WO2024168287A2 - Methods and compositions for treating inflammatory eye disease - Google Patents

Abstract

Compositions, methods, and kits are provided for treating inflammatory eye diseases. In particular, methods of treating intraocular inflammation with a semaphorin 7A peptide or peptidomimetic are provided.

Description

METHODS AND COMPOSITIONS FOR TREATING INFLAMMATORY EYE DISEASE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. § 119(e) of provisional application 63/444,840, filed February 10, 2023, which application is hereby incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF A SEQUENCE LISTING

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML file, “STAN-STAN- 2073WO S22-508” created on February 1 , 2024 and having a size of 8,901 bytes. The contents of the Sequence Listing XML file are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

[0003] Uveitis is characterized by intraocular inflammation of the uvea in the eye and represents a major cause of visual impairment and blindness worldwide, accounting for approximately 10-15% of blindness in developed countries. The current standard of care for non-infectious uveitis involves the use of corticosteroids as local or systemic therapy, which however has frequent therapy-limiting side effects, such as steroid-induced glaucoma or cataracts. Therefore, there is an unmet medical need for alternative immunosuppressive therapies for the eye.

SUMMARY OF THE INVENTION

[0004] Compositions, methods, and kits are provided for treating inflammatory eye diseases. In particular, methods of treating intraocular inflammation with a semaphorin 7A (SEMA7A) peptide or peptidomimetic are provided. The SEMA7A peptide or peptidomimetic comprises a binding domain for binding to plexin C1 (PLXNC1 ). The disclosed compositions and methods provide an alternative to current immunosuppressive therapies for treating inflammatory ocular diseases, including non- infectious or autoimmune uveitis.

[0005] In one aspect, a method of treating intraocular inflammation or inflammation-induced vascular leakage in a subject is provided, the method comprising administering a therapeutically effective amount of a SEMA7A peptide or peptidomimetic to the subject, wherein the SEMA7A peptide or peptidomimetic binds to PLXNC1 .

In certain embodiments, the SEMA7A peptide or peptidomimetic lacks an RGD motif. [0006] In certain embodiments, the SEMA7A peptide or peptidomimetic comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9, or an amino acid sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto, wherein the peptide is capable of binding to PLXNC1 , reducing numbers of one or more immune cells (e.g., neutrophils, macrophages, dendritic cells, lymphocytes, or a combination thereof) in the eye, reducing or eliminating intraocular inflammation, and or reducing or preventing inflammation-induced vascular leakage.

[0007] In certain embodiments, the intraocular inflammation is acute.

[0008] In certain embodiments, the intraocular inflammation is caused by an autoimmune disease or an infection.

[0009] In certain embodiments, the subject has non-infectious uveitis or autoimmune uveitis.

[0010] In certain embodiments, the subject has anterior uveitis, intermediate uveitis, posterior uveitis, or panuveitis.

[0011 ] In certain embodiments, the SEMA7A peptide or peptidomimetic is administered intravitreally, intraocularly, juxtasclerally, subconjunctivally, intracamerally, or retrobulbarly.

[0012] In certain embodiments, the SEMA7A peptide or peptidomimetic is administered locally to an eye of the subject at a site of inflammation. In some embodiments, the SEMA7A peptide or peptidomimetic is administered locally to the vitreous, anterior chamber, iris, ciliary body, retina, or choroid.

[0013] In certain embodiments, multiple cycles of treatment are administered to the subject. In some embodiments, the SEMA7A peptide or peptidomimetic is administered daily or intermittently.

[0014] In certain embodiments, the subject is a mammal. In some embodiments, the mammal is human.

[0015] In certain embodiments, the method further comprises administering an additional antiinflammatory agent or immunosuppressive agent.

[0016] In another aspect, a composition comprising a SEMA7A peptide or peptidomimetic for use in a method of treating intraocular inflammation or inflammation-induced vascular leakage is provided.

[0017] In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0018] In certain embodiments, the composition further comprises an additional anti-inflammatory agent or immunosuppressive agent.

[0019] In certain embodiments, the composition is formulated for intravitreal, intraocular, juxtascleral, subconjunctival, intracameral, or retrobulbar administration. [0020] In certain embodiments, the composition is formulated for administration locally to the vitreous, anterior chamber, iris, ciliary body, retina, or choroid of the eye.

[0021] In another aspect, use of a SEMA7A peptide or peptidomimetic in the manufacture of a medicament or pharmaceutical composition for treating intraocular inflammation or inflammation- induced vascular leakage in a subject in need thereof, optionally in combination with an additional anti-inflammatory agent or immunosuppressive agent is provided.

[0022] In another aspect, a method of suppressing an immune response of one or more immune cells in an eye of a subject is provided, the method comprising administering an effective amount of a SEMA7A peptide locally to the eye of the subject, wherein the SEMA7A peptide or peptidomimetic binds to PLXNC1 .

[0023] In certain embodiments, the one or more immune cells comprise a neutrophil, a macrophage, a hyalocyte, a dendritic cell, a monocyte, a lymphocyte, or a combination thereof.

[0024] In certain embodiments, administration of the SEMA7A peptide or peptidomimetic decreases numbers of the one or more immune cells in the eye of the subject.

[0025] In certain embodiments, the one or more immune cells are in an iris, a ciliary body, a retina, or a choroid, or a combination thereof.

[0026] In another aspect, a method of reducing or preventing inflammation-induced vascular leakage in an eye of a subject is provided, the method comprising administering an effective amount of a semaphorin 7A (SEMA7A) peptide locally to the eye of the subject, wherein the SEMA7A peptide or peptidomimetic binds to plexin C1 (PLXNC1 ).

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGS. 1A-1 F. TEMPO provides evidence for neuro-immunological interactions involved in the immune privilege of the human eye. (FIG. 1A) TEMPO (Tracing Expression of Multiple Protein Origins) integrates high- resolution proteomics of aqueous humor (AH) liquid biopsies with cell level transcriptomics of all known cell types in the human eye (and blood and liver cells) to trace each AH protein back to its cell origins. Cell-cell interaction analyses were performed based on known receptor-ligand interactions only considering interactions where the ligand was detectable on the protein level in the AH. (FIG. 1 B) Heatmap visualizing the expression of genes encoding for 888 immune-related proteins detected in AH. Cell types are shown in columns. Proteins are grouped by well-established categories of immune proteins (based on Gene Ontology annotation). Proteins included in “immune response” and in a curated list of immunosuppressive proteins that don’t appear in one of the established categories are categorized under “other”. RPE: retinal pigment epithelium, c.: corneal, TMW: trabecular meshwork. (FIG. 1C) Proteins of all categories in (FIG. 1 B) in AH compared to plasma. Immunosuppressive proteins are shown in color and are subclassified based on the comparison between AH and plasma (see legend). Absolute number of proteins are shown in parenthesis. (FIG. 1 D) Heatmap showing 187 immunosuppressive proteins that are increased in AH compared to plasma (blue in C). 142 of these proteins significantly decrease in AH during active intraocular inflammation (uveitis and endophthalmitis) and normalized when the inflammation was inactive (inactive uveitis), demonstrating that the immune privilege is disturbed. (FIG. 1 E) Cell-cell interaction analyses revealed the cellular source of the 187 enriched immunosuppressive proteins in the eye. Classical immune proteins include the proteins from the well- established categories of immune proteins from (FIG. 1 B) and non-classical immune proteins are proteins categorized under "other” in (FIG. 1 B). The middle panel shows the immune cells that are targeted by the immunosuppressive proteins originating from the retina. The heatmap shows the top 10 retinal immunosuppressive proteins falling under “other”. (FIG. 1 F) Cell-cell interactions for 4 of the top 10 proteins. The source retinal cells are shown in color, the target immune cells are shown in grey. The line thickness corresponds to the number of interactions.

[0028] FIGS. 2A-2F. TEMPO identifies SEMA7A as a natural immunosuppressive protein in the human eye. (FIG. 2A) Cell-cell interactions for the ligand SEMA7A, showing the source cells at the top and the target cells on the right. Expression magnitude values are shown multiplied by 1 *101 , interaction specificity values are shown multiplied by 1*104. C.: cell. (FIG. 2B) Violin plot visualizing the distribution of Pearson’s correlation coefficients between SEMA7A and each protein in aqueous humor (AH). Negative correlation corresponds to a decrease of the protein with increasing SEMA7A levels. The analysis is based on 27 healthy AH liquid biopsies (Table S1). On the right, a pathway enrichment is shown for proteins with negative correlation with SEMA7A levels (negative R and p < 0.05). Proteins involved in one of these pathways are shown as color matched points in the violin plot. (FIG. 2C) SEMA7A level in normal plasma compared to normal AH as well as in AH from patients with active (uveitis, endophthalmitis) and inactive (inactive uveitis) intraocular inflammation. The right panel shows the fractional increase of proteins which were negatively correlated with SEMA7A levels in healthy AH (SEMA7A-suppressed proteins) in active and inactive inflammation. (FIG. 2D) Intravitreal injection of 1 ng LPS in Sema7a+/- mice confirms an immunosuppressive effect of SEMA7A in the eye. Number of cells in the anterior and posterior chamber were quantified manually in whole eye histology sections. Each point represents one eyeball (2 eyeballs each from n = 5 C57BL/6 wildtype (WT) littermate mice and n = 9 SEMA7A+/- mice, two independent experiments). (FIG. 2E) A 19 amino acid SEMA7A peptide (or a non-targeting control peptide) was injected intravitreally in both eyes of C57BL/6 wildtype mice (with or without 200ng LPS). After 24h, mice were sacrificed, and eyes were enucleated. Both eyes of each mouse were lysed and the TNF alpha protein concentration was measured using an ELISA and normalized by total protein concentration determined by a BCA assay. Each point represents two pooled eyeballs from one mouse (n = 8 mice per group, two independent experiments). (FIG. 2F) Whole eye lysates were investigated using an aptamer-based proteomics assay. The protein levels for SEMA7A are shown (more data in FIG. 30). Each point represents one eyeball. LPS: lipopolysaccharide, RFU: relative fluorescence unit. *: p<0.05, **: p<0.01 , ***: p<0.001 , ns: p>0.05.

[0029] FIGS. 3A-3C. A SEMA7A peptide broadly suppresses acute immune response in the eye. (FIG. 3A) Histology of murine eyeballs 24h after intravitreal injections. Left top panel: experimental design, d: day, EIU: endotoxin (LPS) induced uveitis, LPS: lipopolysaccharide. Left bottom panel: hematoxylin and eosin-stained murine whole eye section (pupil to optic nerve). The magnifications on the right correspond to the dashed boxes labeled with a and b. Cells in the anterior chamber and the vitreous in a whole eye section were quantified manually. (FIG. 3B) Immunophenotyping in the iris/ciliary body and the retina using flow cytometry. Intravitreal injections were performed in one eyeball per mouse and after perfusion, eyeballs were enucleated and dissected after 24h. Each point represents one eye from one mouse (control and SEMA7A: n = 5 mice per group, LPS + control and LPS + SEMA7A: n = 10 mice per group, two independent experiments). *: p<0.05, **: p<0.01 , ***: p<0.001 , ns: p>0.05. (FIG. 3C) The proteomic profile of EIU whole eye lysates was investigated using an aptamer-based proteomics assay. Heatmap visualizing LPS-induced proteins rescued by the SEMA7A peptide. Proteins rescued by SEMA7A are shown on the right. Each column corresponds to one eyeball and each row to one protein. The z-score represents the deviation from a protein’s mean abundance in standard deviation units. The number of proteins is shown within the heatmap. On the right, a functionally grouped network analysis of the enriched Gene ontology (GO)/pathway terms reveals the molecular pathways affected by the SEMA7A peptide in LPS- induced inflammation in the eye in vivo. Enriched terms are visualized as nodes being linked based on the associated proteins. The node size represents the term’s enrichment significance. The pie charts visualize the percentage of proteins induced by LPS and rescued by SEMA7A (green) or not rescued by SEMA7A (red). Functionally related terms are circled and labeled.

[0030] FIGS. 4A-4E: Re-engineered SEMA7A peptide demonstrates improved rescue of vascular leakage in vivo. (FIG. 4A) Cryogenic electron microscopy (CryoEM) structure of human SEMA7A mimic (PDBID: 6VXK) in complex with human Plexin-C1 (PLXNC1) demonstrating that the RGD motif of the SEMA7A peptide is not involved in binding of PLXNC1. Peptide sequence of the SEMA7A peptide (SEQ ID NOU ), the re-engineered version without RGD motif (SEQ ID NO:3), and the control peptide (SEQ ID NO:2) without interaction motifs for PLXNC1 and integrins. (FIG. 4B) Gene expression of the SEMA7A receptors PLXNC1 and ITGB1 in immune and vascular cells in the human eye (based on TEMPO). Each point represents one cell type per tissue (data from macrophages are e.g., from the RPE/choroid and the ciliary body). (FIG. 4C) The effect of the reengineered SEMA7A peptide on LPS-induced retinal vascular leakage was investigated in vivo based on the amount of a systemically administered fluorescent tracer in retinal tissue lysate (n = 8 eyes from 4 mice per group, two independent experiments). Vascular leakage was normalized to eyes injected with the control peptide. Retinal FA: fluorescence angiogram visualizing LPS-induced retinal vascular leakage. (FIG. 4D) Illustration regarding the mechanism of reduced vascular leakage by the optimized SEMA7A peptide. (FIG. 4E) Human neutrophils were isolated from whole blood from 5 different donors. In cell culture, neutrophils were pretreated with either control peptide, SEMA7A peptide, or the re-engineered SEMA7A peptide without RGD, and subsequently stimulated with LPS (100 ng/ml). After 4h, the supernatant was obtained and prepared for cytokine analysis using a customized Luminex panel. The panel included cytokines that are known to promote vascular leakage. Lines connect data points from the same patient. *: p<0.05, ***: p<0.001.

[0031] FIGS. 5A-5D: Re-engineered SEMA7A peptide demonstrates enhanced anti-inflammatory activity. (FIG. 5A) Shorter versions of the 19-mer SEMA7A peptide (SEQ ID NO:1 ) were created that only include the PLXNC1 binding motif (shown in bold). The three panels in the center show the docking of the 15-, 12-, and 11 -mer peptides (SEQ ID NOS:4-6) to the binding pocket of human PLXNC1 . The efficacy of the peptides was tested in vivo using the endotoxin-induced uveitis mouse model. The bar plot on the right is based on number of cells in the anterior chamber and the vitreous in whole eye histology sections 24h after 200ng LPS. Data was normalized to the LPS group and is shown as percent of the LPS effect (n = 4 eyes from 2 mice per group). (FIG. 5B) Point mutations were introduced in the 11 -mer peptide (SEQ ID NO:6) with the goal to achieve enhanced interaction with human PLXNC1 . The sequences of three peptides with mutations (SEQ ID NOS:7-9) are shown. The bar plot on the right shows efficacy in vivo (as described above, n = 4-8 eyes per group). (FIG. 5C) Effect of the peptides in vivo at lower peptide doses and in comparison to dexamethasone (1 mg/ml) (n = 8 eyes from 4 mice for each peptide and each dose, n = 12 eyes for dexamethasone). **: p<0.01 , ***: p<0.001 , ns: p>0.05. (FIG. 5D) Dose response of the re-engineered 11 -02 peptide in vivo (n = 14 eyes per dose for doses 0.02 - 2 pM, n = 8 eyes per dose for 8 - 160 pM).

DETAILED DESCRIPTION OF THE INVENTION

[0032] Compositions, methods, and kits are provided for treating inflammatory eye diseases. In particular, methods of treating intraocular inflammation with a SEMA7A peptide or peptidomimetic are provided. The SEMA7A peptide or peptidomimetic comprises a binding domain for binding to PLXNC1. The disclosed compositions and methods provide an alternative to current immunosuppressive therapies for treating inflammatory ocular diseases, including non-infectious or autoimmune uveitis.

[0033] Before the present compositions, methods, and kits are described, it is to be understood that this invention is not limited to particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0034] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0036] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0037] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a drug" includes a plurality of such drugs and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof, e.g., peptides or proteins known to those skilled in the art, and so forth. [0038] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Definitions

[0039] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and/or symptom(s). Those in need of treatment include those already inflicted (e.g., those with uveitis, those with ocular inflammation, etc.) as well as those in which prevention is desired (e.g., those with a genetic predisposition for developing uveitis, those with increased susceptibility to uveitis, those with an increased likelihood of ocular inflammation, those suspected of having uveitis, etc.).

[0040] A therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration. In some embodiments, the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment. In some embodiments, the subject is suspected of having an increased likelihood of becoming inflicted.

[0041] By "therapeutically effective dose or amount" of a SEMA7A peptide or peptidomimetic is intended an amount that, when administered brings about a positive therapeutic response, such as decreasing intraocular inflammation and/or reducing or preventing inflammation-induced vascular leakage. A therapeutically effective dose or amount" of a SEMA7A peptide or peptidomimetic may also reduce eye pain, eye redness, floaters, and blurred vision, and prevent, delay, or reduce the risk of developing permanent loss of vision, uveitic glaucoma, retinal detachment, optic nerve damage, or cataracts. Additionally, a therapeutically effective dose or amount of a SEMA7A peptide or peptidomimetic may suppress the immune response and reduce numbers of one or more types of immune cells in the eye, including, but not limited to, neutrophils, macrophages, dendritic cells, and lymphocytes. A therapeutically effective dose can be administered in one or more administrations.

The term “intraocular inflammation” includes inflammation in any part of the eye such as, but not limited to, the vitreous, anterior chamber, iris, ciliary body, retina, or choroid. The term encompasses intraocular inflammation caused by an autoimmune disease or an infection, and includes anterior uveitis, intermediate uveitis, posterior uveitis, or panuveitis. Intraocular inflammation may be caused by various diseases such as, but not limited to, sympathetic ophthalmia, Behget disease, Crohn's disease, Fuchs heterochromic iridocyclitis, granulomatosis with polyangiitis, HLA-B27 related uveitis, spondyloarthritis, juvenile idiopathic arthritis, tubulointerstitial nephritis and uveitis syndrome, enthesitis, ankylosing spondylitis, juvenile rheumatoid arthritis, psoriatic arthritis, reactive arthritis, Behpet's disease, inflammatory bowel disease, Whipple's disease, systemic lupus erythematosus, polyarteritis nodosa, Kawasaki's disease, chronic granulomatous disease, sarcoidosis, multiple sclerosis, Vogt-Koyanagi-Harada disease, subretinal abscess in tubercular posterior uveitis, bartonellosis, tuberculosis, brucellosis, herpesviruses (herpes zoster ophthalmicus - shingles of the eye), leptospirosis, presumed ocular histoplasmosis syndrome, syphilis, toxocariasis, toxoplasmic chorioretinitis, Lyme disease, Zika fever; white dot syndromes such as, but not limited to, acute posterior multifocal placoid pigment epitheliopathy, birdshot chorioretinopathy, multifocal choroiditis and panuveitis, multiple evanescent white dot syndrome, punctate inner choroiditis, serpiginous choroiditis, and acute zonal occult outer retinopathy; and drug-induced uveitis or intraocular inflammation associated with drug-related side effects such as caused by drugs, including, but not limited to, rifabutin, pamidronate, alendronate, etanercept, infliximab, adalimumab, fluoroquinolones, diethylcarbamazine, metipranolol, brimonidine, prostaglandin analogues, ranibizumab, bevacizumab, triamcinolone acetate, moxifloxacin, sulfonamides and vaccines such as, but not limited to, the measles, mumps, and rubella (MMR) vaccine, influenza vaccine, hepatitis B virus (HBV) vaccine, and the varicella vaccine.

[0042] The term "about," particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

[0043] The terms “individual”, “subject”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Mammals include human and non-human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; primates, and transgenic animals.

[0044] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including, but not limited to, compounds comprising amino and/or imino groups. The terms also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Both full-length proteins and fragments thereof are encompassed by the definition. No particular size is implied by use of the terms "polypeptide," "peptide" and "protein" and these terms are used interchangeably. The terms also include post-expression modifications and other modifications of the peptide, for example, glycosylation, acetylation, phosphorylation, hydroxylation, myristoylation, lipidation, methylation, PEGylation (modification with polyethylene glycol (PEG), and the like.

[0045] Included within the definition are, for example, peptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), peptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic). Thus, synthetic oligopeptides, dimers, multimers (e.g., tandem repeats, linearly-linked peptides), cyclized, branched molecules and the like, are included within the definition. The terms also include molecules comprising one or more peptoids (e.g., N-substituted glycine residues) and other synthetic amino acids or peptides. (See, e.g., U.S. Patent Nos. 5,831 ,005; 5,877,278; and 5,977,301 ; Nguyen et al. (2000) Chem Biol. 7(7):463-473; and Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89(20):9367-9371 for descriptions of peptoids). Non-limiting lengths of peptides suitable for use include peptides of 10 to 19 residues in length (or any integer therebetween), 20 to 30 residues in length (or any integer therebetween), 40 to 50 residues in length (or any integer therebetween), 10 to 50 residues in length (or any integer therebetween), 15 to 100 (or any integer therebetween), or polypeptides of greater than 100 residues in length. Typically, peptides useful in the subject methods have a maximum length suitable for the intended application. Preferably, the peptide is between about 10 and 40 residues in length, including any length within this range such as 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 residues in length. Further, peptides, as described herein, for example synthetic peptides, may include additional molecules such as labels or other chemical moieties. Such moieties may further enhance interaction of the peptides with plexin C1 , reduce intraocular inflammation, increase immunosuppression of immune cells such as neutrophils, macrophages, dendritic cells, lymphocytes in the eye, and/or further detection of the peptides. [0046] Thus, references to peptides also include derivatives of the amino acid sequences of the invention including one or more non-naturally occurring amino acids. A first peptide is "derived from" a second peptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide encoding the second peptide, or (ii) displays sequence identity to the second peptide as described herein. Sequence (or percent) identity can be determined as described below. Preferably, derivatives exhibit at least about 50% percent identity, more preferably at least about 80%, and even more preferably between about 85% and 99% (or any value therebetween) to the sequence from which they were derived. Such derivatives can include modifications of the peptide, for example, glycosylation, acetylation, phosphorylation, hydroxylation, myristoylation, lipidation, methylation, PEGylation (modification with polyethylene glycol (PEG), and the like.

[0047] Amino acid derivatives can also include modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature), so long as the peptide maintains the desired activity (e.g., reduces intraocular inflammation). These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PGR amplification. Furthermore, modifications may be made that have one or more of the following effects: increase affinity of binding to plexin C1 , reduce intraocular inflammation, increase immunosuppression of immune cells such as neutrophils, macrophages, dendritic cells, and lymphocytes in the eye, and facilitate purification, delivery, or cell processing. Peptides can be made recombinantly, synthetically, or in tissue culture.

[0048] The term “peptidomimetic” refers to synthesized peptides as well as peptoids, semipeptoids, muteins, and other peptide analogs. The term includes peptide derivatives, which may include unnatural amino acids and/or modifications and/or additions of a chemical function to an amino acid side chain, without a chemical change in the peptidic backbone and analogues with modification and/or addition of a chemical function within the peptidic backbone. Such modifications may include, for example, an N-terminal or C-terminal modification or a peptide bond modification. In some cases, a peptidomimetic may comprise modifications, e.g., to enhance biological activity or improve stability or penetration into cells. In some embodiments, the peptidomimetic is modified with an acyl group such as, but not limited to, CH₃-CO, CO-(CH₂)3-CO₂H, CO-(CH₂)₂-CO₂H, or CO-(CH₂)₂-CO-NH. In some embodiments, the peptidomimetic includes a crosslinkable moiety such as, but not limited to, an amine, thiol, or carboxylate group. In some embodiments, the peptidomimetic comprises a modified sequence of a structural domain or a biologically active fragment of a natural protein.

[0049] The terms “variant,” “analog” and “mutein” refer to biologically active derivatives of the reference molecule that retain desired activity, such the ability to bind to plexin C1 , reduce intraocular inflammation, immunosuppress immune cells such as neutrophils, macrophages, dendritic cells, and lymphocytes in the eye, as described herein. In general, the terms “variant” and “analog” refer to compounds having a native peptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity, and which are “substantially homologous” to the reference molecule as defined below. In general, the amino acid sequences of such analogs will have a high degree of sequence homology to the reference sequence, e.g., amino acid sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned. Often, the analogs will include the same number of amino acids but will include substitutions, as explained herein. The term “mutein” further includes peptides having one or more amino acid-like molecules including but not limited to compounds comprising only amino and/or imino molecules, peptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), peptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules and the like. The term also includes molecules comprising one or more N-substituted glycine residues (a “peptoid”) and other synthetic amino acids or peptides. (See, e.g., U.S. Patent Nos. 5,831 ,005; 5,877,278; and 5,977,301 ; Nguyen et al., Chem Biol. (2000) 7:463-473; and Simon et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371 for descriptions of peptoids). Preferably, the analog or mutein has at least the same biological activity as the native molecule. Methods for making peptide analogs and muteins are known in the art and are described further below.

[0050] As explained above, analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1 ) acidic - aspartate and glutamate; (2) basic - lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar - glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. For example, the peptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5- 25, so long as the desired function of the molecule remains intact. One of skill in the art may readily determine regions of the molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte-Doolittle plots, well known in the art.

[0051] By “derivative” is intended any suitable modification of the peptidomimetic or peptide of interest, a fragment of a native polypeptide, or of their respective analogs, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or other addition of foreign moieties, as long as the desired biological activity is retained. Methods for making peptidomimetics, peptides, polypeptide fragments, analogs, and derivatives are generally available in the art.

[0052] The term “derived from” is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.

[0053] In some embodiments, peptides or peptidomimetics comprising variations of the enumerated sequences comprising one or more amino acid substitutions are provided. In some implementations, an amino acid substitution may be a conservative substitution with an amino acid having one or more similar properties. In some implementations, a small amino acid is substituted with another small amino acid, for example wherein alanine, cysteine, glycine, proline, serine, and threonine are considered small amino acids. In some implementations, an amino acid is substituted with another amino acid having similar charge, for example, aspartate and glutamate are negatively charged; arginine and lysine are positively charged; and asparagine, glutamine, histidine, serine, threonine, and tyrosine are neutral. In some implementations, a hydrophobic amino acid may be substituted with another hydrophobic amino acid. In some implementations, a hydrophilic amino acid may be substituted with another hydrophilic amino acid. In some implementations, an amino acid having an aromatic side chain may be substituted with another amino acid having an aromatic side chain. In some implementations, an amino acid having an aliphatic region may be substituted with another amino acid having an aliphatic region. Exemplary substitutions include the following: substitution of alanine with cysteine, glycine, proline, serine or threonine; substitution of isoleucine, valine, and methionine with each other; substitution of phenylalanine with tyrosine; substitution of tryptophan with phenylalanine, tryptophan, or tyrosine; substitution of tyrosine with phenylalanine; substitution of arginine with lysine; Substitution of lysine with arginine; substitution of aspartate with glutamate or asparagine; substitution of glutamate with glutamine or asparagine; substitution of asparagine with aspartate. Some of these substitutions could be used to stabilize a peptide or peptidomimetic by forming intra-peptide hydrogen bonds or salt bridges, or they could be used to make the peptide more bioavailable.

[0054] In some embodiments, modified peptides are provided, for example peptides comprising unnatural amino acids. Exemplary modifications include glycosylation, N-terminal modification, C- terminal modification, cyclization, formation of disulfide bridges, “stapling,” and other modifications known in the art. Exemplary non-natural amino acids include, for example, hydroxyproline, ornithine, citrulline, homoserine, homocysteine, 2,3-diaminopropionic acid, norleucine, and thyroxine. In some implementations, the peptide is a [3-peptide. In some implementations, one or more amino acids of the peptide or peptidomimetic are a D-isomer.

[0055] The terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" are used herein to include a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded DNA, as well as triple-, double- and single-stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide. More particularly, the terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule," and these terms are used interchangeably.

[0056] The term “SEMA7A” or “semaphorin 7A” as used herein encompasses all forms of SEMA7A and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain biological activity (e.g., binding to plexin C1 , ability to reduce intraocular inflammation and/or reduce or prevent inflammation-induced vascular leakage).

[0057] A SEMA7A polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. A number of SEMA7A nucleic acid and protein sequences are known. Representative SEMA7A sequences, including sequences containing the plexin C1 binding domain are listed in the National Center for Biotechnology

Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NM 001108153, NM 001146030, NM_001146029, NM_003612, NM_011352, NG_011733, NM_001 199749, XM 062061138, XM_062061137, XM_061999932, XM_010205672, XM_061917107,

XM 061917106, XM 061721795, XM_061675912, XM_061675911 , XM_061595368,

XM 061447327, XM_061394841 , XM_061221832, XM_061159008, XM_034409537,

NM 0011 14885, XM_054378999, XM_054332566, XM_010817088, XM_057724951 ,

XM 038442930, XM_038442929, NP_001101623, NP_035482, NP_001139502, NP_001 139501 , NP 003603, NP 001186678, XP_061917122, XP_010203974, XP_061451350, XP_061250825, XP 061077816, XP 061014991 , XP_060891410, XP_034265428, XP_060474048, XP 027812996, XP_021509171 , NP_001108357, XP_054234974, XP_054188541 ,

XP 047289133, XP_010815390, XP_006273216, XP_038298858, and XP_038298857; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used to construct a SEMA7A peptide comprising a binding domain for binding to its receptor, plexin C1 , as described herein. In one embodiment, the SEMA7 peptide comprises or consists of the reference amino acid sequence of SEQ ID NO:1. It is to be understood that the corresponding positions in SEMA7A obtained from other species are also intended to be encompassed by the present invention as long as the SEMA7A peptide binds to plexin C1 and reduces intraocular inflammation.

[0058] By "fragment" is intended a molecule consisting of only a part of the intact full-length sequence and structure. The fragment can include a C-terminal deletion an N- terminal deletion, and/or an internal deletion of the polypeptide. Active fragments of a particular protein or polypeptide will generally include at least about 5-20 contiguous amino acid residues of the full length molecule, but may include at least about 15-25 contiguous amino acid residues of the full length molecule, and can include at least about 20-50 or more contiguous amino acid residues of the full length molecule, or any integer between 5 amino acids and the full length sequence, provided that the fragment in question retains biological activity (e.g., a fragment of the SEMA7A protein containing the binding domain for binding to its receptor, plexin C1 , having the ability to bind to plexin C1 , reduce intraocular inflammation, and immunosuppress immune cells such as neutrophils, macrophages, dendritic cells, and lymphocytes in the eye).

[0059] “Isolated” refers to an entity of interest that is in an environment different from that in which it may naturally occur. “Isolated” is meant to include entities that are within samples that are substantially enriched for the entity of interest and/or in which the entity of interest is partially or substantially purified.

[0060] "Substantially purified" generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and peptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density. [0061] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0062] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0063] "Homology" refers to the percent identity between two polynucleotide or two polypeptide molecules. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence.

[0064] In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353 358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in AppL Math. 2:482 489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wl) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

[0065] Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs are readily available.

[0066] Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

Production of SEMA7A Peptides and Peptidomimetics

[0067] Intraocular inflammation can be suppressed by a SEMA7A peptide comprising a plexin C1 (PLXNC1) binding domain. In certain embodiments, the peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9, or an amino acid sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto, wherein the peptide is capable of binding to PLXNC1 and suppressing intraocular inflammation. In some embodiments, the SEMA7A peptide lacks an RGD motif.

[0068] A SEMA7A peptide can be prepared in any suitable manner (e.g., recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms (e.g., native, fusions, labeled, lipidated, amidated, hydroxylated, methylated, acetylated, PEGylated, etc.). The SEMA7A peptide may include naturally occurring peptides, recombinantly produced peptides, synthetically produced peptides, or peptides produced by a combination of these methods. Means for preparing peptides are well understood in the art. Peptides are preferably prepared in substantially pure form (i.e. substantially free from other host cell or non-host cell proteins).

[0069] SEMA7A nucleic acid and protein sequences may be derived from any source. A number of

SEMA7A nucleic acid and protein sequences are known. Representative SEMA7A sequences, including sequences containing the plexin C1 binding domain are listed in the National Center for

Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NM 001108153, NM 001146030, NM_001 146029, NM_003612, NM_01 1352, NG_011733, NM 001199749, XM 062061138, XM 062061137, XM 061999932, XM 010205672,

XM 061917107, XM 061917106, XM_061721795, XM_061675912, XM_061675911 ,

XM 061595368, XM_061447327, XM_061394841 , XM_061221832, XM_061 159008,

XM 034409537, NM_001114885, XM_054378999, XM_054332566, XM_010817088,

XM 057724951 , XM_038442930, XM_038442929, NP_001101623, NP_035482, NP_001 139502, NP 001139501 , NP 003603, NP_001186678, XP_061917122, XP_010203974, XP_061451350, XP 061250825, XP 061077816, XP 061014991 , XP 060891410, XP 034265428, XP 060474048, XP_027812996, XP_021509171 , NP_001108357, XP_054234974, XP 054188541 , XP_047289133, XP_010815390, XP_006273216, XP_038298858, and

XP 038298857; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used to construct a SEMA7A peptide comprising a binding domain for binding to its receptor, plexin C1 , as described herein. In some embodiments, the SEMA7 peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9. It is to be understood that the corresponding positions in SEMA7A obtained from other species are also intended to be encompassed by the present invention as long as the SEMA7A peptide binds to plexin C1 and reduces intraocular inflammation and/or reduces or prevents inflammation-induced vascular leakage.

[0070] In one embodiment, a SEMA7A peptide is generated using recombinant techniques. One of skill in the art can readily determine nucleotide sequences that encode the desired peptides using standard methodology and the teachings herein. Oligonucleotide probes can be devised based on the known sequences and used to probe genomic or cDNA libraries. The sequences can then be further isolated using standard techniques and, e.g., restriction enzymes employed to truncate the gene at desired portions of the full-length sequence. Similarly, sequences of interest can be isolated directly from cells and tissues containing the same, using known techniques, such as phenol extraction and the sequence further manipulated to produce the desired truncations. See, e.g., Sambrook et aL, supra, for a description of techniques used to obtain and isolate DNA.

[0071] The sequences encoding peptides can also be produced synthetically, for example, based on the known sequences. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. The complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981 ) Nature 292:756; Nambair et al. (1984) Science 223:1299: Jay et al. (1984) J. Biol. Chem. 259:6311 ; Stemmer et al. (1995) Gene 164:49-53.

[0072] Recombinant techniques are readily used to clone sequences encoding peptides that can then be mutagenized in vitro by the replacement of the appropriate base pair(s) to result in the codon for the desired amino acid. Such a change can include as little as one base pair, effecting a change in a single amino acid, or can encompass several base pair changes. Alternatively, the mutations can be effected using a mismatched primer that hybridizes to the parent nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See, e.g., Innis et al, (1990) PCR Applications: Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie- McFarland et al. Proc. Natl. Acad. Sci USA (1982) 79:6409.

[0073] Once coding sequences have been isolated and/or synthesized, they can be cloned into any suitable vector or replicon for expression. (See, also, Examples). As will be apparent from the teachings herein, a wide variety of vectors encoding modified peptides can be generated by creating expression constructs which operably link, in various combinations, polynucleotides encoding peptides having deletions or mutations therein.

[0074] Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage A (E coli), pBR322 (E coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFRI (gram-negative bacteria), pME290 (non-E co// gram-negative bacteria), pHV14 (E co// and Bacillus subtilis), pBD9 Bacillus), plJ61 (Streptomyces), pUC6 (Streptomyces), Ylp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells). See, generally, DNA Cloning-. Vols. I & II, supra; Sambrook et al., supra; B. Perbal, supra.

[0075] Insect cell expression systems, such as baculovirus systems, can also be used and are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA ("MaxBac" kit).

[0076] Plant expression systems can also be used to produce the SEMA7A peptide. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For a description of such systems, see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221 ; and Hackland et al., Arch. Virol. (1994) 139:1 -22.

[0077] Viral systems, such as a vaccinia-based infection/transfection system, as described in Tomei et al., J. Virol. (1993) 7:4017-4026 and Selby et al., J. Gen. Virol. (1993) 74:1103-11 13, will also find use with the present invention. In this system, cells are first transfected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the DNA of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA that is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation product(s).

[0078] The polynucleotide comprising the coding sequence encoding the SEMA7A peptide can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired peptide is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. With the present invention, both the naturally occurring signal peptides or heterologous sequences can be used. Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431 ,739; 4,425,437; 4,338,397. Such sequences include, but are not limited to, the TPA leader, as well as the honeybee mellitin signal sequence.

[0079] Other regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

[0080] The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.

[0081 ] In some cases, it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the proper reading frame. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g., Sambrook et al., supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

[0082] The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells e.g., Hep G2), Vero293 cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

[0083] Depending on the expression system and host selected, the peptides are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The selection of the appropriate growth conditions is within the skill of the art.

[0084] In one embodiment, the transformed cells secrete the SEMA7A peptide product into the surrounding media. Certain regulatory sequences can be included in the vector to enhance secretion of the protein product, for example using a tissue plasminogen activator (TPA) leader sequence, an interferon (yor a) signal sequence or other signal peptide sequences from known secretory proteins. The secreted SEMA7A peptide product can then be isolated by various techniques described herein, for example, using standard purification techniques such as but not limited to, hydroxyapatite resins, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.

[0085] Alternatively, the transformed cells are disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the recombinant peptides substantially intact. Intracellular peptides can also be obtained by removing components from the cell wall or membrane, e.g., by the use of detergents or organic solvents, such that leakage of the peptides occurs. Such methods are known to those of skill in the art and are described in, e.g., Protein Purification Applications: A Practical Approach, (Simon Roe, Ed., 2001 ).

[0086] For example, methods of disrupting cells for use with the present invention include but are not limited to: sonication or ultrasonication; agitation; liquid or solid extrusion; heat treatment; freezethaw; desiccation; explosive decompression; osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium dodecylsulphate, Triton, NP40 and CHAPS. The particular technique used to disrupt the cells is largely a matter of choice and will depend on the cell type in which the peptide is expressed, culture conditions and any pre-treatment used.

[0087] Following disruption of the cells, cellular debris is removed, generally by centrifugation, and the intracellularly produced peptides are further purified, using standard purification techniques such as but not limited to, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.

[0088] For example, one method for obtaining intracellular peptides involves affinity purification, such as by immunoaffinity chromatography using antibodies (e.g., previously generated antibodies), or by lectin affinity chromatography. Particularly preferred lectin resins are those that recognize mannose moieties such as but not limited to resins derived from Galanthus nivalis agglutinin (GNA), Lens culinaris agglutinin (LCA or lentil lectin), Pisum sativum agglutinin (PSA or pea lectin), Narcissus pseudonarcissus agglutinin (NPA) and Allium ursinum agglutinin (AUA). The choice of a suitable affinity resin is within the skill in the art. After affinity purification, the peptides can be further purified using conventional techniques well known in the art, such as by any of the techniques described above. [0089] A SEMA7A peptide can be conveniently synthesized chemically, for example by any of several techniques that are known to those skilled in the peptide art. See, e.g., Fmoc Solid Phase Peptide Synthesis: A Practical Approach (W. C. Chan and Peter D. White eds., Oxford University Press, 1 ^st edition, 2000) ; N. Leo Benoiton, Chemistry of Peptide Synthesis (CRC Press; 1^st edition, 2005); Peptide Synthesis and Applications (Methods in Molecular Biology, John Howl ed., Humana Press, 1 ^st ed., 2005); and Pharmaceutical Formulation Development of Peptides and Proteins (The Taylor & Francis Series in Pharmaceutical Sciences, Lars Hovgaard, Sven Frokjaer, and Marco van de Weert eds., CRC Press; 1^st edition, 1999); herein incorporated by reference.

[0090] In general, these methods employ the sequential addition of one or more amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected, under conditions that allow for the formation of an amide linkage. The protecting group is then removed from the newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support, if solid phase synthesis techniques are used) are removed sequentially or concurrently, to render the final peptide. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis (Pierce Chemical Co., Rockford, IL 1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide Synthesis, (Springer-Verlag, Berlin 1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, Vol. 1 , for classical solution synthesis. These methods are typically used for relatively small polypeptides, i.e., up to about 50-100 amino acids in length, but are also applicable to larger polypeptides.

[0091] Typical protecting groups include t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl); biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, o- bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o-nitrophenylsulfonyl and the like.

[0092] Typical solid supports are cross-linked polymeric supports. These can include divinylbenzene cross-linked-styrene-based polymers, for example, divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers and divinylbenzene- benzhydrylaminopolystyrene copolymers.

[0093] The SEMA7A peptide can also be chemically prepared by other methods such as by the method of simultaneous multiple peptide synthesis. See, e.g., Houghten Proc. Natl. Acad. Sci. USA (1985) 82:5131 -5135; U.S. Patent No. 4,631 ,21 1.

[0094] SEMA7A peptidomimetics can be chemically prepared using solution and solid-phase synthesis strategies (see, e.g., Abdildinova et al. (2021 ) Asian Journal of Organic Chemistry 10 (9):2300-2317). The Ugi reaction, which involves condensation of an aldehyde or ketone, a carboxylic acid, an amine, and an isocyanide, is commonly used to synthesize peptidomimetics and can be used to prepare linear, cyclic, or polycyclic peptidomimetics (see, e.g., Liu et al. (2023) Chemistry Dec 20:e202303597). Alternatively, SEMA7A peptidomimetics can be prepared using cell-free biosynthesis, which allows incorporation of non-canonical residues into peptidomimetic molecules using non-canonical residues attached covalently to a transfer RNA (see, e.g., Lee et al. (2023) BiotechnoL Bioprocess Eng. Feb 3:1 -17).

Pharmaceutical Compositions

[0095] A SEMA7A peptide or peptidomimetic for treating intraocular inflammation can be formulated into pharmaceutical compositions optionally comprising one or more pharmaceutically acceptable excipients. Exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0096] A composition can also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0097] An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the agent, or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0098] A surfactant can be present as an excipient. Exemplary surfactants include: polysorbates, such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.

[0099] Acids or bases can be present as an excipient in the composition. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.

[00100] The amount of the SEMA7A peptide or peptidomimetic (e.g., when contained in a drug delivery system) in the composition will vary depending on a number of factors but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial). A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint.

[00101] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred. These foregoing pharmaceutical excipients along with other excipients are described in "Remington: The Science & Practice of Pharmacy", 19^th ed., Williams & Williams, (1995), the "Physician’s Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3^rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.

[00102] The compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid pharmaceutical compositions, solutions and suspensions are envisioned. Additional preferred compositions include those for oral, intravenous, intramuscular, vaginal, intrathecal, intraspinal, or localized delivery such as by injection into the myometrium to inhibit contractions.

[00103] The pharmaceutical preparations herein can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use. Preferably, the compositions comprising the agent are in unit dosage form, meaning an amount of a conjugate or composition of the invention appropriate for a single dose, in a premeasured or pre-packaged form.

[00104] The compositions herein may optionally include one or more additional agents, such as other drugs for treating intraocular inflammation such as anti-inflammatory or immunosuppressive agents, or other medications. For example, compounded preparations may include a SEMA7A peptide or peptidomimetic and one or more other drugs, including, without limitation, anti-inflammatory or immunosuppressive agents such as, but not limited to, glucocorticoid steroids including, without limitation, prednisolone, methylprednisolone, iluvien, ozurdex, retisert, and triamcinolone; T-cell inhibitors including, without limitation, calcineurin inhibitors such as cyclosporine, tacrolimus and voclosporin, and mTOR inhibitors such as everolimus and sirolimus; antimetabolites including, without limitation, purine antagonists such as azathioprine, dihydrofolate reductase (DHFR) inhibitors such as methotrexate, and inosine monophosphate dehydrogenase (IMPDH) inhibitors such as mycophenolate mofetil; anti-TNF agents including, without limitation, adalimumab, certolizumab, golimumab, infliximab, and etanercept; biologic agents including, without limitation, efalizumab, rituximab, abatacept, alemtuzumab, anakinra, canakinumab, gevokizumab, daclizumab, tocilizumab, secukinumab, interferon a/p, fingolimod, aflibercept, bevacizumab, ranibizumab, and intravenous immunoglobulin (IVIG); alkylating agents including, without limitation, chlorambucil and cyclophosphamide; and cycloplegic agents including, without limitation, atropine and homatropine; or a combination thereof. For treatment of bacterial uveitis, compositions may comprise, for example, one or more antibiotics such as, but not limited to, vancomycin, ceftazidime, amikacin, gentamycin, moxifloxacin, and cephalosporins such as cefacetrile (cephacetrile), cefadroxil (cefadroxyl; Duricef), cefalexin (cephalexin; Keflex), cefaloglycin (cephaloglycin), cefalonium (cephalonium), cefaloridine (cephaloradine), cefalotin (cephalothin; Keflin), cefapirin (cephapirin; Cefadryl), cefatrizine, cefazaflur, cefazedone, cefazolin (cephazolin; Ancef, Kefzol), cefradine (cephradine; Velosef), cefroxadine, ceftezole, cefaclor (Ceclor, Distaclor, Keflor, Raniclor), cefonicid (Monocid), cefprozil (cefproxil; Cefzil), cefuroxime (Zefu, Zinnat, Zinacef, Ceftin, Biofuroksym, Xorimax), cefuzonam, cefmetazole, cefotetan, cefoxitin, loracarbef (Lorabid), cefbuperazone, cefmetazole (Zefazone), cefminox, cefotetan (Cefotan), cefoxitin (Mefoxin), cefotiam (Pansporin), cefcapene, cefdaloxime, cefdinir (Sefdin, Zinir, Omnicef, Kefnir), cefditoren, cefetamet, cefixime (Fixx, Zifi, Suprax), cefmenoxime, cefodizime, cefotaxime (Claforan), cefovecin (Convenia), cefpimizole, cefpodoxime (Vantin, PECEF, Simplicef), cefteram, ceftibuten (Cedax), ceftiofur (Naxcel, Excenel), ceftiolene, ceftizoxime (Cefizox), ceftriaxone (Rocephin), cefoperazone (Cefobid), ceftazidime (Meezat, Fortum, Fortaz), latamoxef (moxalactam), cefclidine, cefepime (Maxipime), cefluprenam, cefoselis, cefozopran, cefpirome (Cefrom), cefquinome, flomoxef, ceftobiprole, ceftaroline, ceftolozane, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium, cefoxazole, cefrotil, cefsumide, ceftioxide, cefuracetime, and nitrocefin; or a combination thereof. For treatment of viral uveitis, compositions may comprise, for example, one or more antiviral agents such as, but not limited to, ganciclovir, acyclovir, foscarnet, valacyclovir, and cidofivir, or a combination thereof. For treatment of fungal uveitis, compositions may comprise, for example, one or more antifungal agents such as, but not limited to, amphotericin B, voriconazole, caspofungin, and fluconazole, or a combination thereof.

Administration

[00105] A SEMA7A peptide or peptidomimetic is administered to a subject for treatment of intraocular inflammation. The intraocular inflammation may occur in any part of the eye such as, but not limited to, the vitreous, anterior chamber, iris, ciliary body, retina, or choroid. The intraocular inflammation may be caused by an autoimmune disease or an infection, including, but not limited to, anterior uveitis, intermediate uveitis, posterior uveitis, or panuveitis. In some embodiments, the intraocular inflammation is caused by a disease such as, but not limited to, sympathetic ophthalmia, Behget disease, Crohn's disease, Fuchs heterochromic iridocyclitis, granulomatosis with polyangiitis, HLA- B27 related uveitis, spondyloarthritis, juvenile idiopathic arthritis, tubulointerstitial nephritis and uveitis syndrome, enthesitis, ankylosing spondylitis, juvenile rheumatoid arthritis, psoriatic arthritis, reactive arthritis, Behget's disease, inflammatory bowel disease, Whipple's disease, systemic lupus erythematosus, polyarteritis nodosa, Kawasaki's disease, chronic granulomatous disease, sarcoidosis, multiple sclerosis, Vogt-Koyanagi-Harada disease, subretinal abscess in tubercular posterior uveitis, bartonellosis, tuberculosis, brucellosis, herpesviruses (herpes zoster ophthalmicus - shingles of the eye), leptospirosis, presumed ocular histoplasmosis syndrome, syphilis, toxocariasis, toxoplasmic chorioretinitis, Lyme disease, Zika fever; white dot syndromes such as, but not limited to, acute posterior multifocal placoid pigment epitheliopathy, birdshot chorioretinopathy, multifocal choroiditis and panuveitis, multiple evanescent white dot syndrome, punctate inner choroiditis, serpiginous choroiditis, and acute zonal occult outer retinopathy; drug-induced uveitis or intraocular inflammation associated with drug-related side effects such as caused by drugs, including, but not limited to, rifabutin, pamidronate, alendronate, etanercept, infliximab, adalimumab, fluoroquinolones, diethylcarbamazine, metipranolol, brimonidine, prostaglandin analogues, ranibizumab, bevacizumab, triamcinolone acetate, moxifloxacin, and sulfonamides; or vaccines such as, but not limited to, the measles, mumps, and rubella (MMR) vaccine, influenza vaccine, hepatitis B virus (HBV) vaccine, and the varicella vaccine.

[00106] At least one therapeutically effective cycle of treatment with a composition comprising a SEMA7A peptide or peptidomimetic will be administered to a subject for treatment of intraocular inflammation. By "therapeutically effective dose or amount" of a SEMA7A peptide or peptidomimetic is intended an amount that, when administered brings about a positive therapeutic response, such as decreasing intraocular inflammation and/or reducing or preventing inflammation-induced vascular leakage. A therapeutically effective dose or amount" of a SEMA7A peptide or peptidomimetic may also reduce eye pain, eye redness, floaters, and blurred vision, and prevent, delay, or reduce the risk of developing permanent loss of vision, uveitic glaucoma, retinal detachment, optic nerve damage, or cataracts. Additionally, a therapeutically effective dose or amount of a SEMA7A peptide or peptidomimetic may reduce numbers of one or more types of immune cells in the eye, including, but not limited to, neutrophils, macrophages, dendritic cells, and lymphocytes. A therapeutically effective dose can be administered in one or more administrations. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular type of agent employed to treat intraocular inflammation, the mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

[00107] In certain embodiments, multiple therapeutically effective doses of compositions comprising a SEMA7A peptide or peptidomimetic and/or one or more other therapeutic agents, such as one or more other drugs for treating intraocular inflammation or other medications. For example, compounded preparations may include a SEMA7A peptide or peptidomimetic and one or more other drugs for treating intraocular inflammation, including, without limitation, anti-inflammatory or immunosuppressive agents such as, but not limited to, glucocorticoid steroids including, without limitation, prednisolone, methylprednisolone, iluvien, ozurdex, retisert, and triamcinolone; T-cell inhibitors including, without limitation, calcineurin inhibitors such as cyclosporine, tacrolimus and voclosporin, and mTOR inhibitors such as everolimus and sirolimus; antimetabolites including, without limitation, purine antagonists such as azathioprine, dihydrofolate reductase (DHFR) inhibitors such as methotrexate, and inosine monophosphate dehydrogenase (IMPDH) inhibitors such as mycophenolate mofetil; anti-TNF agents including, without limitation, adalimumab, certolizumab, golimumab, infliximab, and etanercept; biologic agents including, without limitation, efalizumab, rituximab, abatacept, alemtuzumab, anakinra, canakinumab, gevokizumab, daclizumab, tocilizumab, secukinumab, interferon a/p, fingolimod, aflibercept, bevacizumab, ranibizumab, and intravenous immunoglobulin (IVIG); alkylating agents including, without limitation, chlorambucil and cyclophosphamide; and cycloplegic agents including, without limitation, atropine and homatropine; or a combination thereof. For treatment of bacterial uveitis, compositions may comprise, for example, one or more antibiotics such as, but not limited to, vancomycin, ceftazidime, amikacin, gentamycin, moxifloxacin, and cephalosporins such as cefacetrile (cephacetrile), cefadroxil (cefadroxyl; Duricef), cefalexin (cephalexin; Keflex), cefaloglycin (cephaloglycin), cefalonium (cephalonium), cefaloridine (cephaloradine), cefalotin (cephalothin; Keflin), cefapirin (cephapirin; Cefadryl), cefatrizine, cefazaflur, cefazedone, cefazolin (cephazolin; Ancef, Kefzol), cefradine (cephradine; Velosef), cefroxadine, ceftezole, cefaclor (Ceclor, Distaclor, Keflor, Raniclor), cefonicid (Monocid), cefprozil (cefproxil; Cefzil), cefuroxime (Zefu, Zinnat, Zinacef, Ceftin, Biofuroksym, Xorimax), cefuzonam, cefmetazole, cefotetan, cefoxitin, loracarbef (Lorabid), cefbuperazone, cefmetazole (Zefazone), cefminox, cefotetan (Cefotan), cefoxitin (Mefoxin), cefotiam (Pansporin), cefcapene, cefdaloxime, cefdinir (Sefdin, Zinir, Omnicef, Kefnir), cefditoren, cefetamet, cefixime (Fixx, Zifi, Suprax), cefmenoxime, cefodizime, cefotaxime (Claforan), cefovecin (Convenia), cefpimizole, cefpodoxime (Vantin, PECEF, Simplicef), cefteram, ceftibuten (Cedax), ceftiofur (Naxcel, Excenel), ceftiolene, ceftizoxime (Cefizox), ceftriaxone (Rocephin), cefoperazone (Cefobid), ceftazidime (Meezat, Fortum, Fortaz), latamoxef (moxalactam), cefclidine, cefepime (Maxipime), cefluprenam, cefoselis, cefozopran, cefpirome (Cefrom), cefquinome, flomoxef, ceftobiprole, ceftaroline, ceftolozane, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium, cefoxazole, cefrotil, cefsumide, ceftioxide, cefuracetime, and nitrocefin; or a combination thereof. For treatment of viral uveitis, compositions may comprise, for example, one or more antiviral agents such as, but not limited to, ganciclovir, acyclovir, foscarnet, valacyclovir, and cidofivir, or a combination thereof. For treatment of fungal uveitis, compositions may comprise, for example, one or more antifungal agents such as, but not limited to, amphotericin B, voriconazole, caspofungin, and fluconazole, or a combination thereof. The compositions comprising the SEMA7A peptide or peptidomimetic are typically, although not necessarily, administered orally, via injection (subcutaneously, intravenously, or intramuscularly), by infusion, topically, or locally. Additional modes of administration are also contemplated, such as intraocular, intravitreal, juxtascleral, subconjunctival, intracameral, or retrobulbar, or localized delivery such as by injection into the eye at a site of inflammation, and so forth.

[00108] The preparations according to the invention are also suitable for local treatment. For example, compositions comprising a SEMA7A peptide or peptidomimetic may be administered locally by injection into the eye. The particular preparation and appropriate method of administration can be chosen to target the SEMA7A peptide or peptidomimetic to a site of inflammation in need of treatment such as the vitreous, anterior chamber, iris, ciliary body, retina, or choroid of the eye. Local treatment may avoid some side effects of systemic therapy.

[00109] The pharmaceutical preparation can be in the form of a liquid solution or suspension immediately prior to administration, but may also take another form such as a syrup, cream, ointment, tablet, capsule, powder, gel, matrix, suppository, or the like. The pharmaceutical compositions comprising a SEMA7A peptide or peptidomimetic and/or other agents may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art.

[00110] In another embodiment, the pharmaceutical compositions comprising the SEMA7A peptide or peptidomimetic and/or other drugs for treating intraocular inflammation, and/or other agents are in a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for drug delivery over time in a continuous, steadystate fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.

[00111] Those of ordinary skill in the art will appreciate which conditions the SEMA7A peptide or peptidomimetic can effectively treat. The actual dose to be administered will vary depending upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered. Therapeutically effective amounts can be determined by those skilled in the art, and will be adjusted to the particular requirements of each particular case.

[00112] In certain embodiments, multiple therapeutically effective doses of a composition comprising a SEMA7A peptide or peptidomimetic will be administered according to a daily dosing regimen or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By “intermittent” administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, once a week, every other week, and so forth. For example, in some embodiments, a composition comprising the SEMA7A peptide or peptidomimetic will be administered once-weekly, twice-weekly or thrice-weekly for an extended period of time, such as for 1 , 2, 3, 4, 5, 6, 7, 8...10...15...24 weeks, and so forth. By “twice-weekly” or “two times per week” is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By “thrice weekly” or “three times per week” is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as “intermittent” therapy. In accordance with the methods of the present invention, a subject can receive intermittent therapy (i.e., once-weekly, twice-weekly or thrice-weekly administration of a therapeutically effective dose) for one or more weekly cycles until the desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below. The amount administered will depend on the potency of the SEMA7A peptide or peptidomimetic and/or other agents administered, the magnitude of the effect desired, and the route of administration.

[00113] The SEMA7A peptide or peptidomimetic (again, preferably provided as part of a pharmaceutical preparation) can be administered alone or in combination with one or more other therapeutic agents, such as other agents for treating intraocular inflammation, or other medications used to treat a particular condition or disease according to a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Preferred compositions are those requiring dosing no more than once a day.

[00114] The SEMA7A peptide or peptidomimetic can be administered prior to, concurrent with, or subsequent to other agents. If provided at the same time as other agents, the SEMA7A peptide or peptidomimetic can be provided in the same or in a different composition. Thus, the SEMA7A peptide or peptidomimetic and one or more other agents can be presented to the individual by way of concurrent therapy. By “concurrent therapy” is intended administration to a subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy. For example, concurrent therapy may be achieved by administering a dose of a pharmaceutical composition comprising the SEMA7A peptide or peptidomimetic and a dose of a pharmaceutical composition comprising at least one other agent, such as another drug for treating intraocular inflammation, which in combination comprise a therapeutically effective dose, according to a particular dosing regimen. Similarly, the SEMA7A peptide or peptidomimetic and one or more other therapeutic agents can be administered in at least one therapeutic dose. Administration of the separate pharmaceutical compositions can be performed simultaneously or at different times (i.e., sequentially, in either order, on the same day, or on different days), as long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.

[00115] Toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD₅o (the dose lethal to 50% of the population) or the LD wo (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in further optimizing and/or defining a therapeutic dosage range and/or a sub-therapeutic dosage range (e.g., for use in humans). The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

Kits

[00116] Also provided are kits comprising any of the compositions described herein. In certain embodiments, the kit comprises a SEMA7A peptide or peptidomimetic. In some embodiments, the SEMA7A peptide or peptidomimetic comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9 or an amino acid sequence having at least about 80-100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9, including any percent identity within this range, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. [00117] Kits may comprise one or more containers of the compositions described herein. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device. The kit may also provide a delivery device pre-filled with a composition comprising the SEMA7A peptide or peptidomimetic.

[00118] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, Blu-ray, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.

Examples of Non-Limiting Aspects of the Disclosure

[00119] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-34 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below.

1 . A method of treating intraocular inflammation or inflammation-induced vascular leakage in a subject, the method comprising administering a therapeutically effective amount of a semaphorin 7A (SEMA7A) peptide or peptidomimetic to the subject, wherein the SEMA7A peptide or peptidomimetic binds to plexin C1 (PLXNC1 ). 2. The method of aspect 1 , wherein the SEMA7A peptide or peptidomimetic lacks an

RGD motif.

3. The method of aspect 1 , wherein the SEMA7A peptide or peptidomimetic comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9.

4. The method of any one of aspects 1 -3, wherein the intraocular inflammation is acute.

5. The method of any one of aspects 1 -3, wherein the intraocular inflammation is caused by an autoimmune disease or an infection.

6. The method of any one of aspects 1-3, wherein the subject has non-infectious uveitis or autoimmune uveitis.

7. The method of aspect 6, wherein the subject has anterior uveitis, intermediate uveitis, posterior uveitis, or panuveitis.

8. The method of any one of aspects 1 -7, wherein the SEMA7A peptide or peptidomimetic is administered intravitreally, intraocularly, juxtasclerally, subconjunctivally, intracamerally, or retrobulbarly.

9. The method of any one of aspects 1 -7, wherein the SEMA7A peptide or peptidomimetic is administered locally to an eye of the subject at a site of inflammation.

10. The method of aspect 9, wherein the SEMA7A peptide or peptidomimetic is administered locally to the vitreous, anterior chamber, iris, ciliary body, retina, or choroid.

11. The method of any one of aspects 1 -10, wherein multiple cycles of treatment are administered to the subject.

12. The method of aspect 11 , wherein the SEMA7A peptide or peptidomimetic is administered daily or intermittently. 13. The method of any one of aspects 1-12, wherein the subject is a mammal.

14. The method of aspect 13, wherein the mammal is human.

15. The method of any one of aspects 1 -14, further comprising administering an additional anti-inflammatory agent or immunosuppressive agent.

16. A composition comprising a SEMA7A peptide or peptidomimetic for use in a method of treating intraocular inflammation or inflammation-induced vascular leakage.

17. The composition of aspect 16, wherein the intraocular inflammation is acute.

18. The composition of aspect 16, wherein the intraocular inflammation is caused by non- infectious uveitis or autoimmune uveitis.

19. The composition of any one of aspects 16-18, wherein the SEMA7A peptide or peptidomimetic comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9.

20. The composition of any one of aspects 16-19, further comprising a pharmaceutically acceptable excipient.

21 . The composition of any one of aspects 16-20, further comprising an additional antiinflammatory agent or immunosuppressive agent.

22. The composition of aspect 21 , wherein the anti-inflammatory agent is a corticosteroid.

23. The composition of any one of aspects 16-22, wherein the composition is formulated for intravitreal, intraocular, juxtascleral, subconjunctival, intracameral, or retrobulbar administration. 24. The composition of any one of aspects 16-23, wherein the composition is formulated for administration locally to the vitreous, anterior chamber, iris, ciliary body, retina, or choroid of the eye.

25. Use of a SEMA7A peptide or peptidomimetic in the manufacture of a medicament or pharmaceutical composition for treating intraocular inflammation or inflammation-induced vascular leakage in a subject in need thereof, optionally in combination with an additional anti-inflammatory agent or immunosuppressive agent.

26. The use of aspect 25, wherein the SEMA7A peptide or peptidomimetic comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9.

27. A method of suppressing an immune response of one or more immune cells in an eye of a subject, the method comprising administering an effective amount of a semaphorin 7A (SEMA7A) peptide locally to the eye of the subject, wherein the SEMA7A peptide or peptidomimetic binds to plexin C1 (PLXNC1 ).

28. The method of aspect 27, wherein the one or more immune cells comprise a neutrophil, a macrophage, a hyalocyte, a dendritic cell, a monocyte, a lymphocyte, or a combination thereof.

29. The method of aspect 27 or 28, wherein said administering the SEMA7A peptide or peptidomimetic decreases numbers of the one or more immune cells in the eye of the subject.

30. The method of any one of aspects 27-29, wherein the one or more immune cells are in an iris, a ciliary body, a retina, or a choroid, or a combination thereof.

31. The method of any one of aspects 27-30, wherein the SEMA7A peptide or peptidomimetic comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOU and SEQ ID NOS:3-9, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOU and SEQ ID NOS:3-9. 32. A method of reducing or preventing inflammation-induced vascular leakage in an eye of a subject, the method comprising administering an effective amount of a semaphorin 7A (SEMA7A) peptide locally to the eye of the subject, wherein the SEMA7A peptide or peptidomimetic binds to plexin C1 (PLXNC1 ).

33. The method of aspect 32, wherein the SEMA7A peptide or peptidomimetic lacks an RGD motif.

34. The method of aspect 32, wherein the SEMA7A peptide or peptidomimetic comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9.

[00120] It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

EXPERIMENTAL

[00121] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[00122] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[00123] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.

Example 1

Treating Inflammatory Eve Disease

Introduction

[00124] A beneficial immune response requires the right balance between a pro-inflammatory response to eliminate a pathogen and anti-inflammatory control to prevent damage to host cells. In immune privileged organs like the brain and the eye, this balance is shifted to favor anti-inflammatory control to protect critical non-regenerative tissues like the retina from inflammatory damage.¹'³ There is evidence that the immune privilege is disturbed during disease ⁴⁵ and this reduced protection may contribute to inflammation-induced tissue damage and irreversible functional impairment. Current evidence suggests that anatomical, cellular, and molecular mechanisms are involved in immune privilege, among them the blood-brain or the blood-retina barrier, cells that directly inhibit immune cells, and about 25 known immunosuppressive proteins.¹'²⁶ However, in contrast to several studies on other aspects of the immune system,⁷⁸ the exact mechanisms of the immune privilege and its disturbance during disease have not been examined using a high-resolution multi-omics approach. Recent re-engineering of natural immune system components to treat various human diseases highlights the opportunity to create novel therapeutics once basic molecular mechanisms are identified.⁹

[00125] Here, we applied a highly sensitive aptamer-based assay to obtain the proteomic profile of microvolume aqueous humor (AH) liquid biopsies from the living human eye with previously unachieved proteomic resolution. We applied TEMPO (Tracing Expression of Multiple Protein Origins),¹⁰ our multi-omics approach that integrates proteomics with single-cell transcriptomics from all known cell types in the human eye, allowing us to trace thousands of AH proteins back to their cells of origin in the eye. We identified 888 immune proteins in AH, among them 187 natural immunosuppressive proteins with strong local enrichment in the AH compared to plasma. TEMPO revealed that many of these proteins originated from neuronal cell types in the retina and targeted receptors on immune cells, providing evidence for a pivotal role of neuro-immunological interactions in the immune privilege of the eye. A key protein was Semaphorin 7A (SEMA7A) originating mainly from rod photoreceptors and binding to PLXNC1 on neutrophils and other innate immune cells. A short SEMA7A peptide demonstrated strong immunosuppressive activity in the endotoxin-induced uveitis (EIU) mouse model in vivo. \Ne then generated re-engineered versions of the peptide that demonstrated improved activity in vitro and in vivo. Using this highly translational approach, we identified hundreds of novel natural immunosuppressive proteins as part of the immune privilege and demonstrated that re-engineering of the key protein SEMA7A led to a promising novel therapeutic to target inflammatory diseases.

Results

TEMPO provides evidence for neuro-immune interactions involved in the immune privilege of the human eye

[00126] We applied TEMPO (Tracing Expression of Multiple Protein Origins)¹⁰ to assess the molecular and cellular mechanisms of the immune privilege of the human eye. The aptamer-based assay identified 4,941 different proteins in eight normal AH liquid biopsies (Table S1 and S2), corresponding to a substantially increased proteomic resolution compared to five previous studies using liquid chromatography-mass spectrometry (LC- MS) that together detected 297 proteins in normal AH (FIG. S1 ).^11-15 By mapping the almost 5000 AH proteins to published single cell RNA sequencing data of all known cell types of the human eye, TEMPO allowed us to trace each AH protein back to its cell origins (FIG. 1A). We first focused our analysis on known immune privilege proteins (Table S3) and detected 15 of the 17 known ligands in AH, including POMC, IL10, and TGFB2 (FIG. S1A). While many of these proteins originated from immune cells, ligands like TGFB2, VIP, and SERPINF1 came from stroma or neuronal cells in the retina (FIG. S1 B-C). We also found that 10 of the ligands decreased in eyes with active intraocular inflammation (uveitis or endophthalmitis) indicating disturbance of the immune privilege during inflammation (FIG. S1 D).

[00127] Our next goal was to assess the ocular immune privilege at larger scale. Using TEMPO, we detected 888 known immune proteins in AH, among them 184 innate immunity proteins, 93 adaptive immunity proteins, 38 overlapping innate and adaptive immunity proteins, 164 cytokines, 44 complement proteins, 72 humoral immune proteins, and 293 immune proteins that did not fall under one of these categories (alternative immune proteins) (FIG. 1 B). As expected, most of the corresponding genes of these 888 immune proteins where highly expressed in immune cells, but we also found many immune proteins originating from non-immune cells, including retinal cells, vascular endothelial cells, and the liver (FIG. 1 B). 350 (39.4%) of the 888 immune proteins had known immunosuppressive functions 16 with the highest percentage seen in alternative immune proteins, adaptive immune proteins, and cytokines (FIG. 1C). We also compared the protein levels in AH to plasma, 17 revealing that 187 immunosuppressive proteins were significantly enriched in normal AH including 107 alternative immune proteins (FIG. 1C and D, Table S4). Strikingly, 142 of the 187 proteins were significantly decreased in eyes with active intraocular inflammation (uveitis and endophthalmitis) and normalized in cases with inactive uveitis (FIG. 1 D). These findings identify 187 immunosuppressive proteins in AH strongly indicating their involvement in immune privilege and its impairment during active inflammation (FIG. 1 D).

[00128] We then applied cell-cell interaction analyses to determine the cellular origins and targets of the 187 immunosuppressive proteins in the eye. We applied a consensus method for receptor-ligand interactions based on single cell RNA sequencing data, 18 and expanded this method by integrating high-resolution AH proteomics data that allowed us to only consider cell-cell interactions where the ligand was detectable on the protein level in the AH. We found that the classical immune proteins (FIG. 1 B) predominantly originated from immune cells, whereas the source shifted to retinal cells for the alternative immune proteins, providing evidence for neuro-immunological interactions involved in the immune privilege of the human eye (FIG. 1 E).

[00129] These interactions mainly targeted neutrophils, macrophages, and T cells. EFNB2, ADM, LGALS1 , and SEMA7A were among the top 10 ligands (FIG. 1 E). While EFNB2 and ADM originated from various retinal cell types, LGALS1 was more specific to ganglion cells and targeted T cells, NK cells, and mast cells. SEMA7A originated from ganglion cells, amacrine cells, cones and rods and targeted neutrophils and monocytes (FIG. 1 F). These results substantially expand our understanding of the molecules and cells involved in ocular immune privilege and point towards a key role of neuro- immune interactions in this process as shown for SEMA7A.

SEMA7A represents a neuro-immune interaction with immunosuppressive functions in the eye

[00130] To further investigate the role of neuro-immune interactions in the immune privilege of the human eye, we focused our analysis on SEMA7A. Our cell interaction analysis revealed that SEMA7A is mainly produced by rod photoreceptors and other retinal cell types (FIG. 2A). Via two different receptors, SEMA7A targets immune cells (via PLXNC1 ) including neutrophils, hyalocytes (vitreous macrophages), and monocytes, but also endothelial cells and pericytes (via ITGA1/ITGB1 ) (FIG. 2A). We then analyzed the correlation of SEMA7A protein levels in AH with all other proteins in 27 normal AH samples (Table S1) revealing that higher SEMA7A levels were mainly associated with lower abundance of these proteins (FIG. 2B). Strikingly, these proteins were enriched in immune-related pathways, further indicating that SEMA7A represents a natural immunosuppressive protein in the human eye. Compared to plasma, SEMA7A protein levels were highly enriched in normal AH, significantly decreased in eyes with active intraocular inflammation and significantly increased in eyes with inactive inflammation (FIG. 2C). Concurrently, proteins with negative correlation with SEMA7A significantly increased in active inflammation (FIG. 2C). These results strongly suggest that SEMA7A is a natural immunosuppressive protein in the human eye playing a key role in the immune privilege. To functionally validate the role of SEMA7A, we applied the endotoxin-induced uveitis (EIU) model in Sema7a+/- mice. Lipopolysaccharide (LPS) was injected intravitreally and after 24h, mice were perfused and cells were manually quantified in the anterior and posterior chamber in whole eye histology sections. Sema7a+/- mice demonstrated significantly increased inflammation compared to Sema7a wildtype mice (p<0.01 ) (FIG. 2D). Intravitreal injections of a 19 amino acid SEMA7A peptide significantly rescued the LPS-induced TNF protein increase in whole eye lysates (p<0.01 ) (FIG. 2E). In addition, endotoxin-induced uveitis resulted in a significant reduction of SEMA7A protein levels in murine eyes (p<0.05), a finding that is in accordance with the SEMA7A decrease observed in human eyes with active inflammation. These results confirm an immunosuppressive role of SEMA7A in the eye, suggest a decline of this function during active inflammation and indicate that the supplementation of SEMA7A could be therapeutically beneficial.

A SEMA7A peptide broadly suppresses intraocular inflammation

[00131] To further investigate the functional role of the SEMA7A peptide, we performed multimodal experiments in the EIU mouse model, including histology, flow cytometry, and proteomic profiling. Histological examination and manual cell quantification revealed a strong LPS-induced infiltration of cells per whole eye section in both the anterior chamber and the vitreous that was significantly reduced by the SEMA7A peptide (p<0.01 and p<0.001 ) (FIG. 3A). To further specify the phenotype of infiltrating cells, iris/ciliary body and retinal tissue from EIU- eyes were analyzed by flow cytometry. LPS mainly induced infiltration of neutrophils, but also of monocytes/macrophages, dendritic cells, and T cells (FIG. 3B). In both iris/ciliary body and retinal tissue, the SEMA7A peptide significantly reduced the number of all CD45+ immune cells (p<0.05 and p<0.01 ), neutrophils (p<0.05 and p<0.01 ), monocytes/macrophages (p<0.05 and p<0.01 ), dendritic cells (p<0.01 and p<0.01 ), and T cells (p=0.10 and p<0.05) (FIG. 3B). We also analyzed the proteomic profile of whole eye lysates using the aptamer-based assay to investigate which proteins and molecular pathways were rescued by the SEMA7A peptide in vivo. LPS affected the level of 336 proteins compared to the control group, including 260 increased and 76 decreased proteins (FIG. 3C). The SEMA7A peptide significantly rescued 77 (29.6%) of the increased and 12 (15.8%) of the decreased proteins and affected most of the LPS-induced signaling pathways, including myeloid cell and neutrophil immunity, NFkB pathway, TLR signaling, hemostasis, proteolysis, and oxidative stress (FIG. 3C and FIG. S3). These multimodal functional validation experiments demonstrate a strong and broad immunosuppressive effect and the therapeutic potential of the SEMA7A peptide in vivo.

Re-engineered SEMA7A peptide demonstrates improved rescue of vascular leakage

[00132] Overlaying the 19 amino acid SEMA7A peptide with the known cryoEM structure of human SEMA7A in interaction with the human receptor PLXNC1 19 revealed that the RGD motif of the SEMA7A peptide is not part of the interaction motif with PLXNC1 (FIG. 4A). We therefore reengineered a SEMA7A peptide that only includes the interaction motif for PLXNC1 , but not the RGD motif, that is known to confer integrin binding.²⁰

[00133] Neutrophils demonstrated the highest expression of PLXNC1 compared to other immune cell types in the human eye, with negligible expression of integrin B1 (ITGB1) (FIG. 4B). We thus hypothesized that the re- engineered SEMA7A peptide controls neutrophil-driven inflammation more effectively compared to the unmodified peptide. Patients with intraocular inflammation often present with retinal vasculitis, characterized by increased vascular leakage, which can cause significant tissue damage, potentially leading to blindness. We investigated the effect of the re-engineered SEMA7A peptide without RGD on retinal vascular leakage in the EIU mouse model, as determined by the amount of systemically administered fluorescent tracer in retinal tissue after 24h. As expected, LPS increased retinal vascular leakage by 4.3 times (±1.2; p<0.001 ) compared to the control group (FIG. 4C). The unmodified SEMA7A peptide reduced retinal vascular leakage to 3.0 times of controls (±1.1 ; p=0.059), whereas the re-engineered peptide demonstrated a significantly stronger rescue effect to levels near the control group (1 .1 ±0.6; p<0.001 ).

[00134] To further investigate the mechanism of this effect, our next hypothesis was that SEMA7A without RGD more effectively controls cytokine release from LPS-activated human neutrophils, thereby reducing vascular leakage (FIG. 4D). The fact that PLXNC1 is strongly expressed in neutrophils but only weakly expressed in endothelial cells (FIG. 4B) suggests that the re-engineered peptide acts via modulation of neutrophils rather than by direct targeting of endothelial cells. Human neutrophils were isolated from the whole blood of 5 donors. Cells were preincubated with either control peptide, unmodified SEMA7A peptide (50nM), or SEMA7A without RGD peptide (50nM) and then stimulated with LPS (100ng/ml) for 4h at 37°C. We customized a multiplex immunoassay to assess the protein level of 8 cytokines in the supernatant that are known to be released by activated neutrophils and to directly affect endothelial cell permeability.21 Indeed, SEMA7A without RGD demonstrated a stronger rescue effect on CCL4 (p<0.05), CXCL2 (p<0.05), and CCL20 (p<0.05) compared to the unmodified SEMA7A peptide, whereas the effect was comparable for CCL3 (p=0.465) (FIG. 4E). The other 4 cytokines were either below the limit of detection (CXCL1 and CXCL6) or none of the two peptides demonstrated a rescue effect (CXCL5 and CXCL8) (data not shown). These results reveal that a re- engineered SEMA7A peptide that only includes the interaction motif for PLXNC1 , but not the RGD motif, demonstrated a significantly stronger rescue effect on inflammation-induced vascular leakage and that this effect may at least partly be driven by reduced cytokine release from neutrophils.

Re-engineered SEMA7A peptides enhance anti-inflammatory activity

[00135] The vascular leakage experiments revealed that replacing the RGD motif improved the potency of the 19-mer SEMA7A peptide. We next hypothesized that a shorter peptide that only includes the PLXNC1 interaction motif would have a stronger rescue effect on infiltrating immune cells in the EIU mouse model. We created three shorter peptides with 15, 12, and 11 amino acids, all covering the PLXNC1 interaction motif, and found that the 1 1 -mer peptide demonstrated the strongest rescue effect in vivo (FIG. 5A). Our next goal was to further optimize the 11-mer peptide by introducing point mutations to create additional interaction with PLXNC1 . In the first version (11 - 01 ), we introduced a L276E mutation that targets K208 of PLXCN1 (FIG. 5B). The V278D mutation of the 11 -02 peptide targets the R213 of PLXNC1 and the S279D mutation of the 11 -03 peptide targets Q218 of PLXNC1. We found that the re-engineered peptide 11 -02 seemed to have the strongest rescue effect in vivo (FIG. 5B). To compare the potency of the re-engineered 11 -02 peptide in comparison to the unmodified 19-mer SEMA7A peptide, we tested the peptides at lower concentrations in the EIU mouse model. While 1 1 -02 and 19-mer SEMA7A were comparably strong at 160pM, the re-engineered 11 -02 peptide demonstrated a more than 6-fold stronger effect at 8pM compared to the 19-mer SEMA7A peptide (p<0.01 ) (FIG. 5C). We also compared the peptides to intravitreal injection of dexamethasone in a clinically used dose (1 mg/ml) revealing that the 19-mer SEMA7A peptide was comparable with steroid at 8 pM (p=0.537) whereas the 11-02 peptide was significantly more efficacious compared to steroid (p<0.001 ) (FIG. 5C). We also assessed the in vivo dose-response relationship for 11 -02 using the EIU mouse model. We tested seven different peptide concentrations between 20 nM and 160 pM (8 - 14 eyes per dose) and determined the in vivo half maximal effective concentration (EC50) to be 37.4 nM (95% Cl: 15.5 - 92.0) (FIG. 5D). These findings reveal that the re-engineered 1 1-02 peptide demonstrated a significantly stronger rescue effect on acute intraocular inflammation, both compared to the unmodified SEMA7A peptide and dexamethasone. The 11 -02 peptide represents a promising novel therapeutic approach to target acute inflammation. Methods

Patients and aqueous humor sample collection

[00136] AH liquid biopsies were collected from 34 patients with uveitis (active and inactive), endophthalmitis, and normal controls undergoing cataract surgery (Table S1 ). After the eye was prepped and draped for surgery, anterior chamber paracentesis was performed using a 30-gauge needle and at least 100 pl of undiluted AH were manually aspirated into a 1 ml syringe. Samples were transferred into a barcoded cryovial and immediately frozen on dry ice in the operating room.22 Samples were stored in a biobank at -80°C until further analysis. The study protocol was approved by the Institutional Review Board for Human Subjects Research (IRB) at Stanford University, was HIPAA compliant, and adhered to the tenets of the Declaration of Helsinki. All subjects underwent informed consent for study participation.

Animal study approval

[00137] All animal experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use committees of Stanford University. Specific pathogen-free 6-8 week-old female C57BL/6J mice were purchased from the Jackson laboratory and maintained in the Animal Care Facilities at Stanford University. Sema7a heterozygote knockout mice were purchased from the Jackson laboratory (Strain #: 005128)²³ and bread and maintained in the Animal Care Facilities at Stanford University. Genotyping was performed using the primers and the protocol provided by the Jackson laboratory.

Peptides

[00138] The following known peptides were used in this study: SEMA7A peptide (CRGDQGGESSLSVSKWNTF (SEQ ID NO:1 ) ²⁴²⁵) and control peptide (KLGFTYVTIRVTYQIRVAG (SEQ ID NO:2) ²⁵). The following novel peptides were generated in this study: SEMA7A peptide without RGD motif (CLGLQGGESSLSVSKWNTF, SEQ ID NO:3), 15-mer peptide (DOGGESSLSVSKWNT, SEQ ID NO:4), 12-mer peptide (QGGESSLSVSKW, SEQ ID NO:5), 11 - mer peptide (QGGESSLSVSK, SEQ ID NO:6), 1 1 -01 peptide (OGGESSESVSK, SEQ ID NO:7), 11 - 02 peptide (OGGESSLSDSK, SEQ ID NO:8), and 1 1 -03 peptide (OGGESSLSVDK, SEQ ID NO:9). Peptides were synthesized by Nai- Pin Lin, PhD, Division of Endocrinology and Diabetes, Department of Pediatrics, School of Medicine, Stanford University, by the Protein and Nucleic Acid (PAN) facility at Stanford University, and by Genscript (New Jersey, USA). Endotoxin-induced uveitis (EIU) mouse model

[00139] Mice were anesthetized via an intraperitoneal injection of a mixture of ketamine (10 pig/pl) and xylazine (0.2 pg/pl) at a dose of 10 pil per g of body weight. Intravitreal injections were performed with borosilicate glass microcapillary needles created in-house and a Femtojet 4i microinjector (Eppendorf, Hamburg, Germany) as described previously.263 pl of phosphate buffered saline (PBS) containing 200 ng lipopolysaccharide (LPS, from Escherichia coli, strain 01 11 :B4, Sigma-Aldrich, Missouri, USA, catalog #: L2630) and 1 pg of SEMA7A peptide (160pM, or an equimolar amount of the other peptides, unless otherwise specified). After 24h,27 mice were perfused with ice cold PBS containing heparin (3 ILJ/ml, Tocris Bioscience, UK, Catalog # 2812) and the eyes were enucleated and processed for further analysis as described below.

Histology

[00140] Murine eyes were obtained from the EIU mouse model, as described above. Immediately after enucleation, eyes were fixed in Excalibur’s Alcoholic z-fix for at least 24 hours (provided by Excalibur pathologies, Norman, USA), as previously described.²⁸ After fixation, the eyes were embedded in paraffin, sectioned at 10 pm, and placed on slides (VWR International, LLC, Radnor, PA, USA). The slides were air-dried and placed in a 60 °C oven overnight. The slides were cooled down, deparaffinized by water and stained with Hematoxylin (Mercedes Scientific, Lakewood Ranch, FL, USA) and Eosin (Fisher Scientific, Hampton, NH, USA). The resulting histological slices were then viewed and processed using a Zeiss microscope (AXIO Scope. A1 , Zeiss, Oberkochen, Germany) and Zen microscopy software. Infiltrate cells were manually quantified in the anterior chamber and the vitreous using Imaged (National Institutes of Health, Bethesda, Maryland) and its cell-counter plug-in.

ELISA

[00141] Murine eyes were obtained from the EIU mouse model, as described above. After enucleation, both eyes of each mouse were transferred to a 5 ml Eppendorf tube on ice containing 250 pl of RIPA buffer (ThermoFisher scientific, Massachusetts, USA) supplemented with 5mM EDTA and protease inhibitor cocktail (Roche). A disposable pellet pestle was then used to homogenize the eye. Homogenized solutions were incubated on ice for 30 minutes and were then centrifuged with maximum speed for 5 minutes to obtain the supernatant. A Quantikine high-sensitivity mouse TNF- alpha ELISA (Cat. #: MHSTA50, R&D Systems, Minnesota, USA) was used to quantify TNF-alpha protein levels. The ELISA assay was conducted according to a protocol provided by the supplier. Samples were run undiluted and as a duplicate. Results were normalized to total protein concentration as determined by BCA assay.

Flow cytometry

[00142] Murine samples were obtained from the EIU mouse model, as described above. Twenty-four hours after intravitreal injection of LPS, mice were perfused transcardially with ice-cold PBS containing 3 ILI/mL heparin. The eyes were enucleated, and eyeballs were dissected under a dissection microscope to isolate the iris/ciliary body and retina. Tissue samples were then digested for 30 minutes at 37°C with constant agitation using 1 mL of pre-warmed digestion buffer (DMEM, 2% FBS, 1 mg/mL collagenase VIII (Sigma Aldrich), and 0.5 mg/mL DNase I), and filtered through a 70pm cell strainer. Enzymes were then neutralized with 1 mL of complete medium (DMEM with 10% FBS). An additional 2 mL of FACS buffer was added, samples were centrifuged at 350 x g for five minutes, and samples were resuspended in FACS buffer with anti-CD16/32 (Biolegend, 101302) and Zombie NIR viability dye (Biolegend 423106) diluted 1 :50 in FACS buffer. Fluorescently conjugated antibodies were added, and incubated for 20 minutes at 4°C. The following antibodies were used (all 1 :50): CD11 b (563, Fisher #741242), CD11 c (737, Fisher #749039), MHCII (E450, Fisher #48-5321 - 82), TCR (71 1 , BD #563135), CD45 (750, Fisher #746947), Ly6g (PerCP, Biolegend #127654), SiglecF (PerCP 5.5, Biolegend #155525), CD64 (PE, Biolegend #139306), CD8 (Spark, Biolegend #303808), ckit (594, Biolegend #135128), CD3 (F640, Biolegend #100270), CD4 (F700, Biolegend #100484), GD (647, Biolegend #118134). Samples were washed in FACS buffer and resuspended in FACS buffer with 133 ng/mL DAPI before running on a Cytek Aurora spectral flow cytometer (Cytek) and analyzed using FlowJo software (Tree Star).

Retinal vascular leakage

[00143] Retinal vascular leakage was assessed in the ElU-mouse model using a modified version of a previously described protocol.²⁹ Briefly, intravitreal injections were performed as described above. After 24h, a fluorescent tracer was injected intraperitoneal (2mM in PBS, 200pl, fluorescein isothiocyanate-dextran, Sigma, FD4) and was allowed to circulate for 2h. The optimal dose and circulation time of the fluorescent tracer were determined in optimization experiments (data not shown). 200pl PBS without tracer were injected intraperitoneal serving as a background control. After 2h, animals were anesthetized and perfused as described above and eyes were enucleated and stored in light protected tubes on ice. The eyeballs were carefully dissected using a dissection microscope and the retinas were isolated. Special attention was paid to isolate the entire retina in one piece. After the dissection, each retina was frozen and stored at -80°C in a light protected tube. The next day, retinal samples were thawed on ice and homogenized in 150pl RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA, catalog #: 89901 ) per retina using a 1.5ml pipette. Samples were incubated on ice for 30 minutes and were then centrifuged at 15,000g at 4°C for 20 min. The supernatant was transferred to a new light protected tube on ice. 50gl of each sample were transferred to a 96 well plate (black bottom, Thermo Fisher Scientific, Waltham, MA, USA, catalog #: 237108) and fluorescence was determined at 480/520nm. Each sample was measured in duplicates. For data analysis, the mean RFU value of 4 background control retinas (without fluorescent tracer, no treatment to the eye) was subtracted from each retina’s RFU value. Background corrected RFU values were then normalized to the control group.

Human neutrophil in vitro experiments

[00144] Peripheral blood (6 ml) was collected in EDTA tubes from 5 different healthy donors (female 38 years, female 60 years, female 64 years, male 38 years, and male 64 years) (kindly provided by the Stanford Blood Center, Palo Alto, CA, USA). Samples were kept at 4°C and processed within 4h after the blood draw. Human neutrophils were isolated using the EasySep Direct Human Neutrophil Isolation Kit (Stemcell Technologies, Vancouver, Canada, catalog #: 19666) following the manufacturer’s instructions, as previously described.³⁰

[00145] The live cell concentration was determined using the Countess II FL Automated Cell Counter (Life Technologies, Carlsbad, CA, USA). Neutrophils were resuspended in X-VIVO 15 Serum-free Hematopoietic Cell Medium (Lonza, Basel, Switzerland, catalog #: 04-418Q) at a concentration of 6.25 x 106 cells/ml. The following experiments were performed in a 96 well plate (flat bottom, Corning, Corning, NY, USA, catalog # 3596) in a total volume of 10Opil per well. 80gl of the neutrophil suspension were transferred to each well and the cells were preincubated with either control peptide, unmodified SEMA7A peptide (0.1 pg/ml, 50nM), or SEMA7A without RGD peptide (0.0978 pg/ml, 50nM) for 20 min at 37°C in a 5% CO2 humidified incubator. Cells were then stimulated with LPS (100 ng/ml) (from Escherichia coli, strain 0111 :B4, Sigma-Aldrich, Missouri, USA, catalog #: L2630) for 4h at 37°C in a 5% CO2 humidified incubator. The final neutrophil concentration was 5.0 x 106 cells/ml. After 4h, the plate was centrifuged at 1000 RPM (233g) for 5 minutes at room temperature and 80pl of cell culture supernatant were transferred to a new 96 well plate (round bottom, Corning, Corning, NY, USA, catalog # 3799) and the plate was immediately frozen at -80°C until further analysis. The samples were analyzed using a customized multiplex immunoassay (Human Luminex Discovery Assay, R&D Systems, Minneapolis, MN, USA, catalog #: LXSAHM), that included the following 8 cytokines: CCL3, CCL4, CCL20, CXCL1 , CXCL2, CXCL5, CXCL6, and CXCL8. These cytokines were selected because they are known to be released by activated neutrophils and to directly affect endothelial cell permeability.21 The assay was run by the Human Immune Monitoring Center (HIMC) at Stanford University (Palo Alto, CA, USA) following the manufacturer’s instructions. Samples were analyzed as technical duplicates and at two-fold dilution with PBS.

Aptamer-based proteomics assay

[00146] Human AH samples as well as murine whole eye tissue lysates were analyzed using an aptamer-based proteomic assay (SomaScan® Assay, 7k, SomaLogic) as previously described.^{31 32} Murine samples were obtained from the EIU mouse model, as described above with the following modification: after enucleation, each eye was transferred to a 5 ml Eppendorf tube on ice containing 250pl of Mammalian Protein Extraction Reagent (M-PER) (Thermo Scientific, Catalog #: 78501 ) supplemented with protease inhibitor cocktail (Roche). A disposable pellet pestle was then used to homogenize the eye. Homogenized solutions were incubated on ice for 30 minutes and were then centrifuged with maximum speed for 5 minutes to obtain the supernatant. Total protein concentration was determined using a BCA assay. An aliquot of 200 pl with a total protein concentration of 2000 pg/ml was prepared to be analyzed by the aptamer-based assay. Human AH samples with a minimum volume of 55 pl were analyzed without further preprocessing.

Bioinformatics

[00147] Aptamer-based assay data were normalized by Somalogic, as described previously.33 Normalized data were imported to R Studio (version 2022.02.0+443, R version 4.1.2). Aptamers’ target annotation and mapping to UniProt accession numbers as well as Entrez gene identifiers were provided by Somalogic. Only human protein targets were retained for subsequent analysis (7,289 out of the 7,596 aptamers). The estimated limit of detection (eLoD) was calculated for each aptamer using a ‘robust estimate’ method as previously described.³³

[00148] Briefly, it was calculated as the median plus 4.9 x median absolute deviation signal of three buffer samples. The eLoD was then subtracted from each intensity value of each aptamer in each sample to obtain the actual protein intensity above the detection limit and values below 0 were replaced by 0. For some proteins, Somalogic provides more than one aptamer. In these cases (611 of expressed proteins) only the aptamer with the highest intensity was retained. Differentially expressed proteins were determined using the limma package 34 with default parameters except using method = “robust” in ImFit. P-values were corrected for multiple testing using Benjamini- Hochberg adjustment implemented in the limma package. Proteins with Iog2 fold change (log2FC) >2 or <-2 and adjusted p value < 0.05 were considered as differentially expressed proteins (DEP). Heatmaps were created with the R package ComplexHeatmap (version 2.10.0).35 The z-score represents a gene’s or a protein’s expression in relation to its mean expression by standard deviation units. Other data visualization was done using the ggplot2 package (version 3.3.5). Functional enrichment analysis was performed with g rofiler as previously specified.^{36 37} Briefly, a list with all identified AH proteins ranked according to their intensity values was uploaded to the g Profiler website. The analysis was run with the following specifications: ordered query, filters on gene annotation data to Gene ontology biological processes and Reactome molecular pathways, no electronic GO annotations, size of functional category to min = 15 and max = 350. In addition, the background was customized to all proteins included in the aptamer-based assay.³⁷ Functional groups of enrichment results were determined and visualized using ClueGO38 in Cytoscape.³⁹

[00149] To investigate the cellular origin of AH proteins in the eye, we applied our approach, TEMPO, as previously reported.¹⁰ Briefly, published single cell RNA sequencing data from the human eye were reanalyzed. Data from the following tissue and cell types were used: cornea,⁴⁰ trabecular meshwork,⁴¹ iris/ciliary body,⁴² lens,⁴³ hyalocytes,⁴⁴⁴⁵ retina,⁴⁶ RPE/choroid, ⁴⁷ blood,⁴⁸ and liver cells.⁴⁹ Cell type annotations were extracted from the original publications. Mean expression for each cell type was calculated for each gene. Expression data were combined and normalized using DESeq2 ⁵⁰ in R.

[00150] Cell interactions were determined between all cell types in the human eye, based on the combined cell level gene expression data (see above). For this calculation, the tool LIANA (Ligandreceptor ANalysis framework) was applied,¹⁸ which combines 16 cell-cell communication inference resources and 7 methods and provides a consensus of the resources’ and methods' predictions. Only interactions with a consensus p-value < 0.05 and with a significant enrichment of the ligand on the protein level in AH were retained. Selected cell interactions were visualized using customized Circle plots ⁵¹ and customized dotplots.¹⁸

[00151] Aptamer-based assay data were also compared to previously published liquid chromatography mass- spectrometry datasets of normal AH 11 -15 and of AH from eyes with AMD,⁵² DR,⁵³ Coat’s disease,⁵⁴ and glaucoma,^55-58 as well as proteins identified using a proximity extension assay (Olink) in patients with uveal melanoma.⁵⁹ Lists of proteins were extracted from each publication. When gene symbols were provided, they were filtered for approved symbols using the HGNC (Human Genome Organization Gene Nomenclature Committee) database.⁶⁰ If no gene symbols were provided in the original publication, symbols were obtained from UniProt database61 and filtered for approved symbols using the HGNC database.60 The AH proteome was compared to the plasma proteome using published SomaScan data.¹⁷ Ten of the plasma samples were randomly selected for further analysis using the sample() function in R. The following samples were selected: X6085.3, X5594.8, X1645.7, X1235.13, X4853.1 , X1574.12, X5343.12, X1466.16, X5249.11 , X1726.5. eLoD corrected AH and plasma data were combined and normalized using the normalizeBetweenArrays function (method: scale) of the limma package.

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Claims

What is claimed is:

1. A method of treating intraocular inflammation or inflammation-induced vascular leakage in a subject, the method comprising administering a therapeutically effective amount of a semaphorin 7A (SEMA7A) peptide or peptidomimetic to the subject, wherein the SEMA7A peptide or peptidomimetic binds to plexin C1 (PLXNC1 ).

2. The method of claim 1 , wherein the SEMA7A peptide or peptidomimetic lacks an RGD motif.

3. The method of claim 1 , wherein the SEMA7A peptide or peptidomimetic comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9.

4. The method of any one of claims 1 -3, wherein the intraocular inflammation is acute.

5. The method of any one of claims 1 -3, wherein the intraocular inflammation is caused by an autoimmune disease or an infection.

6. The method of any one of claims 1 -3, wherein the subject has non-infectious uveitis or autoimmune uveitis.

7. The method of claim 6, wherein the subject has anterior uveitis, intermediate uveitis, posterior uveitis, or panuveitis.

8. The method of any one of claims 1 -7, wherein the SEMA7A peptide or peptidomimetic is administered intravitreally, intraocularly, juxtasclerally, subconjunctivally, intracamerally, or retrobulbarly.

9. The method of any one of claims 1 -7, wherein the SEMA7A peptide or peptidomimetic is administered locally to an eye of the subject at a site of inflammation.

10. The method of claim 9, wherein the SEMA7A peptide or peptidomimetic is administered locally to the vitreous, anterior chamber, iris, ciliary body, retina, or choroid.

11. The method of any one of claims 1-10, wherein multiple cycles of treatment are administered to the subject.

12. The method of claim 11 , wherein the SEMA7A peptide or peptidomimetic is administered daily or intermittently.

13. The method of any one of claims 1 -12, wherein the subject is a mammal.

14. The method of claim 13, wherein the mammal is human.

15. The method of any one of claims 1 -14, further comprising administering an additional anti-inflammatory agent or immunosuppressive agent.

16. A composition comprising a SEMA7A peptide or peptidomimetic for use in a method of treating intraocular inflammation or inflammation-induced vascular leakage.

17. The composition of claim 16, wherein the intraocular inflammation is acute.

18. The composition of claim 16, wherein the intraocular inflammation is caused by non- infectious uveitis or autoimmune uveitis.

19. The composition of any one of claims 16-18, wherein the SEMA7A peptide or peptidomimetic comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9.

20. The composition of any one of claims 16-19, further comprising a pharmaceutically acceptable excipient.

21. The composition of any one of claims 16-20, further comprising an additional antiinflammatory agent or immunosuppressive agent.

22. The composition of claim 21 , wherein the anti-inflammatory agent is a corticosteroid.

23. The composition of any one of claims 16-22, wherein the composition is formulated for intravitreal, intraocular, juxtascleral, subconjunctival, intracameral, or retrobulbar administration.

24. The composition of any one of claims 16-23, wherein the composition is formulated for administration locally to the vitreous, anterior chamber, iris, ciliary body, retina, or choroid of the eye.

25. Use of a SEMA7A peptide or peptidomimetic in the manufacture of a medicament or pharmaceutical composition for treating intraocular inflammation or inflammation-induced vascular leakage in a subject in need thereof, optionally in combination with an additional anti-inflammatory agent or immunosuppressive agent.

26. The use of claim 25, wherein the SEMA7A peptide or peptidomimetic comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9.

27. A method of suppressing an immune response of one or more immune cells in an eye of a subject, the method comprising administering an effective amount of a semaphorin 7A (SEMA7A) peptide locally to the eye of the subject, wherein the SEMA7A peptide or peptidomimetic binds to plexin C1 (PLXNC1 ).

28. The method of claim 27, wherein the one or more immune cells comprise a neutrophil, a macrophage, a hyalocyte, a dendritic cell, a monocyte, a lymphocyte, or a combination thereof.

29. The method of claim 27 or 28, wherein said administering the SEMA7A peptide or peptidomimetic decreases numbers of the one or more immune cells in the eye of the subject.

30. The method of any one of claims 27-29, wherein the one or more immune cells are in an iris, a ciliary body, a retina, or a choroid, or a combination thereof.

31 . The method of any one of claims 27-30, wherein the SEMA7A peptide or peptidomimetic comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9.

32. A method of reducing or preventing inflammation-induced vascular leakage in an eye of a subject, the method comprising administering an effective amount of a semaphorin 7A (SEMA7A) peptide locally to the eye of the subject, wherein the SEMA7A peptide or peptidomimetic binds to plexin C1 (PLXNC1 ).

33. The method of claim 32, wherein the SEMA7A peptide or peptidomimetic lacks an RGD motif.

34. The method of claim 32, wherein the SEMA7A peptide or peptidomimetic comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NOS:3-9.