that K14-positive cells do not show the Fc receptor.

[0170] In addition, an experiment was performed to determine whether ACPA in tears is reactive to NET-citrullinated proteins. Dot blot assays were performed using a Bio-Dot microfiltration apparatus (Bio-Rad, #170-6545) with a nitrocellulose membrane (company, catalog number). The manufacturer's instructions are attached after the overall protocol. The membrane was pre-wetted in TBS; it was rehydrated if necessary. 1 μg / 100 μL of total protein from the lysate was applied to the membrane and incubated for one hour under vacuum in a ventilated chamber to allow the protein to pass through the membrane under gravity. 200 μL of blocking solution (1% BSA-TBS) was added to each well, and the membrane was filtered under gravity and then washed. 200 μL of washing solution (0.05% Tween-TBS) was added and the chamber was evacuated, and the process was repeated once more. 100 μL of 1:80 diluted patient tears (diluted with 1% BSA-TTBS) was added and filtered under gravity, and the membrane was then washed with 200 μL of TTBS. The membrane was incubated with 100 μL of HRP-bound human IgG antibody (#A112P, EMD). The blot was then incubated with enhanced chemiluminescence SuperSignal West Femto maximum sensitivity substrate (34095, Thermo Fisher), and the signal was detected using an ImageQuant LAS 4000 system (GE Lifesciences Inc.). The intensity of the blot was determined using ImageJ software.

[0171] Calcium ionophores were used to simulate neutrophils for 3 hours, as it induced hypercitrullination and RPMI within the same time period, serving as a control. Following stimulation, neutrophils were lysed to extract total protein, which was then immobilized on a membrane. Figure 11 As shown, ACPA in tears was found to react with NET protein, thus confirming that neutrophilically citrullinated protein may be a source of ACPA stimulation. Results are expressed as fold ratios calculated using the following formula: Fold = Intensity of citrullinated protein / Intensity of wild-type protein. ACPA-positive tears showed a higher reactivity to stimulated NET protein (92.8 ± 7.85) than unstimulated reticular protein (54.1 ± 5.46), while ACPA-negative tears showed no difference (45.0 ± 3.19 vs. 49.3 ± 4.79). In conclusion, ACPA in tears is reactive to NET protein.

[0172] Another experiment was performed to determine the presence of polyclonal ACPA in tears. Dot blot assays were performed using a direct detection assay free card (#DDAC00010-GR) obtained from Millipore (Billiton, MA). 2 μL of recombinant protein (1 μg) was applied to the membrane and allowed to dry at room temperature (RT) for 20 min. The membrane was then blocked for 30 min at room temperature with 10% BSA and 0.05% Tween-20 prepared in 1X PBS (pH 7.4). The membrane was rinsed with wash buffer (1% BSA and 0.05% Tween 20 in 1X PBS) and probed with a patient tear sample (1:40 dilution with sterile water) and incubated at room temperature for 30 min. The membrane was washed three times with wash buffer and then incubated with a human IgG antibody (#A112P, EMD) bound to HRP for 30 min at room temperature. The membrane was washed three times with wash buffer. The blots were incubated with an enhanced chemiluminescent SuperSignal West Femto maximum sensitivity substrate (34095, Thermo Fisher) and the signal was detected using an ImageQuant LAS 4000 system (GE Lifesciences Inc.). The intensity of the blots was determined using ImageJ software. The results shown in Table 2 are expressed as fold ratios calculated using the following formula: fold ratio = intensity of citrullinated protein / intensity of wild-type protein (Table 2).

[0173] like Figure 12 As shown, ACPA-positive tears indicate polyclonal activity, and ACPA reacts with several citrullinated proteins. In patients with Sjögren's syndrome and confirmed oGVHD, the fold change of citrullinated fibrinogen or enolase relative to wild type was greater than 1.5. In patients with Sjögren's syndrome, citrullinated vimentin showed a 1.5-fold change compared to its wild type, while in confirmed oGVHD, citrullinated histone H4 was greater than wild type. The tears of two affected patients showed polyclonal ACPA in their tears, while neurogenic and healthy tears were negative for ACPA. In conclusion, ACPA-positive tears exhibit polyclonal activity. (+):>1.5.

[0174] Table 2: fold change of citrullinated proteins relative to wild-type proteins

[0175]

[0176] Example 7: ACPA H4R3cit antiserum in ocular surface diseases

[0177] Next, the presence of a specific autoantibody, ACPA (H4R3cit antiserum), in the ocular surface was examined in an experimental model to determine whether it caused ocular surface disease. Specifically, the experiment investigated the presence of fH4R3cit on mucosal cell aggregates from patients. Mucosal cell aggregates (MCAs) were collected from patients' eyes using sterile jeweler's forceps and transferred to sterile 0.2 mL PCR tubes containing RefreshOptive and stored in a refrigerator. Fresh MCA samples were then embedded in... Inoculated with OCT compound (Sakura Finetek, Torrance, CA, #4583) and rapidly frozen. Frozen sections were cut to 10 μm thickness using a cryostat (ThermoScientific, CryoStar NX50, #957130). Specific MCAs were collected from patients diagnosed with confirmed oGHVD. For immunostaining, slides were air-dried and then fixed with 4% PFA for 20 minutes. Samples were then infiltrated with 0.025% Triton X-100 (Fisher Scientific, #BP151-100) and impregnated with freshly prepared 10% donkey serum and 1% bovine serum albumin (Gemini Bio-Products, #700-100P) for 2 hours. Samples were incubated with primary antibody overnight at 4°C. The following primary antibodies were 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 with 1X PBS, gently agitated for 5 minutes, and incubated with secondary antibodies diluted in retardation buffer at room temperature for 1.5 hours. The following secondary antibodies were used: Alexa Fluor 594 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). Wash the slide twice with 1X PBS, counterstain with Hoechst 33342 (2 μl / mL) (Thermo Scientific, #62249) for 10 minutes, then wash rapidly with PBS and smear with one drop of ProLong. TMGold anti-fading agent (Invitrogen, #P36930) was applied. Images were captured at 100x magnification using a Zeiss LSM 710 confocal microscope (Leica, UIC Ophthalmology Core device) and analyzed using Zeiss LSM Image Software.

[0178] To confirm the presence of H4R3cit in the ocular surface of patients, MCAs were collected from patients diagnosed with confirmed oGVHD and analyzed by immunofluorescence staining. Immunofluorescence staining revealed a large number of neutrophils with multifidus nuclei, confirmed by positive staining for neutrophil elastase. It is known in the art that H4R3cit is expressed in the perinuclear region (pernuclear region), such as… Figure 6 As shown. Figure 13 As shown, H4R3cit is immunolocalized at H on the ocular surface, and dot pattern analysis using tear fluid from oGVHD patients showed a reaction with H4cit. Figure 13 The study also revealed the presence of H4R3cit on the patient's mucosal cell aggregates, with positive staining, while the isotype control showed negative staining. In conclusion, the patient's mucosal cell aggregates possess the H4R3cit antigen, which may be the source of the ACPA reaction.

[0179] To investigate whether ACPA antibodies induce ocular surface disease, 8-10 week old Thy1-YFP mice were used in the treatment experiment. Thy1-YFP mice (n=5 / group) were anesthetized by a combination of intraperitoneal injection of ketamine (20 mg / kg; Phoenix Scientific, St. Joseph, MO) and xylazine (6 mg / kg; Phoenix Scientific). To assess the pathological effects of ACPA, ACPA antibodies were applied to the mouse cornea, and the degree of corneal surface disease induced by the antibody was determined and compared with that induced by non-ACPA antibody application. The following conditions were used: 1) Non-ACPA antibody: anti-histone H4 antibody (100 ng / mL; CellSignaling#2592); 2) ACPA antibody #1: anti-histone H4 (citrulline R3) (H4R3cit) antiserum (100 ng / mL; Abcam#ab81797); 3) ACPA antibody #2: anti-histone H3 (citrulline R2+R8+R17) antiserum (100 ng / mL; NovusBiologicals#NB100-57135SS). These were applied to the surface of mouse corneas (10 uL / cornea) and incubated for 40 minutes for 7 consecutive days. Because ACPA antibodies were used in rabbit antiserum, normal rabbit serum (100 ng / mL; Abcam#ab7487) was used as a control. All antibody dilutions were performed in Refresh Optive. For the fluorescein staining assay, 10 μl of 0.2% fluorescein was instilled onto the mouse cornea for 1 minute and washed twice with 1X PBS. Fluorescein staining was detected using cobalt blue illumination and a yellow blocking filter, and images were taken with a slit lamp (Haag Streit, Bern, Switzerland). Images were collected for each group (n=5), and the intensity of fluorescein staining was measured using the Metamorph imaging system (Metamorph, Universal Imaging, Downington, PA).

[0180] Applying H4R3cit antiserum to the cornea of ​​mice demonstrated a significant increase in ocular surface disease, as evidenced by fluorescein staining of the cornea. In several controls (rabbit serum, H3cit antiserum, and wild-type H4 antibody), fluorescein staining was significantly increased. Figure 14 ).like Figure 14 As shown in panels A1 and A5, continuous application of non-ACPA antibodies to the mouse cornea for 7 days did not induce corneal epithelial disease, which was confirmed in the absence of fluorescein staining on day 7 (2.59 x 10⁻⁶). 6 ±5.41x10 4Of the two ACPA antibodies used, only one caused corneal epithelial disease, as evidenced by a significant increase in corneal fluorescein staining on day 7. The ACPA antibody that caused the corneal epithelial disease was ACPA#1-H4R3cit antiserum (with 3.00 x 10⁻⁶ serum on day 0). 6 ±6.05x10 4 Compared to 1.85 x 10⁻⁶, the fluorescence intensity on day 7 was 1.85 x 10⁻⁶. 7 ±3.19x10 6 p = 0.0006; Figure 14 A2 and A6), however, ACPA#2-H3cit antiserum did not cause corneal epithelial disease (compared to day 0 (3.36x10). 6 ±3.05x10 5 Compared to 10⁻⁶, the fluorescence intensity on day 7 was 3.34 x 10⁻⁶. 6 ±4.90x10 5 p = 0.97; Figure 14 (A3 and A7). The mediator control also did not induce corneal epithelial disease, as evidenced by the lack of fluorescein staining on day 7 (3.11 x 10⁻⁶). 6 ±3.49x10 5 ; Figure 14 (A4 and A8). In summary, these data indicate that ACPA antibodies cause corneal epithelial disease, but not all ACPA antibodies do so.

[0181] Table 3: Fluorescence measurements from fluorescein staining of mouse cornea

[0182]

[0183] An experiment was also conducted to determine whether ACPA antibodies could induce NETosis. Thy1-YFP mice aged 8–10 weeks were used for the treatment experiment. Thy1-YFP mice (n = 5 / group) were anesthetized by a combination of intraperitoneal injection 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 as a control group, 2) ACPA antibody #1: H4R3cit antiserum, or 3) ACPA antibody #2: H3cit antiserum. 10 μL of each treatment was applied to the mouse cornea. Treatment was continued for 7 days. At the end of the experiment, blot cytology of the mouse cornea was performed using filter paper staining. Adhesive labels (EMS, #76760) were affixed to microscope slides. Filter paper (Millipore, #JHWP02500) was cut to the optimal size. While the mice were anesthetized, one drop of procaine was applied to the eyeball for one minute, then gently removed with a Kimwipe. A piece of filter paper was placed on the cornea and gently pressed for 30 seconds. It was then gently lifted and placed on a viscous faucet with the touched area facing upwards. All subsequent steps, except for the washing step, were performed in a humid indoor environment. A waterproof layer was drawn with a Pasteur pen, and the filter paper was fixed with 4% paraformaldehyde (PFA) for 20 minutes. The filter paper was washed with 1X PBS with gentle agitation for 5 minutes, then saturated with PBS-T (0.025% Triton in 1X PBS) for 10 minutes. The filter paper was washed with 1X PBS with gentle agitation for 5 minutes, then blocked with 2.5% donkey serum (secondary host bacteria) and 1% BSA in 1X PBS for at least 1 hour. The filter paper was then incubated with the primary antibody 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 filter paper was washed three times with 1X PBS and gently shaken for 5 minutes, and then incubated with the secondary antibody at room temperature for 1 hour.The secondary antibodies used were as follows: Alexa Fluor 594 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 Immu-noResearch Lab, #715-547-003). After incubation, filter paper was washed three times with 1X PBS for 5 minutes each time, gently agitated in the dark. The slides were dried and treated with ProLong containing DAPI. TM Fixation was performed using Gold anti-fading fixative (Invitogen, P36931). A coverslip was placed and sealed with nail polish. The slides were completely dry before imaging. Images were captured at 63x magnification using a Zeiss LSM710 confocal microscope (Leica, UIC Ophthalmology Core device) and analyzed using Zeiss LSM image software. After treatment for 7 days with 1) RPMI, 2) ACPA#2-H3cit antiserum, or 3) ACPA#1-H4R3cit antiserum, the mouse corneal epithelium was lifted onto filter paper to investigate other pathological effects.

[0184] like Figure 15 As shown, 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); NETs were confirmed by neutrophil elastase staining. Figure 15 (A2). This indicates that H4R3cit antiserum induced NETosis. In summary, ACPA#1 antibody induced NETosis and citrullination in mouse corneal epithelial cells. These data suggest that ACPA antibody contributes to the feedforward cycle of citrullination to further amplify citrullination-related ocular pathologies.

[0185] Example 8: Blocking interaction of ACPA (H4R3cit) - Elimination of ocular surface disease caused by ACPA-H4R3cit using an Fc receptor blocking strategy

[0186] 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 in the treatment experiment. Peptides that block all Fc receptors were used, followed by ACPA antibodies. 10 μL of either 1) an azide-free Fc receptor blocker (Innovex Biosciences #NB355) or 2) a disordered peptide was applied to the mouse cornea and incubated for 30 minutes. Pre-incubation with the peptide was performed without anesthesia. Thy1-YFP mice (n=5 / group) were then anesthetized, and 10 μL of LACPA#1-H4R3cit antiserum was administered simultaneously to both the Fc receptor blockade group and the disordered peptide blockade group, incubated for 40 minutes for 10 consecutive days. Diluted in Refresh Optive. The mouse corneas were stained with fluorescein, monitored, and imaged with a slit lamp. Fluorescein intensity was measured using a Metamorph imaging system. To investigate whether competitive blockade of the Fc receptor would produce the same results as the peptide that blocks the Fc receptor, mouse IgG and ACPA antibody were added together. For the IgG competition assay, 10 μL of a 1:1 mixture of ACPA antibody (H4R3cit antiserum) and mouse IgG (Abcam#ab188776) was applied to mouse corneas and incubated for 40 minutes for 40 consecutive days. In the control assay, only ACPA antibody was used. The mouse corneas were stained with fluorescein, monitored, and imaged with a slit lamp. Fluorescein intensity was measured using a Metamorph imaging system. After 10 days, the corneas were harvested, lysed, and subjected to Luminex assays to detect cytokines.

[0187] like Figure 16 As shown, both strategies significantly reduced H4R3cit-induced ocular surface disease. Analysis of corneal cytokines showed that IL-2 was significantly increased under ACPA conditions, but not in the control group. Figure 16 Blocking Fc receptors with peptide blockers elicited a counteracting effect of H4R3cit antiserum. Corneal tissue was collected at the end of the experiment, lysed, and analyzed for cytokines. IL-2 levels in the corneas of both competitive and peptide-blocked mice were significantly lower than in unblocked mice. In summary, mouse IgG competes with ACPA antibodies for binding to Fc receptors, while Fc receptor blockers block all Fc receptors. This competitive and peptide-based blocking action eliminates the pathological effects of ACPA on the cornea, suggesting that methods preventing the interaction of ACPA antibodies with Fc receptors may be a potential therapeutic strategy for treating citrullination-related ocular diseases.

[0188] Example 9: ACPA-H4R3cit induces NETosis in vitro.

[0189] To determine whether ACPA could induce NETosis in vitro, peripheral blood was collected via venipuncture in a BD vacuum container heparin tube (BD Biosciences, #367878) and immediately transported to the laboratory for neutrophil isolation. Neutrophils were isolated using MACSxpress magnetic beads (MACSxpress Neutrophil Isolation Kit, Miltenyi Biotech, #130-104-434) by immunomagnetic consumption of non-target cells, according to the manufacturer's instructions. Residual red blood cells were removed using the MACSxpress Erythrocyte Removal Kit (Miltenyi Biotec, #130-094-183). The isolated neutrophils were resuspended in 3 mL of serum-free, phenol red-free RPMI-1640 medium (GIBCO, #11835-030). After cell count, 1.0 x 10⁶ cells were transferred to a 10⁻¹² plate. 6 Cells were plated at 1 / mL on glass slides (Millipore, #PEZGS0416) and incubated with the following antibodies and controls: 1) RPMI as a negative control, 2) 1 nM PMA as a positive control, 3) 100 ng / mL normal rabbit serum (Abcam, ab7487), 4) 100 ng / mL H3cit antiserum (Novusbio, NB100-57135SS), 5) 100 ng / mL H4R3cit antiserum (Abcam, ab81797), 6) 100 ng / mL citrullinated fibrinogen antibody (Cayman Chemical, 17088), 7) 100 ng / mL citrullinated vimentin antibody (Cayman Chemical, 22054), 8) 100 ng / mL citrullinated α-enolase antibody (Cayman Chemical, 23000) or 9) CCP antibody (Bioss, bs-1053R). Neutrophils were incubated overnight and stained with 1 M Sytox Green (Molecular Probes, Invitrogen, catalog number S7020) and 5 μM Hoechst (Fisher Scientific, Pittsburgh, PA, #33342). They were immediately imaged at 20X with a Zeiss Observer Z1.

[0190] like Figure 17As shown, in vitro experiments using isolated human neutrophils demonstrated that ACPA-H4R3cit stimulation resulted in a significant increase in extracellular DNA strands, but not in controls (other ACPAs). PMA stimulation alone, ACPA-H4R3cit stimulation, and CCP ACPA stimulation alone produced significant extracellular DNA strands. In conclusion, ACPA-H4R3cit induces NETosis in vitro.

[0191] Example 10: Determining the cytotoxicity of ocular surface immunoglobulin (OSIG) on human corneal epithelial cells under normal or stress conditions.

[0192] The cytotoxic effects of OSIG on human corneal epithelial cells were determined using primary human corneal epithelial cells under normal or stress conditions. Primary human corneal epithelial cells were purchased from EMD Millipore (EMD, #SCCE016). Cells were cultured in EpiGRO... TMHuman ocular epithelium was grown entirely in MedUM (EMD, #SCMC001). One day prior to wound scraping, 20,000 cells / well were seeded in 96-well ImageLock plates (Essen Bioscience, #4379) and allowed to grow for 18 hours to achieve monolayer confluence. ImageLock plates are specially modified plates whose technology is achieved through reference markers on the bottom of the plate, providing points for precise reference to image locations. Wound scraping (700–800 μm wide) was performed using an IncuCyte 96-needle wound scraper (Essen Bioscience, #4493). After scraping, cells were washed twice with 100 μL of phenol red-free RPMI-1640 medium (Gibco #11835030). For the OSIG (Flebogamma 5% DIF) dose-dependent assay, 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 the IncuCyte Zoom live cell analysis system (Essen Bioscience). Images were captured every 3 hours. The relative wound density (%) at 69 hours was determined using IncuCyte Zoom software. This metric was based on the spatial cell density in the wound region relative to the spatial cell density outside the wound region at each time point. It was designed to be 0% at t=0 and 100% when the cell density inside the wound was the same as the initial cell density outside the wound. It was independent of the discovery of cell boundaries. The cytotoxicity of HCE-T cells was determined by the LDH (lactose dehydrogenase) cytotoxicity assay (Thermo Scientific, #88954). Collect the cell culture supernatant, mix 50 μL of the supernatant with 50 μL of the reaction mixture and incubate for 30 minutes, then transfer the mixture to a 96-well plate. Measure the absorbance at wavelengths (490-680 nm) using a Cytation 5 microplate reader.

[0193] Epithelial scraping assays were performed using a human corneal epithelial (HCE-T) cell line transformed with SV40-adenovirus vector (RIKEN Cell Bank RCB2280, Tsukuba, Japan). Cells were grown in DMEM medium (GIBCO, #11965-092) supplemented with 10% FBS (Invitrogen, #26140-079) and 1% antibiotic and antifungal 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 at 37°C in a tissue culture incubator with 5% CO2. The day before wound scraping, 30,000 cells / well were seeded into 96-well ImageLock plates (Essen Bioscience, #4379) and allowed to grow for 18 hours to achieve monolayer confluence. ImageLock plates are specially modified plates whose technology utilizes reference markers on the bottom of the plate, providing points for precise reference to image locations. Wound scraping (700–800 μm wide) was performed using an IncuCyte 96-needle wound scraper (Essen Bioscience, #4493). After scraping, the cells were washed twice with 100 μL of phenol red-free RPMI-1640 medium (Gibco #11835030). For the OSIG (Flebogamma 5% DIF) dose-dependent assay, the culture medium was replaced with 200 μL of the following conditioned medium: (1) Under normal conditions (Fig. B): (i) complete medium (CM); (ii) 4 mg / mL OSIG or (iii) 10 mg / mL OSIG; (2) Under stress conditions (Fig. C): (i) RPMI; (ii) 4 mg / mL OSIG or (iii) 10 mg / mL. Plates were incubated in the IncuCyte Zoom live cell analysis system (Essen Bioscience). Images were captured every 3 hours. Relative wound density (%) was determined using IncuCyte Zoom software over 12 hours. This metric is based on the spatial cell density in the wound region relative to the spatial cell density outside the wound region at each time point. It was designed to be 0% at t=0 and 100% when the cell density inside the wound was the same as the initial cell density outside the wound. It is independent of the discovery of cell boundaries. The cytotoxicity of HCE-T cells was determined by an LDH (lactose dehydrogenase) cytotoxicity assay (Thermo Scientific, #88954). Cell culture supernatant was collected, and 50 μL of the supernatant was mixed with 50 μL of the reaction mixture and incubated for 30 minutes. The mixture was then transferred to a 96-well plate.Absorption rates were measured using a Cytation 5 microplate reader at wavelengths (490-680 nm). 10 mg / mL OSIG showed toxicity under all three conditions, while 4 mg / mL was non-toxic and can be used as a working concentration (effective and non-toxic). Figure 18 As shown, the toxicity remained unchanged regardless of whether the cells were in a normal or stressed state. In conclusion, OSIG at its highest concentration of 4 mg / mL showed no toxicity.

[0194] Example 11: The ACPA reaction on the ocular surface is a reaction resulting from the production of active ocular antibodies.

[0195] This study investigated the number of ocular surface diseases patients who had elevated ACPA levels in their tears despite the absence of these ACPA antibodies in serum and negative rheumatoid factor. Of the 166 patients, 49% were negative for cyclic citrullinated peptide (CCP) in serum but positive for ACPA in their tears. 69% of these patients were negative for rheumatoid factor. This suggests that the ACPA response on the ocular surface is an active, localized antibody-generating response, rather than a passive exudation of ACPA from serum, and therefore does not require an accompanying systemic immune disease.

[0196]

[0197] Example 12: An example of a patient with elevated autoantibodies (ACPA) in the tear film and severe ocular surface disease.

[0198] The first patient was a 38-year-old woman with Sjögren's syndrome. Figure 19 As shown, the patient had elevated levels of autoantibodies (ACPA) in their tears and corneal melting in the right eye. Despite treatment with steroid eye drops, serum tear solutions, and other standard DED therapies, the patient continued to experience severe symptoms. This case demonstrates an association between high autoantibody levels and severe ocular surface disease.

[0199] APCA levels in tear flushing fluid (normal level <5.0)

[0200] 6 / 2018 10 / 2018

[0201] OD: 121.8 104.2

[0202] OS: 121.4 142.6

[0203] The second patient was a 39-year-old woman who was diagnosed with severe ocular discomfort in May 2018. Examination revealed superior limbal keratoconjunctivitis (SLK) 3+ in both eyes. She began taking methylprednisolone eye drops. At a follow-up in June 2018, despite treatment, her ocular discomfort persisted. ACPA levels in her tears were high. Serum ACPA and rheumatoid arthritis tests were negative. ACPA levels were again high when measured in July 2018, and the patient still had severe symptoms. At an examination in October 2018, although ACPA levels remained high, the patient's symptoms had improved, but ACPA levels had decreased. Figure 20 As shown, this case demonstrates that autoantibodies (ACPA) can still be present in the eye even in the absence of autoantibodies in the blood or systemic autoimmune diseases such as rheumatoid arthritis. This indicates that the autoantibodies are produced locally in the eye tissue.

[0204] APCA in tear flushing solution

[0205] 6 / 2018 7 / 2018 10 / 2018

[0206] OD: 151.9 174.3 56.4

[0207] OS: 164.8 174.3 50.6

[0208] The third patient was a 47-year-old woman with asymmetric ocular disease. She was diagnosed in February 2017 with severe dry eye and ocular discomfort. Examination revealed more severe ocular surface disease in the right eye than the left. Tear production in the right eye was significantly less than in the left eye (1 mm in Schirmer I cases compared to 12 mm in the left). Figure 21 As shown, the right cornea staining ratio is 8 / 15, and the left eye staining ratio is 2 / 15. The right conjunctiva staining ratio is 6 / 6, and the left eye staining ratio is 3 / 6. ACPA in the tear film is high only in the right eye, and not in the left eye. Higher levels of autoantibodies (ACPA) in the tears indicate more severe ocular surface disease. This suggests that higher ACPA levels are associated with more severe ocular disease.

[0209] Example 13: Other Patient Case Studies (Case Studies 1-6)

[0210] In the following case studies, patients were treated with commercially available IVIG (Flebogamma 10%), a solution prepared to represent OSIG. The commercially available IVIG (10%) was diluted with ophthalmic excipient NaCl solution to a final concentration of 4 mg / ml IgG (0.4%) at 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 a dropper. OSIG is not currently available and can therefore be prepared using commercially available IVIG (Flebogamma 10%), but any commercially available IgG or plasma-derived conjugated human immunoglobulin G can be used to prepare the OSIG formulation. These case studies provide clinically significant results relating to the ophthalmic formulations presented herein.

[0211] Case Study 1 involves a 56-year-old woman suffering from severe tear deficiency and severe ocular surface disease due to graft-versus-host disease. Figure 22 The right eye had an OSDI of 77.7 and corneal staining of 6 / 15. VBR showed ocular hyperemia in the right eye at age 70. Corneal scarring was present without any corneal neovascularization. OSIG eye drops (0.4%) were started twice daily. After two weeks of treatment, the patient reported a significant reduction in ocular discomfort (OSDI decreased to 30.5), a significant reduction in corneal staining (2 / 15), and a significant reduction in VBR (50). In the following two weeks of treatment, VBR decreased to 30 and corneal staining decreased to 1 / 15. Extracellular DNA in the tear film of the right eye was 56 μg / mL, which decreased to 12.3 μg / mL after OSIG treatment. In this case, OSIG eye drops alleviated the symptoms and signs of severe dry eye and reduced inflammatory biomarkers in the tear film.

[0212] Case 2 is a 31-year-old woman who suffers from severe tear deficiency and severe ocular surface disease due to ocular graft-versus-host disease. Figure 23 This patient presented with severe conjunctival keratosis outside the area covered by the PROSE contact lens, but without any corneal neovascularization, and with severe ocular discomfort. OSIG eye drops (0.4%) were started twice daily. After one month of treatment, conjunctival keratosis decreased, and the patient reported “significant improvement” in subjective ocular symptoms. In this case, OSIG eye drops reduced keratosis caused by severe dry eye and significantly alleviated ocular discomfort.

[0213] Case Study 3 involved a 74-year-old male who developed neurotrophic keratitis due to a history of LASIK surgery on his left eye. Figure 24The left eye corneal staining was 2 / 15, and the patient's ocular discomfort intensity was 4 / 10. Faint scarring was observed at the edge of the LASIK flap, with no corneal neovascularization. Overall, the examination was consistent with the diagnosis of dissociated symptoms and signs (DED). OSIG eye drops (0.4%) were started twice daily. After one month of treatment, the patient reported subjective improvement (SGA), with symptom intensity decreasing to 3. Left eye corneal staining disappeared (0 / 10). In this case, OSIG eye drops were beneficial in addressing postoperative complications leading to dissociated symptoms and signs.

[0214] Case Study 4 involved a 39-year-old male with severe tear deficiency and a severe ocular surface disease, ocular cicatricial pemphigoid. SPK staining of the cornea was 1 / 15 in the right eye and 3 / 15 in the left eye, with no corneal neovascularization. The patient reported an ocular discomfort intensity of 3 / 10. He was initially treated with 0.4% OSIG eye drops twice daily. After one month of treatment, discomfort in both eyes decreased to 2 / 10, and corneal staining disappeared (0 / 10). This example demonstrates the beneficial effect of OSIG eye drops in ocular cicatricial pemphigoid.

[0215] Case Study 5 presents a 33-year-old patient with severe tear deficiency and severe ocular discomfort, diagnosed as discontinuous symptom-sign (DED). There was no corneal staining or corneal neovascularization. The ocular discomfort intensity was 6 / 10. The patient was initially treated with 0.4% OSIG eye drops twice daily. After one month of treatment, the discomfort intensity decreased to 3 / 10 in the right eye and 4 / 10 in the left eye. This case demonstrates the beneficial effect of OSIG eye drops in alleviating ocular discomfort in patients with discontinuous symptom-sign.

[0216] Case Study 6 involves a 29-year-old male suffering from tear deficiency and severe ocular surface disease due to Steven Johnson syndrome. Figure 25 The OSDI was 54.7, and the symptom intensity was 8 / 10 (severe, terrifying discomfort). OSIG eye drops (0.4%) were started three times daily in both eyes. After two weeks of treatment, the patient reported significant reduction in eye discomfort (OSDI decreased to 14.2), and the symptom intensity decreased to 0 / 10 (no discomfort). Prior to OSIG treatment, the patient had consistently experienced photosensitivity and a gritty sensation in their eyes. After OSIG treatment, photosensitivity was only "sometimes," and there was no gritty sensation at all. OSIG treatment also reduced eye redness. In this case, following Steven Johnson syndrome, OSIG eye drops significantly reduced the signs and symptoms of dry eye.

Claims

1. An ophthalmic preparation capable of reducing the amount or harmful effects of autoantibodies in ocular tissue, said ophthalmic preparation comprising: (a) One or more pharmaceutically acceptable ophthalmic excipients; and (b) Combined human plasma, The ophthalmic preparation contained immunoglobulin G (IgG) at a concentration of 0.01% to 10%.

2. An ophthalmic preparation capable of reducing the amount or harmful effects of autoantibodies in ocular tissue, said ophthalmic preparation comprising: (a) One or more pharmaceutically acceptable ophthalmic excipients; (b) Combined plasma-derived human immunoglobulin G (IgG) or its antigen-binding fragments; and (c) Combined human plasma proteins, combined human lipids, or a combination thereof, The ophthalmic preparation contained immunoglobulin G (IgG) at a concentration of 0.01% to 10%.

3. An ophthalmic preparation capable of reducing the amount or harmful effects of autoantibodies in ocular tissue, said ophthalmic preparation comprising: (a) One or more pharmaceutically acceptable ophthalmic excipients; and (b) Combined plasma-derived human immunoglobulin G (IgG) or its antigen-binding fragments, The ophthalmic preparation contained immunoglobulin G (IgG) at a concentration of 0.01% to 10%.

4. The ophthalmic preparation according to any one of claims 1-3, further comprising a pharmaceutically active compound selected from: steroids; anti-inflammatory agents; mucolytics; PAD enzyme inhibitors or NETs degrading agents; Fab-targeting antibody fragments; Fc receptor blocking peptides; Fc receptor blocking antibodies; recombinant peptides containing pathogenic epitopes; conventionally synthesized DMARDs; TNF-α targeted therapeutic agents; B-cell targeted therapeutic agents; T-cell targeted therapeutic agents; interleukin targeted therapeutic agents; growth and differentiation factors; JAK pathway inhibitors; and combinations thereof.

5. The ophthalmic preparation according to claim 4, wherein the PAD enzyme inhibitor is selected from paclitaxel, glucocorticoids, and Cl-amidine.

6. The ophthalmic preparation according to claim 4, wherein the NETs decomposing agent is selected from DNase and heparin.

7. The ophthalmic preparation according to claim 4, wherein the conventionally synthesized DMARD is selected from methotrexate, leflunomide, teriflunomide, sulfasalazine, chloroquine, and hydroxychloroquine.

8. The ophthalmic preparation according to claim 4, wherein the TNF-α targeted therapeutic agent is selected from infliximab, adalimumab, etanercept, golimumab, and sertozumab.

9. The ophthalmic preparation according to claim 4, wherein the B-cell targeted therapeutic agent is selected from rituximab, ofamumumab, belimumab, asceticip and taberucizumab.

10. The ophthalmic preparation according to claim 4, wherein the T-cell targeted therapeutic agent is selected from abatacept and berazip.

11. The ophthalmic preparation according to claim 4, wherein the interleukin-targeting therapeutic agent is selected from tocilizumab, anaerobicin, canananurumab, linazapril, and secukinumab.

12. The ophthalmic preparation according to claim 4, wherein the growth and differentiation factors are selected from denosumab and malvastatin.

13. The ophthalmic preparation according to claim 4, wherein the JAK pathway inhibitor is selected from tofacitinib, baricitinib, and felotinib.

14. The ophthalmic preparation according to any one of claims 1-13, wherein the preparation is capable of reducing the amount or harmful biological effects of autoantibodies on or within the ocular surface, wherein the autoantibodies are generated in response to citrullinated proteins, hypercitrullinated proteins, or natural proteins.

15. The ophthalmic preparation according to any one of claims 1-14, wherein one or more pharmaceutically acceptable ophthalmic excipients are selected from: cyclodextrins; carbomers or acrylic polymers; poloxamer; polyols; prazolam; xyloglucan; methylcellulose; hydroxypropyl methylcellulose; pseudo-latex; cellulose acetate phthalate; gellan gum; alginate; carrageenan; hyaluronic acid; sodium acetate; disodium edetate; acetic acid; ethanol; alginate; Amerchol-cab; antipyrine; benzalkonium chloride; benzalkonium bromide; boric acid; caffeine; calcium chloride; sodium carboxymethyl cellulose; hydroxyethyl cellulose; cetyl alcohol; chlorobutanol; citric acid; citric acid monohydrate; citrate buffer; creatine; styrene-divinylbenzene copolymer; ethylene-vinyl acetate copolymer; glycerol; glyceryl stearate; lanolin; lorazine; lauroyl sarcosine; Magnesium chloride; Methylparaben; Mineral oil; Nonoxynol-9; Octylphenyl polyol-40; Petrolatum; Phenylephrine; Phenymercuric acetate; Phenymercuric nitrate; Phosphate buffer; Poloxamer; Poloxamer; Polycarboflavone; Polyethylene glycol 35 castor oil; Polyethylene glycol 40 hydrogenated castor oil; Polyethylene glycol 40 stearate; Polysorbate 20; Polyvinyl alcohol; Potassium chloride; Potassium sorbate; Povidones; Propylene glycol; Propylparaben; Soda ash; sodium acetate; sodium bisulfate; sodium borate; sodium borate decahydrate; sodium carbonate; sodium chloride; sodium citrate; sodium metabisulfite; sodium nitrate; sodium sulfate; sodium sulfite; sodium thiosulfate; sorbic acid; sorbitol; stable oxychloride complex; sulfuric acid; thimerosal; titanium dioxide; tococelen; dehydrated trisodium citrate; Tris buffer; tromethamine; tyloxapine; vegetable oil; xanthan gum; zinc chloride; or combinations thereof.

16. The ophthalmic preparation according to any one of claims 1, 2 or 4-15, wherein the preparation comprises 0.1% to 99% of combined plasma proteins.

17. The ophthalmic preparation according to any one of claims 1, 2 or 4-15, wherein the preparation comprises 0.01 mg / mL to 1 g / mL of combined IgG by weight.

18. The ophthalmic preparation according to any one of claims 2-15, wherein the amount of combined IgG present in the ophthalmic preparation ranges from 0.4% to 4%.

19. The ophthalmic preparation according to any one of claims 1-3, wherein the ophthalmic preparation comprises 5% conjugated IgG.

20. Use of the composition in the preparation of a medicament for treating a clinical condition in a patient in need, said composition comprising an ophthalmic preparation according to any one of claims 1-19, wherein said clinical condition is an inflammatory ocular surface disease or intraocular disease, and / or an immune ocular surface disease or intraocular disease.

21. Use of the composition in the preparation of a medicament for reducing or alleviating ocular discomfort in patients with clinical symptoms, said composition comprising an ophthalmic preparation according to any one of claims 1-19, wherein said clinical symptoms are inflammatory ocular surface diseases or intraocular eye diseases, and / or immune ocular surface diseases or intraocular eye diseases.

22. The use according to claim 21, wherein the eye discomfort includes one or more of the following: foreign body sensation, pain, photosensitivity, stinging, irritation, soreness, dryness, burning, redness, itching, or tingling.

23. The use according to any one of claims 20-22, wherein the patient has autoantibodies present in a biological sample.

24. The use according to claim 23, wherein the autoantibody present in the biological sample is an anti-citrullinated protein antibody.

25. The use according to claim 23 or 24, wherein the biological sample is an eye drop.

26. The use according to any one of claims 20-25, wherein the clinical symptom is an inflammatory or immune-mediated eye disease comprising: Ocular graft-versus-host disease; Steven Johnson syndrome; ocular cicatricial pemphigoid; Mild, moderate, and severe tear-deficiency dry eye disease; Meibomian gland disorders; styes; ocular rosacea; Blepharitis; superior keratoconjunctivitis; blepharochalasis syndrome; neurotrophic eye disease; dry eye disease with discontinuous symptoms and signs; neuropathic pain; thyroid eye disease; rheumatoid arthritis-related eye disease; lupus-related eye disease; Sjögren's syndrome; Rosacea; Allergic keratoconjunctivitis; Tegsen's keratitis; retinal gliosis; aniridia; keratitis or postoperative / post-traumatic ocular symptoms; ocular symptoms associated with posterior ocular surface reconstruction surgery, use of anti-metabolites on the ocular surface, pterygium surgery, cataract surgery, refractive surgery, artificial corneal surgery, or radiation, chemical, or traumatic injury; keratitis; scleritis; uveitis; and glaucoma.

27. The use according to any one of claims 20-25, wherein the clinical symptom is an inflammatory eye disease or an immune eye disease comprising: Superficial scleritis; Marginal ulcerative keratitis; Eye symptoms associated with glaucoma surgery; And secondary Sjögren's syndrome.

28. The use according to any one of claims 20-27, wherein the ophthalmic preparation is administered in the form of an eye drop, topical liquid, gel, emulsion, suspension, ointment or injectable preparation capable of penetrating the cornea and intraocularly.

29. The use according to any one of claims 20-27, wherein the ophthalmic preparation, agent, or composition is administered to the patient at least once daily, or wherein the ophthalmic preparation, agent, or composition is administered to the patient at least once every week to three weeks.

30. The use according to any one of claims 20-29, wherein the ophthalmic preparation is administered intraocularly as an intraocular injection.

31. A reagent kit comprising: (i) an ophthalmic preparation according to any one of claims 1-19; and (ii) Instructions for use of the kit and for administering the ophthalmic preparation.

32. A diagnostic kit, the kit comprising: A testing apparatus for determining the concentration of anti-citrullinated protein autoantibodies in ocular fluid; and A dosing device for indicating a therapeutic dose and regimen sufficient to treat and / or reduce the damaging effects of the concentration of the anti-citrullinated protein autoantibody in the ocular fluid.

Images