Methods and compositions for treating diabetic retinopathy

Abstract

The present disclosure provides a method for treating diabetic retinopathy and ocular inflammatory diseases using anti-ceramide antibodies and antibody fragments. Also provided is a method for treating subjects who have previously been treated for diabetic retinopathy. In some embodiments, the present disclosure further provides a method for treating diabetic retinopathy using a single dose of an anti-ceramide antibody or its antigen-binding fragment.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 907,287, filed September 27, 2019, the contents of which are incorporated herein by reference in their entirety. Sequence Listing Statement The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. Computer readable format copy of the Sequence Listing: Filename: CERA-011_01WO_SeqList_ST25.txt, Recorded on: September 28, 2020, File size: 32.9 KB. FIELD OF THE DISCLOSURE The present disclosure relates to anti-ceramide compositions and methods of use thereof for treating diabetic retinopathy. [Background technology]

[0002] Diabetic retinopathy (DR) affects the blood vessels of the light-sensitive tissue lining the back of the eye, called the retina. It is the most common cause of blindness in patients with diabetes and the leading cause of visual impairment and blindness in working-age adults. Diabetic retinopathy can result in swelling of a part of the retina called the macula, a condition called diabetic macular edema (DME). Existing treatments for DR and DME include anti-VEGF immunotherapy, which has shown some efficacy in treating both neovascular diabetic retinopathy (DR) and diabetic macular edema (DME). However, several large-scale clinical studies have shown that approximately 40% of patients do not respond to anti-VEGF therapy. Furthermore, anti-VEGF treatment is intended for the very late stages of the disease, when complete recovery from retinal damage is difficult. There is an unmet need for safe and effective treatments for DR and DME. [Brief explanation of the drawings]

[0003] [Figure 1]FIG. 1 shows an exemplary experimental design for measuring the effect of intravitreal anti-ceramide scFv administration in a mouse model of diabetic retinopathy with ischemia-reperfusion (I / R) injury. [Figure 2A-2B] Figure 2 shows the results of intravitreal anti-ceramide scFv administration in retinal ischemia-reperfusion (I / R) injured mice in a mouse model of diabetic retinopathy. Figure 2A shows the changes in expression of TNFα, IL-1β, IL6, ICAM-1, VCAM-1, and MCP1 relative to cyclophilin A in the following order: control eyes (white bars), I / R eyes (black bars), and I / R eyes with anti-ceramide scFv administration (gray bars). Figure 2B shows retinal vascular permeability as indicated by fluorescence microscopy images and quantitative bar graph analysis in control eyes (left panel, left bar), I / R eyes (center panel, center bar), and I / R eyes with anti-ceramide scFv administration (right panel, right bar). Scale bars in the images represent 50 μm. [Figure 3] FIG. 1 shows an exemplary experimental design for measuring the effect of intravitreal anti-ceramide scFv administration in a mouse model of diabetes. [Figure 4] FIG. 1 shows the results of anti-ceramide scFv administration in diabetic rats. [Figure 5] FIG. 1 shows an exemplary experimental design for comparing the effects of intravitreal and systemic anti-ceramide scFv administration in a mouse model of diabetic retinopathy. [Figure 6] FIG. 1 shows an exemplary experimental design for comparing the effects of anti-ceramide scFv and DHA administration in a mouse model of diabetic retinopathy. [Figure 7] FIG. 1 shows acellular capillaries in DHA-treated control mice. Summary of the Invention

[0004] The present disclosure relates to a method for treating diabetic retinopathy, comprising administering an anti-ceramide antibody or antigen-binding fragment thereof to the eye. Also provided are methods for treating a subject who has previously received treatment for diabetic retinopathy. In some embodiments, the present disclosure further provides a method for treating diabetic retinopathy with a single dose of an anti-ceramide antibody or antigen-binding fragment thereof. Additionally, methods for treating diabetic retinopathy with two or more doses of an anti-ceramide antibody or antigen-binding fragment thereof spaced at least two weeks to at least one year apart are provided. Another aspect of the present disclosure relates to a method for treating an ocular inflammatory disease using an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the present disclosure provides a method of treating or preventing diabetic retinopathy in a subject in need thereof, comprising ocularly administering to the subject an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). In some embodiments, the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the limbus, intralimbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration. In some embodiments, the administration is intravitreal administration.

[0005] In some embodiments, the subject has undergone prior treatment for diabetic retinopathy. In some embodiments, the subject has not responded to prior treatment for diabetic retinopathy. In some embodiments, the prior treatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroids and anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered as a single dose. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least two weeks, at least three weeks, or at least four weeks apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at about two weeks to about four weeks apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, or at least eleven months apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at about one month to about six months apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least one year apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative phase of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative phase of diabetic retinopathy.

[0006] In some embodiments, the present disclosure provides a method for treating or preventing diabetic retinopathy in a subject in need thereof, comprising administering an anti-ceramide antibody or antigen-binding fragment thereof to the subject, wherein the subject has received prior treatment for diabetic retinopathy. In some embodiments, the subject has not responded to prior treatment for diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). In some embodiments, the administration is ocular administration. In some embodiments, the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the limbus, intralimbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration. In some embodiments, the ocular administration is intravitreal administration. In some embodiments, the prior treatment was vitrectomy, laser surgery, steroids, and / or anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

[0007] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered as a single dose. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least two weeks, at least three weeks, or at least four weeks apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at about two weeks to about four weeks apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, or at least eleven months apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at about one month to about six months apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least one year apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative phase of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative phase of diabetic retinopathy.

[0008] In some embodiments, the present disclosure provides a method of treating or preventing diabetic retinopathy in a subject, comprising administering a single dose of an anti-ceramide antibody or antigen-binding fragment thereof to the subject. In some embodiments, the present disclosure provides a method for treating or preventing diabetic retinopathy in a subject, comprising administering two or more doses of an anti-ceramide antibody or antigen-binding fragment thereof to the subject, wherein the two or more doses are separated by at least two weeks. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses at least two weeks, at least three weeks, or at least four weeks. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses at intervals of about two weeks to about four weeks. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, or at least 11 months. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses at intervals of about one month to about six months. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least one year apart.

[0009] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). In some embodiments, the administration is ocular administration. In some embodiments, the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the limbus, intralimbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration. In some embodiments, the ocular administration is intravitreal administration. In some embodiments, the subject has undergone prior treatment for diabetic retinopathy. In some embodiments, the subject has not responded to prior treatment for diabetic retinopathy. In some embodiments, the prior treatment has been vitrectomy, laser surgery, steroids, and / or anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

[0010] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative phase of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative phase of diabetic retinopathy. In some embodiments, the present disclosure provides a method of treating an ocular inflammatory disease, comprising administering an anti-ceramide antibody or antigen-binding fragment thereof to the eye. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). In some embodiments, the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the limbus, intralimbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration. In some embodiments, the ocular inflammatory disease is selected from the group consisting of retinal neovascularization, choroidal neovascularization, corneal neovascularization, macular degeneration, age-related macular degeneration, diabetic retinopathy, vitreous hemorrhage, retinal hemorrhage, choroiditis, neovascular glaucoma, choroidal disease, telangiectasia, retinal artery occlusion, retinal vein occlusion, chorioretinitis, epiretinal membrane, choroidal neoplasm, retinopathy of prematurity, cystoid macular edema, papilledema, recurrent ischemia, ocular hemorrhage, and proliferative vitreoretinopathy.

[0011] In some embodiments, the ocular administration is intravitreal administration. In some embodiments, the subject has undergone prior treatment for diabetic retinopathy. In some embodiments, the subject has not responded to prior treatment for diabetic retinopathy. In some embodiments, the prior treatment was a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroids and anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered as a single dose. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least two weeks, at least three weeks, or at least four weeks apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at about two weeks to about four weeks apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, or at least eleven months apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at about one month to about six months apart. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least one year apart.

[0012] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of an ocular inflammatory disease, hi some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of an ocular inflammatory disease. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1), an HCDR2 comprising the amino acid sequence of YNYPRDGSTKYNEKFKG (SEQ ID NO: 2), and an HCDR3 comprising the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3), L comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of RASKSISKYLA (SEQ ID NO: 4), an LCDR2 comprising the amino acid sequence of SGSTLQS (SEQ ID NO: 5), and an LCDR3 comprising the amino acid sequence of QQHNEYPWT (SEQ ID NO: 6). Hcomprises the amino acid sequence of SEQ ID NO: 7, and V L comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the 6B5 antibody. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the 6B5 scFv.

[0013] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a humanized 6B5 (h6B5) antibody. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is an h6B5 scFv. In some embodiments, the h6B5 antibody, scFv, or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises a heavy chain complementarity-determining region 1 (HCDR1) comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 and 43, an HCDR2 comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 44-47, and an HCDR3 comprising or consisting of the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3), and the VL comprises a light chain complementarity-determining region 1 (LCDR1) comprising or consisting of the amino acid sequence of RASKSISKYLA (SEQ ID NO: 4), an LCDR2 comprising or consisting of the amino acid sequence of SGSTLQS (SEQ ID NO: 5), and an LCDR3 comprising or consisting of the amino acid sequence of QQHNEYPWT (SEQ ID NO: 6). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1), and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYNEKFQG (SEQ ID NO: 44). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1), and HCDR2 comprises or consists of the amino acid sequence of YNYPREGSTKYNEKFQG (SEQ ID NO: 45). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1), and HCDR2 comprises or consists of the amino acid sequence of YNYPRDVSTKYNEKFQG (SEQ ID NO: 46). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1), and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYAEKFQG (SEQ ID NO: 47).In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43), and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYNEKFQG (SEQ ID NO: 44). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43), and HCDR2 comprises or consists of the amino acid sequence of YNYPREGSTKYNEKFQG (SEQ ID NO: 45). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43), and HCDR2 comprises or consists of the amino acid sequence of YNYPRDVSTKYNEKFQG (SEQ ID NO: 46). In some embodiments, HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43), and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYAEKFQG (SEQ ID NO: 47). In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55.In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 49, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 49, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 49, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 50, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 50, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 50, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55.In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 51, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 51, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 51, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 52, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 52, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 52, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55.In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 48, and the VL comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 48, and the VL comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 50, and the VL comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 50, and the VL comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 51, and the VL comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 52, and the VL comprises the amino acid sequence of SEQ ID NO: 53.

[0014] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V Hcomprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of GYTFTNYWMH (SEQ ID NO: 33), an HCDR2 comprising the amino acid sequence of AIYPGDSDTSYNQKFKG (SEQ ID NO: 34), and an HCDR3 comprising the amino acid sequence of GLYYGYD (SEQ ID NO: 35), L comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of KSSQSLIDSDGKTFLN (SEQ ID NO: 36), an LCDR2 comprising the amino acid sequence of LVSKLDS (SEQ ID NO: 37), and an LCDR3 comprising the amino acid sequence of WQGTHFPYT (SEQ ID NO: 38). H comprises the amino acid sequence of SEQ ID NO: 39, and V L comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the 2A2 antibody. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the 2A2 scFv.

[0015] In some embodiments, preventing diabetic retinopathy or ocular inflammatory disease comprises delaying the onset of diabetic retinopathy or ocular inflammatory disease. In some embodiments, one or more symptoms of diabetic retinopathy or ocular inflammatory disease are reduced in a subject compared to a control subject or compared to a subject before treatment with an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the one or more symptoms of diabetic retinopathy or ocular inflammatory disease are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemia-reperfusion injury, retinal leakage area, and occlusive breakdown. In some embodiments, the one or more symptoms of diabetic retinopathy or ocular inflammatory disease are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject before treatment with an anti-ceramide antibody or antigen-binding fragment thereof.

[0016] In some embodiments, the expression level of one or more inflammatory markers in the eye is reduced compared to the expression level in a control subject's eye or compared to the expression level in the subject's eye before treatment with an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the one or more inflammatory markers are selected from a cytokine, a growth factor, and an adhesion molecule. In some embodiments, the cytokine is selected from TNFα, IL-1β, IL-6, or MCP1. In some embodiments, the growth factor is VEGF. In some embodiments, the adhesion molecule is ICAM-1 or VCAM-1. In some embodiments, the expression level of one or more inflammatory markers in the subject's eye is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the expression level in a control subject's eye or compared to the expression level in the subject's eye before treatment with an anti-ceramide antibody or antigen-binding fragment thereof.

[0017] In some embodiments, one or more visual parameters are increased in the subject compared to the visual parameters of a control subject, or compared to the visual parameters of the subject before treatment with the anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the one or more visual parameters are selected from peripheral vision, night vision, color vision, distance vision, near vision, and visual clarity. In some embodiments, retinal vascular permeability in the subject's eye is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to retinal vascular permeability in a control subject's eye or compared to the expression level in the subject's eye before treatment with the anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the mean Early Treatment Diabetic Retinopathy Study (ETDRS) grade score in the subject group is reduced by at least 0.2, at least 0.5, at least 1, at least 1.5, at least 2, or at least 2.5 compared to the mean ETDRS grade score in a control subject group, or compared to the mean ETDRS grade score in the subject group before treatment with the anti-ceramide antibody or antigen-binding fragment thereof. DETAILED DESCRIPTION OF THE INVENTION

[0018] Overview The present disclosure relates to compositions and methods for treating diabetic retinopathy. In some embodiments, compositions of anti-ceramide antibodies and their antigen-binding fragments (e.g., scFvs) and methods of use in the treatment or prevention of diabetic retinopathy are provided. Such compositions and methods can be used in other treatments for diabetic retinopathy, for example, in patients who have previously failed anti-VEGF antibody therapy. Additionally, methods of administering anti-ceramide scFvs intravitreally are provided. definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. As used herein, the term "and / or" is used in this disclosure to mean either "and" or "or," unless otherwise indicated.

[0019] Throughout this specification, unless the context indicates otherwise, the term "comprise", or variations such as "comprises" or "comprising", will be understood to mean the inclusion of a stated element or integer, or group of elements or integers, but not the exclusion of other elements or integers, or groups of elements or integers. As used in this application, the terms "about" and "approximately" are used as equivalents. Numerals used in this application, whether about or not, are meant to cover any normal variations understood by one of ordinary skill in the relevant art. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 10% or less in either direction (greater or less) of the stated reference value, unless otherwise stated or clear from the context (except where such number exceeds 100% of the possible values ​​or falls below 0% of the possible values). The term "sample" refers to a biological composition (e.g., a portion of a cell or tissue) that is subjected to analysis and / or modification. In some embodiments, a sample is a "primary sample" in that it is obtained directly from a subject; in some embodiments, a "sample" is the result of processing a primary sample, for example, to remove certain components and / or to isolate or purify certain components of interest.

[0020] The term "subject" includes animals, such as mammals. In some embodiments, the mammal is a primate. In some embodiments, the mammal is a human. In some embodiments, the subject is a livestock animal, such as a cattle, sheep, goat, cow, or pig; or a domestic animal, such as a dog or cat. In some embodiments (e.g., particularly in research settings), the subject is a rodent (e.g., a mouse, rat, hamster), rabbit, primate, or pig, such as an inbred pig. The terms "subject" and "patient" are used interchangeably herein. In some embodiments, the subject may be a newborn, infant, or adult. Of particular interest are mammalian subjects. Mammalian species that can be treated with the present methods include dogs and cats; horses; cows; sheep, etc., and primates, particularly humans. Animal models, particularly small mammals (e.g., mice, rats, guinea pigs, hamsters, rabbits, etc.), can be used for experimental investigations. As used herein, the terms "treatment," "treating," or "ameliorating" refer to either therapeutic or prophylactic / preventative treatment. Treatment is therapeutic if at least one symptom of the disease in the individual receiving treatment improves, or if treatment can slow the worsening of progressive disease in the individual or prevent the onset of additional related diseases.

[0021] As used herein, the term "effective amount" refers to the minimum amount of a drug or composition required to produce a specific physiological effect. The effective amount of a specific drug can be expressed in various ways based on the nature of the drug, including mass / volume, number of cells / volume, particles / volume, (mass of drug) / (mass of subject), number of cells / (mass of subject), or particles / (mass of subject). The effective amount of a specific agent can also be expressed as the half-maximal effective concentration (EC), which refers to the concentration of the agent that produces a specific physiological response that is half the magnitude between the baseline level and the maximum response level. 50 ) can also be expressed as The term "antibody" refers to an immunoglobulin (Ig) molecule capable of binding to a specific target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, via at least one epitope recognition site located in the variable region of the Ig molecule. As used herein, the term encompasses intact polyclonal or monoclonal antibodies and antigen-binding fragments thereof. For example, a natural immunoglobulin molecule is composed of two heavy chain polypeptides and two light chain polypeptides. Each heavy chain polypeptide is linked to a light chain polypeptide by an interchain disulfide bond between the heavy and light chain polypeptides to form two heterodimeric proteins or polypeptides (i.e., proteins composed of two heterologous polypeptide chains). The two heterodimeric proteins are then linked by an additional interchain disulfide bond between the heavy chain polypeptides to form an immunoglobulin protein or polypeptide.

[0022] The term "antigen-binding fragment," as used herein, refers to a polypeptide fragment containing at least one complementarity-determining region (CDR) of an immunoglobulin heavy and / or light chain that binds to at least one epitope of an antigen of interest. In this regard, the antigen-binding fragment of an antibody described herein can contain one, two, three, four, five, or all six CDRs of the variable heavy (VH) and variable light (VL) chain sequences from an antibody that specifically binds to ceramide. Antigen-binding fragments include proteins containing portions of full-length antibodies, generally the antigen-binding or variable regions thereof, e.g., Fab, F(ab'), Fab', Fv fragments, minibodies, diabodies, single-chain antibodies (dAbs), single-chain variable fragments (scFvs), multispecific antibodies formed from antibody fragments, and other modified configurations of immunoglobulin molecules that contain an antigen-binding site or fragment of the required specificity. In certain embodiments of the present disclosure, antigen-binding fragments, rather than intact antibodies, are used to increase tissue or tumor penetration. In other embodiments, the antigen-binding fragment is further modified to increase serum half-life.

[0023] "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from a portion of an antibody capable of binding to cellular Fc receptors and / or the C1q component of complement, thereby mediating the antibody's effector functions. Fc refers to "fragment crystallizable," a fragment of an antibody that readily forms protein crystals. Distinct protein fragments, first described by proteolytic digestion, can define the overall structure of immunoglobulin proteins. As originally defined in the literature, the Fc region is a homodimeric protein comprising two polypeptides linked by disulfide bonds, each containing a hinge region, a CH2 domain, and a CH3 domain. However, more recently, the term has been applied to a single-chain monomeric component consisting of at least a portion of the CH3, CH2, and hinge, sufficient to form a disulfide-linked dimer with a second such chain. Therefore, depending on the context, the use of the terms "Fc region" or "Fc domain" herein refers to either the dimeric form or the individual monomers that combine to form the dimeric protein. For a review of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol. 31:169-217, 1994. As used herein, the term Fc domain includes naturally occurring sequence variants.

[0024] The term "immunoglobulin constant region" or "constant region" refers to a peptide or polypeptide sequence corresponding to or derived from part or all of one or more constant domains (e.g., CH1, CH2, CH3) of an immunoglobulin. In certain embodiments, the constant region does not include a CH1 domain. In certain embodiments, the constant domains making up the constant region are human. The terms "light chain variable region" (also called "light chain variable domain" or "VL") and "heavy chain variable region" (also called "heavy chain variable domain" or "VH") refer to the variable binding regions derived from antibody light and heavy chains, respectively. The variable binding regions are composed of discrete, well-defined subregions known as "complementarity-determining regions" (CDRs) and "framework regions" (FRs). The term "immunoglobulin light chain constant region" (also referred to as "light chain constant region" or "CL") is the constant region derived from an antibody light chain. The term "immunoglobulin heavy chain constant region" (also referred to as "heavy chain constant region" or "CH") refers to the constant region derived from an antibody heavy chain. Depending on the antibody isotype, the CH can be further divided into CH1, CH2, and CH3 domains (IgA, IgD, IgG), or CH1, CH2, CH3, and CH4 domains (IgE, IgM).

[0025] The term "F(ab)" refers to two protein fragments resulting from proteolytic cleavage of an IgG molecule with the enzyme papain. Each F(ab) comprises a covalently linked heterodimer of a VH chain and a VL chain and contains an intact antigen-binding site. Each F(ab) is a monovalent antigen-binding fragment. The term "Fab'" refers to a fragment derived from F(ab')2 and may contain only a portion of Fc. Each Fab' fragment is a monovalent antigen-binding fragment. The term "F(ab')2" refers to the protein fragment of IgG produced by proteolytic cleavage with the enzyme pepsin. Each F(ab')2 fragment contains two F(ab') fragments and is therefore a bivalent antigen-binding fragment. An "Fd fragment" comprises the VH and CH1 domains. "Fv fragment" refers to a non-covalent VH::VL heterodimer containing an antigen-binding site that retains most of the antigen recognition and binding ability of an intact antibody molecule, but lacks the CH1 and CL domains contained within the Fab. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096. In some embodiments, Fv fragments can be generated by preferential proteolytic cleavage of IgM, and rarely IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art.

[0026] A "dAb fragment" (Ward et al., Nature 341:544 546, 1989) comprises a VH domain. A "single-chain antibody" or "scFv" is a fusion protein of the variable regions of an immunoglobulin heavy chain (VH) and light chain (VL) linked with a short linker peptide of 10 to about 25 amino acids. The linker can be either glycine-rich for solubility, serine- or threonine-rich for solubility, and connects the N-terminus of the VH to the C-terminus of the VL, or vice versa. scFvs retain the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker. In this disclosure, any reference to an antibody or antibody fragment or its uses is intended to include scFv molecules and their uses. The term "minibody" refers to a fusion protein comprising an scFv linked to a CH3 domain, and is also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, for example, Ward, E.S. et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); PCT / US92 / 09965; WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; see S. Hu et al., Cancer Res., 56, 3055-3061, 1996).

[0027] The term "diabody" refers to bispecific antibodies in which the VH and VL domains are expressed in a single polypeptide chain with a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993) and Poljak et al., Structure 2:1121-23 (1994)). The term "nanobody" or "single-domain antibody" refers to an antigen-binding fragment consisting of a single monomeric variable antibody domain. The Nanoclone method is a method for generating nanobodies against desired targets based on automated high-throughput selection of B cells. (See WO 2006 / 079372). The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0028] The term "chimeric antibody," as used herein, refers to a monoclonal antibody in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as the desired biological activity is exhibited. The term "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of immunoglobulin heavy (VH) and light (VL) chains linked by a short linker peptide of 10 to approximately 25 amino acids. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. The linker can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Numerous methods have been described for identifying chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules, which fold into a three-dimensional structure substantially similar to that of the antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al. and U.S. Patent No. 4,946,778 to Ladner et al.

[0029] As used herein, the term "CDR" refers to the "complementarity-determining region" of an immunoglobulin (antibody) molecule. CDRs are parts of the variable domain in an antibody that bind to its specific antigen. There are three CDRs per variable domain (i.e., CDR1, CDR2, and CDR3 in the light chain variable domain, and CDR1, CDR2, and CDR3 in the heavy chain variable domain). Within the variable domain, CDR1 and CDR2 are found in the variable (V) region of the polypeptide chain, and CDR3 shows the greatest variability because it is encoded by VJ recombination in the light chain region and VDJ in the heavy chain region. An "isolated antibody" is an antibody that is (1) free from naturally associated components, including other naturally associated antibodies, that naturally accompany it; (2) free from other proteins from the same species; (3) expressed by a heterologous cell; or (4) not occurring in nature. The term "human antibody" includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In preferred embodiments, all of the variable and constant domains are derived from human immunoglobulin sequences (fully human antibodies). These antibodies can be prepared in a variety of ways, as described below.

[0030] As used herein, the term "humanized" refers to an antibody or antigen-binding fragment thereof derived from a non-human species that retains the antigen-binding properties of the original non-human antibody. In some embodiments, antibody binding fragments (e.g., light and heavy chain variable regions, Fab, scFv) are humanized. Non-human antigen-binding fragments can be engineered using techniques such as CDR grafting (Jones et al., Nature 321:522 (1986)) and modifications thereof, such as "reshaping" (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al., Bio / Technol 1991 9:266-271), hyperchimerization (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol 148:1149-1154), and techniques known as "veneering" (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies." In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312). When derived from non-human sources, other regions of the antibody, such as the hinge region and constant region domains, can also be humanized.

[0031] As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that generally do not produce allergic or other serious side effects when administered using routes known in the art. Molecular entities and compositions approved by federal or state regulatory agencies or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, more particularly humans, are considered "pharmaceutically acceptable." The terms "prevent," "prophylaxis," and "prophylactically" refer to the administration of a compound, e.g., an anti-ceramide antibody or antigen-binding fragment thereof, before the onset of a disease (e.g., before the onset of certain symptoms of a disease). Preventing a disease includes reducing the likelihood of a disease occurring, delaying the onset of a disease, ameliorating long-term symptoms, or slowing the eventual progression of a disease. As used herein, the term "specifically binds" means to bind to a molecule that binds to at least 10 5 M -1 This refers to the ability of an antibody or antigen-binding fragment thereof to bind to a target antigen with a binding affinity (Ka) of at most 100 kJ, while not significantly binding to other components or antigens present in the mixture. References herein to anti-ceramide antibodies refer to antibodies or antigen-binding fragments thereof that specifically bind to ceramide.

[0032] As used herein, the term "sequence identity" refers to the relationship between two or more polynucleotide sequences or two or more polypeptide sequences.If a position in one sequence is occupied by the same nucleic acid base or amino acid residue at the corresponding position in the comparison sequence, the sequences are said to be "identical" at that position.The percent sequence identity is calculated by determining the number of positions where the same nucleic acid base or amino acid residue exists in both sequences to obtain the number of identical positions.The number of identical positions is then divided by the total number of positions within the comparison window and multiplied by 100 to obtain the percent sequence identity.The percent sequence identity is determined by comparing two optimally aligned sequences over the comparison window. A comparison window for polynucleotide sequences can be, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more nucleic acids in length. A comparison window for polypeptide sequences can be, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids in length. To optimally align sequences for comparison, the portion of the polynucleotide or polypeptide sequence within the comparison window can contain additions or deletions, called gaps, while the reference sequence is held constant. An optimal alignment is one that produces the greatest possible number of "identical" positions between the reference and comparison sequences, even if it contains gaps.The percent "sequence identity" between two sequences can be determined using the version of the program "BLAST 2 Sequences" available from the National Center for Biotechnology Information as of September 1, 2004, which incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing "BLAST 2 Sequences," the default parameters as of September 1, 2004, can be used for other required parameters, including, but not limited to, word size (3), open gap penalty (11), extended gap penalty (1), gap dropoff (50), expectation value (10), and matrix options. Two nucleotide or amino acid sequences are considered to have "substantially similar sequence identity" or "substantial sequence identity" if the two sequences have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to each other.

[0033] "Angiogenesis" refers to the formation of new blood vessels. As used herein, the term "eye" or "to the eye" refers to the administration of a drug to the eye and surrounding tissues.For example, in some embodiments, ocular administration comprises topical administration (for example, administration to the surface of the eye, such as the sclera), intraocular administration, subconjunctival administration (such as under the bulbar conjunctiva or under the inner layer of the conjunctiva of eyelid administration), intracameral administration, injection into the anterior chamber through the limbus, intralimbic administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space or intravitreal administration.

[0034] Anti-ceramide antibodies, antibody fragments, and derivatives The present disclosure relates to anti-ceramide antibodies and antigen-binding fragments thereof for treating diabetic retinopathy. Ceramides are a family of waxy lipid molecules. They are composed of sphingosine and fatty acids. Ceramide is found in high concentrations in the plasma membrane of eukaryotic cells because it is a component lipid of sphingomyelin, one of the major lipids in lipid bilayers. Ceramide is involved in various cell signaling pathways, including regulating cell differentiation, proliferation, and programmed cell death (PCD). As a bioactive lipid, ceramide is involved in various physiological functions, including apoptosis, cell growth arrest, differentiation, cellular senescence, cell migration, and adhesion. The role of ceramide and its downstream metabolites has also been suggested in many pathological conditions, including cancer, neurodegeneration, diabetes, microbial pathogenesis, obesity, and inflammation.

[0035] The sequences and properties of exemplary anti-ceramide antibodies are also disclosed in U.S. Patent Application Publication Nos. 2010 / 0239572 and 2017 / 0335014, each of which is incorporated herein by reference. Exemplary anti-ceramide antibody sequences are shown in Table 1. However, any anti-ceramide antibody or antigen-binding fragment thereof can be used in accordance with the disclosed methods and uses.

[0036] [Table 1] TIFF0007822310000002.tif252170 TIFF0007822310000003.tif120170

[0037] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is humanized 6B5 (h6B5). In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. In some embodiments, the sequence of the h6B5 antibody is provided in U.S. Provisional Application No. 62 / 991,232, filed March 18, 2020, the contents of which are incorporated by reference in their entirety for all purposes. The sequences of each CDR of the h6B5 antibody or antigen-binding fragment thereof are disclosed throughout this specification and summarized in Table 2 below.

[0038] [Table 2]

[0039] In some embodiments, the anti-ceramide antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a recombinant antibody, or a synthetic antibody. In some embodiments, the anti-ceramide antigen-binding antibody fragment is any one of the above antigen-binding fragments. In some embodiments, the anti-ceramide antigen-binding antibody fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, an Fd fragment, a dAb fragment, a diabody, an scFv, or the like. In some embodiments, the anti-ceramide antibody and its antigen-binding fragments are produced using recombinant DNA technology. Procedures for expression and purification of recombinant proteins are well established in the art. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). In some embodiments, the scFv includes the CDR sequences and / or variable chain sequences of the 2A2, h2A2, 6C8, 7B10, 9H10, h6B5, or 6B5 antibody.

[0040] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the 2A2 antibody or antigen-binding fragment thereof described in U.S. Patent Application Publication No. 2010 / 0239572. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the 6B5 antibody or antigen-binding fragment thereof described in U.S. Patent Application Publication No. 2017 / 0335014. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the h6B5 antibody or antigen-binding fragment thereof of the present disclosure. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is an scFv. In some embodiments, the scFv comprises the CDR sequences of any of the antibodies disclosed in Table 1. In some embodiments, the scFv comprises the CDR sequences of 2A2. In some embodiments, the scFv comprises the CDR sequences of 6B5. In some embodiments, the scFv comprises the CDR sequences of h6B5. In some embodiments, the scFv comprises the variable heavy and light chain sequences of h2A2. In some embodiments, the scFv comprises the variable heavy and light chain sequences of 6B5. In some embodiments, the scFv comprises the variable heavy and light chain sequences of h6B5.

[0041] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof has any immunoglobulin isotype. Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. The IgG isotype is divided into subclasses in certain species, such as IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof contains one or more modifications in the Fc region. Certain modifications may provide desired effector functions or serum half-life. In some embodiments, with an appropriate Fc region, a naked antibody bound to a cell surface can induce cytotoxicity, for example, via antibody-dependent cellular cytotoxicity (ADCC), by recruiting complement in complement-dependent cytotoxicity (CDC), by recruiting nonspecific cytotoxic cells that express one or more effector ligands that recognize the bound antibody on the target cell and subsequently trigger phagocytosis of the target cell in antibody-dependent cell-mediated phagocytosis (ADCP), or by some other mechanism. Certain other Fc regions can be used if it is desirable to remove or reduce effector functions to minimize side effects or therapeutic complications. The Fc region of an antibody can be modified to increase binding affinity to FcRn and thus increase serum half-life. Alternatively, the Fc region can be conjugated to PEG or albumin to increase serum half-life, or some other conjugation that produces a desired effect.

[0042] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a detectable label or tag. Exemplary detectable labels include fluorescent tags, affinity tags, radioisotopes, luminescent markers, particle labels, chromophores, phosphorescent markers, and enzyme labels. Exemplary fluorescent labels include GFP, RFP, and YFP. Exemplary enzyme labels include horseradish peroxidase and alkaline phosphatase. Exemplary peptide tags include His-tags, MBP, and streptavidin. The means of detection will be determined by the label selected. The appearance of the label or its reaction product can be obtained visually if the label is particulate and accumulates at an appropriate level, or by using instruments such as spectrophotometers, luminometers, fluorometers, or by ELISA or Western blot.

[0043] diabetic retinopathy In some embodiments, the present disclosure provides methods and compositions for treating diabetic retinopathy. Diabetic retinopathy refers to a condition in which the retina is damaged by diabetes mellitus. Chronic hyperglycemia caused by diabetes is associated with damage to small blood vessels in the retina, leading to diabetic retinopathy. In diabetic retinopathy, leakage and bleeding of retinal blood vessels can lead to decreased vision. In the most advanced stages, new abnormal blood vessels grow on the surface of the retina, causing retinal scarring and cell loss. In some embodiments, the disclosed methods and compositions can be used to treat different stages of diabetic retinopathy, which can progress through a non-proliferative phase, also called early, and a proliferative phase, also called late.

[0044] In some embodiments, the present disclosure provides methods and compositions that can be used to treat diabetic retinopathy during the non-proliferative stage. Non-proliferative diabetic retinopathy (NPDR) can be mild, moderate, or severe. Mild NPDR is characterized by microaneurysms in retinal blood vessels. These microaneurysms can leak fluid into the retina. As the disease progresses to the moderate NPDR stage, retinal blood vessels become distorted and lose their ability to transport blood, resulting in characteristic changes in the appearance of the retina and may contribute to diabetic macular edema. In the severe NPDR stage, more blood vessels become blocked, cutting off the blood supply to areas of the retina. These areas then secrete pro-angiogenic growth factors. Ocular angiogenic molecules include VEGF, FGF, PIGF, TGF-alpha, TGF-beta, IGF, PDGF, MMP, HGF / SF, TNF-alpha, CTGF, IL-1, IL-8, MCP-1, leptin, integrins, and angiogenin. In some embodiments, the method is characterized by preventing or reducing one or more markers of diabetic retinopathy. In some embodiments, the method can prevent, reduce, inhibit, or lower retinal microaneurysms, retinal fluid leakage, diabetic macular edema, and / or levels of retinal pro-angiogenic growth factors. In some embodiments, one or more of VEGF, FGF, PIGF, TGF-alpha, TGF-beta, IGF, PDGF, MMP, HGF / SF, TNF-alpha, CTGF, IL-1, IL-8, MCP-1, integrins, and angiogenin are reduced by the method. In some embodiments, the present disclosure provides methods and compositions that can be used to treat diabetic retinopathy during the proliferative stage. Proliferative diabetic retinopathy (PDR) is the advanced stage of the disease. At this stage, proangiogenic growth factors secreted by the retina cause the growth of new blood vessels that proliferate along the inner surface of the retina and enter the vitreous gel. The new blood vessels are fragile and prone to leakage and bleeding. The associated scar tissue contracts, which can cause retinal detachment (i.e., separation of the retina from the underlying tissue), leading to permanent vision loss. In some embodiments, the present methods can prevent, reduce, inhibit, or decrease the level of retinal neovascularization, retinal hemorrhage, retinal scarring, retinal detachment, and vision loss.

[0045] In some embodiments, the present disclosure provides methods and compositions that can be used to prevent diabetic retinopathy by administering them to diabetic subjects before the onset of diabetic retinopathy, for example, before the onset of one or more symptoms thereof. Among the most consistent risk factors, the duration of diabetes is a strong predictor of the onset and progression of retinopathy. The prevalence of early-onset diabetes patients is estimated to be approximately 8% at 3 years, 25% at 5 years, 60% at 10 years, and 80% at 15 years. In some embodiments, the disclosed methods and compositions can be used to delay the onset of diabetic retinopathy, for example, for more than 3 years, more than 5 years, more than 10 years, or more than 15 years after the onset of diabetes. In some embodiments, the disclosed methods and compositions can be used to prevent diabetic retinopathy in subjects at risk of developing diabetic retinopathy, for example, subjects with one or more risk factors associated with the development of diabetic retinopathy.Hyperglycemia, hypertension, hyperlipidemia, and renal disease are significant risk factors.Being male, having a high severity of diabetes (as shown by comparing insulin and oral antidiabetic drugs alone or tablet-only with no treatment), having a high mean systolic blood pressure, and having a high hemoglobin A1c are additional factors that should be considered.

[0046] Symptoms and Detection In some embodiments, the method may affect one or more symptoms and / or detectable markers of diabetic retinopathy. The early, non-proliferative stage of diabetic retinopathy is typically asymptomatic. In some embodiments, the method and composition are intended for use in diabetic patients before the onset of diabetic retinopathy symptoms. The disease often progresses unnoticed until it affects vision. Retinal blood vessels bleed early in the disease, causing "floating" spots that may resolve spontaneously. Without prompt treatment, bleeding often recurs, increasing the risk of permanent vision loss. Diabetic macular edema can cause blurred vision. In some embodiments, the composition and method may reduce the incidence, severity, or level of floating spots, retinal hemorrhage, vision loss, and / or blurred vision. In some embodiments, the method may improve one or more parameters of vision, including, but not limited to, overall vision, peripheral vision, night vision, color vision, distance vision, near vision, and visual acuity.

[0047] Diabetic retinopathy and diabetic macular edema may be detected during a comprehensive eye exam, including a visual acuity test (a fundus examination to measure visual acuity at various distances), tomometry (measuring intraocular pressure), pupil dilation, and optical coherence tomography (OCT). During such an exam, a doctor may check for one or more of the following: changes in retinal blood vessels, such as new blood vessel formation, swelling, and bleeding; retinal blood vessel leakage, or warning signs of leaking blood vessels, such as fatty deposits, weakened blood vessel walls, and bulging blood vessel walls; swelling of the macula; changes in the lens, including changes in curvature or cataract formation; and damage to nerve tissue. In some embodiments, the compositions and methods can be used to treat a subject with any one of these symptoms of diabetic retinopathy. In some embodiments, the compositions and methods can prevent, treat, or ameliorate any one of these symptoms. If diabetic macular edema or severe diabetic retinopathy is suspected, fluorescein angiography can be used to check for damaged and leaking blood vessels. In this test, a fluorescent dye is injected into the bloodstream into a vein in the arm. Once the dye reaches the eye, an image of the retinal blood vessels is taken. In some embodiments, the compositions and methods can be used to prevent or reduce the incidence, prevalence, or severity of damaged and / or leaking blood vessels in the eye. Existing treatments for diabetic retinopathy include anti-VEGF therapy, steroids, laser surgery, and vitrectomy.

[0048] Anti-VEGF Injection Therapy. Anti-VEGF drugs are injected into the vitreous gel to block the action of the pro-angiogenic growth factor VEGF. Blocking VEGF can reverse abnormal retinal blood vessel growth and reduce fluid accumulation. Anti-VEGF drugs include Avastin® (bevacizumab), Lucentis® (ranibizumab), and Eylea® (aflibercept). Lucentis® (ranibizumab) and Eylea® (aflibercept) are approved by the U.S. Food and Drug Administration (FDA) for the treatment of diabetic macular edema. Avastin® (bevacizumab) is FDA-approved for the treatment of cancer but is commonly used to treat eye diseases such as diabetic macular edema. Most people require monthly anti-VEGF injections for the first six months of treatment. After that, injections are required less frequently: typically, three to four times in the first six months after treatment, about four times in the second year after treatment, two times in the third year, one time in the fourth year, and none in the fifth year. Recent studies have shown that anti-VEGF treatments are effective in treating diabetic macular edema and slowing the progression of diabetic retinopathy, including PDR. Anti-VEGF treatments are increasingly being used as first-line treatments for PDR. However, several large-scale clinical studies have shown that approximately 40% of patients do not respond to anti-VEGF therapy. Furthermore, anti-VEGF treatments are reserved for the very late stages of the disease, when complete recovery from retinal damage is difficult.

[0049] Laser Surgery. Focal / Grid Laser Surgery for the Macular Diabetics involves the application of several to hundreds of small laser burns to leaking blood vessels in the edematous area near the center of the macula. Laser burns for diabetic macular edema slow fluid leakage and reduce retinal swelling. This procedure is usually completed in a single session, although some patients may require more than one treatment. Focal / Grid Laser Surgery can be used in conjunction with anti-VEGF therapy. For example, focal / Grid Laser Surgery is sometimes performed before anti-VEGF injections, sometimes on the same day or several days after anti-VEGF injections, and sometimes only when diabetic macular edema does not adequately improve after six months of anti-VEGF therapy. PDR can also be treated with scatter laser surgery, also known as panretinal laser surgery or panretinal photocoagulation. The procedure involves creating 1,000 to 2,000 tiny laser burns in an area of ​​the retina away from the macula. These laser burns are intended to shrink abnormal blood vessels. The procedure can be completed in one session, but two or more sessions may be required. While central vision can be preserved, scatter laser surgery may result in a slight decrease in side (peripheral), color, and night vision. Scatter laser surgery is most effective before the fragile new blood vessels begin to bleed.

[0050] Corticosteroids. Corticosteroids may be used alone or in combination with other medications or laser surgery to treat diabetic macular edema. Corticosteroids can be injected or implanted into the eye. Ozurdex® (dexamethasone) implants are used short-term, while Iluvien® (fluocinolone acetonide) implants are used long-term. Both are biodegradable and release a sustained dose of corticosteroid to control diabetic macular edema. Vitrectomy. A vitrectomy is a surgical procedure to remove the vitreous gel in the center of the eye. This procedure is used to treat severe bleeding into the vitreous and is performed under local or general anesthesia. A clear salt solution is gently pumped into the eye through one or more ports to maintain intraocular pressure during surgery and to replace the removed vitreous. The same instruments used in a vitrectomy can also be used to remove scar tissue or repair a detached retina.

[0051] The role of ceramide and ASM in diabetic retinopathy Without wishing to be bound by any one theory, it is believed that the present treatment can downregulate retinal inflammatory signaling by selectively inhibiting ceramide in the retina through treatment of the eye with an anti-ceramide antibody or antigen-binding fragment thereof. Sphingolipids are major components of membrane microdomains, and ceramide-rich microdomains are thought to be a prerequisite for inflammatory cytokine signaling. Acid sphingomyelinase (ASM) and neutral sphingomyelinase (NSM) are key regulatory enzymes in sphingolipid metabolism, promoting sphingomyelin hydrolysis to proinflammatory ceramide. ASM is a key early responder in inflammatory cytokine signaling. The sphingomyelinase pathway is critical for inflammatory signaling in human retinal endothelial cells (HRECs), a resident vasculature affected by diabetic retinopathy. The inflammatory cytokines TNFα and IL-1β induce a rapid increase in cell adhesion molecule (CAM) expression and ASM and NSM activity in HRECs.

[0052] Previous studies have shown the importance of ASM activity in diabetic retinopathy, for example, using ASM- / - mouse models and ASM inhibitors such as DHA and imipramine.Such gene knockout models and anti-ASM inhibitor administration reduce retinal inflammatory markers.See Fox et al., Diabetes 2006;55(12):3573-80; Opreanu et al., Diabetes 2011;60(9):2370-8; Opreanu et al., Investigative Ophthalmology & Visual Science 2010;51(6):3253-63; and Chakravarthy et al., Stem Cells 2016;34:972-83, each of which is incorporated herein by reference in its entirety. However, ASM is an essential enzyme, and its deficiency or inhibition can cause Niemann-Pick disease and can be fatal.Thus, ASM gene knockout and direct inhibition of enzyme are not viable therapeutic strategies for the treatment of diabetic retinopathy.In contrast, the administration of anti-ceramide antibody and antigen-binding fragment herein provides the surprising result of selectively inhibiting the inflammatory markers, angiogenesis and other symptoms of diabetic retinopathy in the eye, without inducing the negative side effects associated with direct inhibition of ASM.

[0053] Additional disorders In some embodiments, the compositions and methods are used to treat diabetic retinopathy. In some embodiments, the compositions and methods are used to treat diabetic macular edema. In some embodiments, the compositions and methods are used to treat macular edema after retinal vein occlusion (RVO). In some embodiments, the compositions and methods are used to treat myopic choroidal neovascularization (mCNV). The present disclosure provides a method for treating diseases or disorders associated with underlying angiogenesis. Exemplary diseases and disorders of the eye include retinal neovascularization, choroidal neovascularization, corneal neovascularization, macular degeneration, age-related macular degeneration, diabetic retinopathy, vitreous hemorrhage, retinal hemorrhage, choroiditis, neovascular glaucoma, choroidal disease, telangiectasia, retinal artery occlusion, retinal vein occlusion, chorioretinitis, epiretinal membrane, choroidal neoplasm, retinopathy of prematurity, cystoid macular edema, papilledema, recurrent ischemia, ocular hemorrhage, and proliferative vitreoretinopathy. Pharmaceutical Compositions, Routes of Administration, Dosages, and Administration Schedules In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-ceramide antibody or antigen-binding fragment thereof for the treatment of diabetic retinopathy.

[0054] For administration, the antibodies or fragments (e.g., anti-ceramide antibodies and their antigen-binding fragments) of the present disclosure can be formulated as pharmaceutical compositions. The pharmaceutical composition may comprise (i) an anti-ceramide antibody or its antigen-binding fragment; and (ii) a pharmaceutically acceptable carrier, diluent, or excipient. Pharmaceutical compositions comprising an anti-ceramide antibody or its antigen-binding fragment, and / or scFv, can be formulated according to known methods for preparing pharmaceutically useful compositions, whereby a therapeutic molecule is combined in a mixture with a pharmaceutically acceptable carrier, diluent, or excipient. Suitable carriers, diluents, or excipients are known to those skilled in the art (see, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may further comprise one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, and the like. In some embodiments, the pharmaceutical composition may be formulated in a dosage form selected from the group consisting of oral unit dosage forms, intravenous unit dosage forms, intranasal unit dosage forms, suppository unit dosage forms, intradermal unit dosage forms, intramuscular unit dosage forms, intraperitoneal unit dosage forms, subcutaneous unit dosage forms, subdural unit dosage forms, sublingual unit dosage forms, intracerebral unit dosage forms, intracameral unit dosage forms, subconjunctival unit dosage forms, subtenon unit dosage forms, retrobulbar unit dosage forms, posterior juxtascleral unit dosage forms, and intravitreal unit dosage forms. In some embodiments, the pharmaceutical composition may be formulated in an intravitreal dosage form. Because anti-ceramide antibodies or antigen-binding fragments thereof are not expected to cross the blood-brain barrier, systemic administration routes are not expected to deliver anti-ceramide antibodies or antigen-binding fragments thereof to the eye.

[0055] The anti-ceramide antibodies and antigen-binding fragments thereof described herein can be administered to a subject by a variety of modes of administration, including, for example, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, oral, topical, intraocular, intracameral, subconjunctival, subtenon, retrobulbar, posterior suprascleral, administration to the suprachoroidal space (SCS), administration to the supracapillary space, and intravitreal routes of administration. In some embodiments, the anti-ceramide antibodies and antigen-binding fragments thereof, and pharmaceutical compositions thereof disclosed herein may be administered directly to the eye by any known route of administration, including topical (e.g., eye drops), topical ocular, intravitreal, intracameral, subconjunctival, subtenon, retrobulbar, and posterior scleral. Many possible delivery modes can be used, including but not limited to intraocular application or topical application. In one embodiment, application is intraocular, including but not limited to subconjunctival injection, intracapillary injection, injection into the anterior cavity via the temporal limb, intrastromal injection, intracorneal injection, subretinal injection, aqueous humor injection, sub-Tenon injection or sustained delivery device, or intravitreal injection (e.g., anterior, middle, or posterior vitreous injection). In one embodiment, application is topical, including but not limited to eye drops into the cornea. In some embodiments, the disclosed antibodies, antigen-binding fragments thereof, and compositions thereof are prepared for intravitreal administration. In some embodiments, the disclosed antibodies, antigen-binding fragments, and compositions thereof are administered intravitreally.

[0056] For prophylactic and therapeutic purposes, anti-ceramide antibodies and antigen-binding fragments thereof can be administered to a subject in a single bolus delivery, via continuous delivery over an extended period of time (e.g., continuous transdermal delivery), or in a repeated administration protocol (e.g., hourly, daily, weekly, monthly, or yearly). In some embodiments, the methods provided herein include administering a therapeutically effective dose of an anti-ceramide antibody or its antigen-binding fragment. A therapeutically effective dose, dosage, or amount, as defined above, refers to the minimum amount of an anti-ceramide antibody or its antigen-binding fragment required to produce a specific physiological effect, such as preventing or ameliorating one or more symptoms of diabetic retinopathy. Determining a therapeutically effective dose in this context is typically based on animal model studies followed by human clinical trials, and is guided by determining an effective dosage and administration protocol that significantly reduces the incidence or severity of diabetic retinopathy in model subjects. The effective dose of the compositions of the present disclosure varies depending on a number of different factors, including the means of administration, the target site, the patient's physiological condition, whether the patient is human or animal, other drugs administered, whether the treatment is preventive or therapeutic, and the specific activity of the composition itself and its ability to elicit the desired response in an individual. Typically, the dosing regimen is adjusted to provide an optimal therapeutic response, i.e., to optimize safety and efficacy.

[0057] In some embodiments, the dose of an anti-ceramide antibody or antigen-binding fragment thereof is about 0.1 μg to 100 mg / kg, or 1 μg / kg to about 50 mg / kg, or 10 μg to 5 mg / kg. In some embodiments, an effective amount of an anti-ceramide antibody or antigen-binding fragment thereof is about 1 μg / kg to about 20 mg / kg, about 10 μg / kg to about 10 mg / kg, or between about 0.1 and about 5 mg / kg. The anti-ceramide antibodies and antigen-binding fragments thereof described herein can also be administered at a dosage of about 0.001 to about 10 milligrams (mg) per kg of body weight, and can be administered in a single dose or in two or more doses. For administration to adult human patients, a therapeutically effective amount can be administered in doses ranging from 0.2 mg to 800 mg per dose, including, but not limited to, 0.2 mg / dose, 0.5 mg / dose, 1 mg / dose, 5 mg / dose, 10 mg / dose, 25 mg / dose, 100 mg / dose, 200 mg / dose, and 400 mg / dose, and one or more doses can be administered over the course of treatment. In some embodiments, the total daily dosage of the anti-ceramide antibodies and antigen-binding fragments thereof described herein can range from about 1 mg to about 2 g, about 100 mg to about 1.5 g, or about 200 mg to about 1200 mg.

[0058] In some embodiments, an anti-ceramide antibody, antigen-binding fragment thereof, or composition comprising the same may be formulated at a concentration of about 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL. The concentration may be 0.1-1 mg / mL, 1-5 mg / mL, 5-10 mg / mL, or 10-50 mg / mL. In some embodiments, the disclosed antibody, fragment, or composition may be administered intravitreally at a dose of about 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1 mg. The dose volume can be about 0.005 mL, 0.01 mL, 0.02 mL, 0.03 mL, 0.04 mL, 0.05 mL, 0.06 mL, 0.07 mL, 0.08 mL, 0.09 mL, or 0.1 mL.

[0059] The anti-ceramide antibodies and antigen-binding fragments thereof described herein can be administered at different times of the day. In some embodiments, a dose can be administered in the evening. In other embodiments, a dose can be administered in the morning. Dosage can be administered in single or multiple doses, including, for example, multiple times per week, once every two weeks, once per month, or once per year. In some embodiments, a single dose of anti-ceramide antibody or antibody fragment is administered to a subject in need thereof. In some embodiments, a patient can receive two or more doses of anti-ceramide antibody treatment spaced at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 1 year apart. In some embodiments, two or more doses may be administered to a patient in need thereof at intervals of about 1 week to about 2 weeks, about 2 weeks to about 4 weeks, about 1 month to about 2 months, about 2 months to about 4 months, about 1 month to about 6 months, about 6 months to about 1 year, or about 1 year to about 2 years. In some embodiments, administration may be irregular as indicated by monitoring clinical symptoms of the disorder.

[0060] The dosage of pharmaceutical compositions containing anti-ceramide antibodies and antigen-binding fragments thereof can be varied by the attending physician to maintain a desired concentration at the target site. High or low concentrations can be selected based on the delivery method. The anti-ceramide antibodies, or antigen-binding fragments thereof, can be administered at any time during the subject's lifetime. In some embodiments, administration occurs before the onset of diabetic retinopathy symptoms. In such embodiments, administration can be used as a prophylactic to prevent or delay the onset of diabetic retinopathy. In some embodiments, administration occurs during early disease. In some embodiments, administration occurs during late disease.

[0061] Treatment Methods and Uses In some embodiments, the present disclosure provides a method of treating, preventing, or ameliorating symptoms of an inflammatory eye disease in a subject, comprising administering to the subject a therapeutically effective amount of an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the present disclosure provides methods for preventing and / or treating diabetic retinopathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-ceramide antibody or anti-ceramide antibody fragment. Previous studies have suggested that the initial metabolic disturbance leading to inflammation and retinal vascular hyperpermeability involves activation of acid sphingomyelinase (ASM), a central enzyme in sphingolipid signaling, which converts sphingomyelin to ceramide. ASM- / - animals were protected from vascular degeneration in a retinal ischemia-reperfusion model, and chimeras with ASM- / - bone marrow transplanted into wild-type mice were also protected from diabetes-induced vascular damage. Several strategies have been implemented to inhibit ASM activity, including administration of docosahexaenoic acid (DHA)-rich fish oil and the tricyclic antidepressant desipramine. Both strategies have shown some efficacy in cell culture models. However, despite improving retinal health in diabetic animals, desipramine was poorly tolerated in long-term studies (see Example 8) and long-term high-dose DHA treatment caused retinal pathology in control animals (see Example 9). Furthermore, ASM is an essential enzyme, and its deficiency causes neurodegenerative diseases (Niemann-Pick syndrome). Therefore, direct inhibition of ASM is not a viable clinical strategy for treating DR. Anti-ceramide antibodies and their antigen-binding fragments are highly effective in binding monomeric ceramide produced on the surface of endothelial and other cells, thereby preventing ASM-induced ceramide-rich platform formation and resulting proinflammatory and apoptotic signaling without affecting essential lysosomal ASM function.

[0062] subject Subjects for treatment by the methods disclosed herein include those who have or are at risk of developing diabetic retinopathy. Subjects with diabetic retinopathy can have early or late stage disease. In some embodiments, the subject has previously received one or more treatments for diabetic retinopathy but has not responded to the previous treatment. In such embodiments, "failed response" refers to the previous treatment not improving and / or ameliorating one or more symptoms of diabetic retinopathy. In some embodiments, the previous treatment may not have resulted in an improvement in vision or retinal vascular health. In some embodiments, the previous treatment may have shown some results but may not have achieved the desired performance or may have ceased to show efficacy after a period of time. Existing treatments for diabetic retinopathy include therapeutic procedures such as vitrectomy and laser surgery, and therapeutic agents such as steroids and anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the subject may have partially responded to a previous diabetic retinopathy treatment: i.e., one or more symptoms of diabetic retinopathy have not improved sufficiently and / or the effects of the previous treatment have not lasted long enough. In some embodiments, the subject has previously received and failed to respond to anti-VEGF treatment, hi some embodiments, the anti-VEGF treatment is selected from Eylea® (aflibercept), Avastin® (bevacizumab), or Lucentis® treatment (ranibizumab).

[0063] Action Readout Treatment and / or prevention of diabetic retinopathy can be measured by various means.In some embodiments, treatment or prevention comprises treating or preventing one or more of the following in diabetic retinopathy in a subject in need thereof: apoptosis, inflammation, acellular capillary formation, angiogenesis, retinal endothelial cell death, retinal vascular permeability, ischemia-reperfusion injury, and occlusion destruction.The method comprises administering an effective amount of an anti-ceramide antibody or its antigen-binding fragment before the onset of diabetic retinopathy in a diabetic patient or after the onset of diabetic retinopathy in a diabetic patient. Methods of treating diabetic retinopathy with anti-ceramide antibodies or antigen-binding fragments thereof are provided. The effectiveness of such treatment can be characterized, assessed, measured, and / or monitored based on several parameters.

[0064] In some embodiments, the methods provided herein result in a reduction in apoptosis and / or endothelial cell death in the eye. Cell death can be monitored according to known methods. Exemplary methods for detecting cell death include nuclear staining techniques such as propidium iodide, Hoechst-33342, 4',6-diamidino-2-phenylindole (DAPI), and acridine orange-ethidium bromide staining. Non-nuclear staining techniques include Annexin V staining. In some embodiments, the methods provided herein reduce the level of apoptosis in an ocular sample subjected to the method by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control ocular sample. In some embodiments, the methods provided herein reduce the level of apoptosis and / or endothelial cell death by at least 10%. In some embodiments, the methods provided herein reduce the level of apoptosis and / or endothelial cell death by at least 20%. In some embodiments, the methods provided herein reduce the level of apoptosis and / or endothelial cell death by at least 30%. In some embodiments, the methods provided herein reduce the level of apoptosis and / or endothelial cell death by at least 50%. In some embodiments, the methods provided herein reduce the level of apoptosis and / or endothelial cell death by at least 70%.

[0065] In some embodiments, the methods provided herein prevent ischemia-reperfusion (IR) injury. Additional methods for detecting IR injury include fluorescein analysis, the fluorescent zinc 2,2'-dipicolylamine coordination complex PSVue® 794, 99mTc glucarate, and electroretinography. In some embodiments, the methods provided herein reduce the level of one or more inflammatory markers. In some embodiments, the one or more inflammatory markers are selected from TNFα, IL-1β, IL-6, VEGF, ICAM-1, VCAM-1, and MCP1. Inflammatory marker levels can be monitored via enzyme-linked immunosorbent assay (ELISA), Luminex, cytokine bead array, Proteo Plex, FAST Quant, etc. In some embodiments, the methods provided herein reduce the level of one or more inflammatory markers in an ocular sample treated with the method by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control ocular sample.

[0066] In some embodiments, the methods provided herein reduce the level of TNFα in an ocular sample subjected to the method by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control ocular sample. In some embodiments, the methods provided herein reduce the level of TNFα by at least 10%. In some embodiments, the methods provided herein reduce the level of TNFα by at least 20%. In some embodiments, the methods provided herein reduce the level of TNFα by at least 30%. In some embodiments, the methods provided herein reduce the level of TNFα by at least 50%. In some embodiments, the methods provided herein reduce the level of TNFα by at least 70%.

[0067] In some embodiments, the methods provided herein reduce the level of IL-1β in an ocular sample subjected to the method by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control ocular sample. In some embodiments, the methods provided herein reduce the level of IL-1β by at least 10%. In some embodiments, the methods provided herein reduce the level of IL-1β by at least 20%. In some embodiments, the methods provided herein reduce the level of IL-1β by at least 30%. In some embodiments, the methods provided herein reduce the level of IL-1β by at least 50%. In some embodiments, the methods provided herein reduce the level of IL-1β by at least 70%.

[0068] In some embodiments, the methods provided herein reduce the level of IL-6 in an ocular sample subjected to the method by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control ocular sample. In some embodiments, the methods provided herein reduce the level of IL-6 by at least 10%. In some embodiments, the methods provided herein reduce the level of IL-6 by at least 20%. In some embodiments, the methods provided herein reduce the level of IL-6 by at least 30%. In some embodiments, the methods provided herein reduce the level of IL-6 by at least 50%. In some embodiments, the methods provided herein reduce the level of IL-6 by at least 70%.

[0069] In some embodiments, the methods provided herein reduce the level of VEGF in an ocular sample subjected to the method by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control ocular sample. In some embodiments, the methods provided herein reduce the level of VEGF by at least 10%. In some embodiments, the methods provided herein reduce the level of VEGF by at least 20%. In some embodiments, the methods provided herein reduce the level of VEGF by at least 30%. In some embodiments, the methods provided herein reduce the level of VEGF by at least 50%. In some embodiments, the methods provided herein reduce the level of VEGF by at least 70%. In some embodiments, the methods provided herein reduce the level of ICAM-1 in an ocular sample subjected to the method by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control ocular sample. In some embodiments, the methods provided herein reduce the level of ICAM-1 by at least 10%. In some embodiments, the methods provided herein reduce the level of ICAM-1 by at least 20%. In some embodiments, the methods provided herein reduce the level of ICAM-1 by at least 30%. In some embodiments, the methods provided herein reduce the level of ICAM-1 by at least 50%. In some embodiments, the methods provided herein reduce the level of ICAM-1 by at least 70%.

[0070] In some embodiments, the methods provided herein reduce VCAM-1 levels in an ocular sample subjected to the method by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control ocular sample. In some embodiments, the methods provided herein reduce VCAM-1 levels by at least 10%. In some embodiments, the methods provided herein reduce VCAM-1 levels by at least 20%. In some embodiments, the methods provided herein reduce VCAM-1 levels by at least 30%. In some embodiments, the methods provided herein reduce VCAM-1 levels by at least 50%. In some embodiments, the methods provided herein reduce VCAM-1 levels by at least 70%.

[0071] In some embodiments, the methods provided herein reduce the level of MCP1 in an ocular sample subjected to the method by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control ocular sample. In some embodiments, the methods provided herein reduce the level of MCP1 by at least 10%. In some embodiments, the methods provided herein reduce the level of MCP1 by at least 20%. In some embodiments, the methods provided herein reduce the level of MCP1 by at least 30%. In some embodiments, the methods provided herein reduce the level of MCP1 by at least 50%. In some embodiments, the methods provided herein reduce the level of MCP1 by at least 70%.

[0072] In some embodiments, the methods provided herein reduce retinal vascular permeability, retinal neovascularization, or other symptoms of retinal health. In some embodiments, the methods provided herein may prevent typical symptoms of diabetic retinopathy in the retinal vasculature or prevent further deterioration. Vascular permeability and other measures of retinal vascular health can be measured, for example, by fluorescein angiography. In some embodiments, the methods provided herein reduce retinal vascular permeability in a subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control subject or compared to the subject before undergoing the disclosed method. In some embodiments, the methods provided herein reduce retinal vascular permeability by at least 10%. In some embodiments, the methods provided herein reduce retinal vascular permeability by at least 20%. In some embodiments, the methods provided herein reduce retinal vascular permeability by at least 30%. In some embodiments, the methods provided herein reduce retinal vascular permeability by at least 50%. In some embodiments, the methods provided herein reduce retinal vascular permeability by at least 70%.

[0073] In some embodiments, the methods provided herein improve one or more visual parameters or prevent the decline of one or more visual parameters.Visual parameters include poor night vision, blurred vision, floating spots, black spots or flashing lights in the visual field, fluctuating vision, color vision impairment, dark or empty areas in the visual field, vision loss, and sudden, severe, painless vision loss.In some embodiments, subjects who undergo treatment according to the methods provided herein may experience one or more of the following effects: improved vision, reduced vision loss, improved night vision, improved low-light vision, improved reading ability, improved peripheral vision, reduced spots in the visual field, reduced flashing lights in the visual field, reduced pain, and improved eye appearance.Many of these parameters can be monitored by routine eye examinations.

[0074] In some embodiments, the methods provided herein improve the Early Treatment Diabetic Retinopathy Study (ETDRS) grade score in subjects with diabetic retinopathy. In some embodiments, the subject is a human. The ETDRS grading scale uses three standard photographs: Standard Photograph 1 indicates mild microaneurysms and hemorrhages, Standard Photograph 2A indicates moderate microaneurysms and hemorrhages, and Standard Photograph 2B indicates severe microaneurysms and hemorrhages. In the ETDRS, a microaneurysm is defined as a red spot measuring 125 μm in its longest dimension with a sharp edge. This is distinct from a hemorrhage, which is defined as a red spot with an irregular edge and dimensions greater than 125 μm. To compare non-proliferative diabetic retinopathy (NPDR) with standard fundus photographs, the extent of the retina covered by microaneurysms and / or hemorrhages was used to describe NPDR. The grade scores for describing NPDR are as follows: Grade 0 = no microaneurysms or hemorrhages; Grade 1 = suspicious microaneurysms or hemorrhages; Grade 2 = clear microaneurysms or hemorrhages less than standard photograph 1; Grade 3 = microaneurysms or hemorrhages greater than or equal to standard photograph 1 but less than standard photograph 2A; Grade 4 = microaneurysms or hemorrhages greater than or equal to standard photograph 2A but less than standard photograph 2B; Grade 5 = microaneurysms or hemorrhages greater than or equal to standard photograph 2B. In some embodiments, the methods provided herein reduce the average ETDRS grade score in subjects with DR by at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.2, at least 1.5, at least 1.7, at least 2, at least 2.2, at least 2.5, at least 2.7, at least 3.0, at least 3.2, at least 3.5, at least 3.7, or at least 4, compared to control subjects or compared to subjects before receiving the disclosed methods. In some embodiments, the methods provided herein reduce the average ETDRS grade score in subjects with DR by at least 0.2. In some embodiments, the methods provided herein reduce the average ETDRS grade score in subjects with DR by at least 0.5.In some embodiments, the methods provided herein reduce the average ETDRS grade score in subjects with DR by at least 1. In some embodiments, the methods provided herein reduce the average ETDRS grade score in subjects with DR by at least 1.5. In some embodiments, the methods provided herein reduce the average ETDRS grade score in subjects with DR by at least 2. In some embodiments, the methods provided herein reduce the average ETDRS grade score in subjects with DR by at least 2.5. In some embodiments, the subject is a human.

[0075] In some embodiments, the method provided herein prevents diabetic retinopathy. This method can be administered to patients at risk of developing diabetic retinopathy. In such subjects, the prevention of diabetic retinopathy can be monitored by maintaining vision or the absence of typical characteristics of diabetic retinopathy. For example, subjects who receive prophylactic administration of anti-ceramide antibody or its antigen-binding fragment may not experience or may experience a reduction in the incidence of one of the following symptoms: microaneurysms, hemorrhage, intraretinal microvascular abnormalities, venous beading, cotton wool spots, the formation of new blood vessels (neovascularization) in other locations and in the optic nerve, fibrous proliferation in other locations and in the optic nerve, preretinal and vitreous hemorrhage, retinal detachment due to the formation of scar tissue, glaucoma, decreased night vision, blurred vision, floating spots, black spots or flashing lights in the visual field, fluctuating vision, color vision impairment, dark or empty areas in the visual field, vision loss, sudden, severe, painless vision loss, traction retinal detachment, macular edema, venous dilation, and intraretinal microvascular abnormalities. In some embodiments, the methods provided herein prevent or reduce macular edema, which can be seen under slit lamp examination as a bulging and blurring of the retinal layers.

[0076] In some embodiments, the methods provided herein delay the onset of diabetic retinopathy. Thus, the disclosed methods can be used to delay the average onset of diabetic retinopathy by more than 5 years, more than 10 years, more than 11 years, more than 12 years, more than 13 years, more than 14 years, more than 15 years, more than 16 years, more than 17 years, more than 18 years, more than 19 years, or more than 20 years after the initial diagnosis of diabetes. In some embodiments, the disclosed methods can be used to reduce, improve, lessen the severity, or reverse one or more symptoms of diabetic retinopathy. In some embodiments, methods of treating diabetic retinopathy with an anti-ceramide antibody or antigen-binding fragment thereof can reduce, improve, lessen the severity, or reverse one or more of the following symptoms: microaneurysms, hemorrhages, intraretinal microvascular abnormalities, venous beading, cotton wool spots, the formation of new blood vessels (neovascularization) elsewhere in the optic nerve, fibrous proliferation elsewhere in the optic nerve, preretinal and vitreous hemorrhage, retinal detachment due to scar tissue formation, vision loss, glaucoma, decreased night vision, blurred vision, floating spots, black spots or flashing lights in the visual field, sudden, severe, painless vision loss, traction retinal detachment, macular edema, venous dilation, and intraretinal microvascular abnormalities.

[0077] In some embodiments, the disclosed methods for treating diabetic retinopathy affect one or more parameters of the retinal vasculature, including, but not limited to, permeability, NFkB levels, inflammatory marker levels, apoptosis incidence, sphingolipid metabolism, and re-endothelialization. In some embodiments, the methods for treating diabetic retinopathy disclosed herein reduce retinal vascular permeability. Retinal vascular permeability can be monitored via fluorescence, tracer dyes, optical examination, and the like. In some embodiments, the methods for treating diabetic retinopathy disclosed herein reduce NFkB and / or other inflammatory marker levels in the retinal vasculature. As discussed above, inflammatory cytokine levels can be measured by conventional means (e.g., ELISA). In some embodiments, the methods for treating diabetic retinopathy disclosed herein reduce the occurrence of apoptosis in the retinal vasculature. As discussed above, apoptosis can be measured by conventional means, such as nuclear and non-nuclear staining techniques. In some embodiments, the methods for treating diabetic retinopathy disclosed herein inhibit or downregulate sphingolipid metabolism. In some embodiments, the methods of treating diabetic retinopathy disclosed herein can increase re-endothelialization of healthy tissue. [Example]

[0078] Example 1 Anti-ceramide scFv inhibits I / R inflammation and retinal vascular permeability in a mouse model of diabetic retinopathy. As shown in Figure 1, a murine ischemia-reperfusion (I / R) model of diabetic retinopathy was used to simulate the damaging effects of diabetic retinopathy on the retinal vasculature. Retinal ischemia-reperfusion (I / R): Each mouse had one I / R eye and one uninjured control eye. Retinal I / R was induced by temporarily increasing intraocular pressure (IOP) to 90 mmHg as follows: Male C57BL / 6J mice weighing 25–30 g were anesthetized. The anterior chamber of one eye was cannulated with a 30-gauge needle attached to a saline infusion line. Intraocular pressure was measured in the mouse eye using a handheld tonometer (TONO Pen; Medtronic Solan, Jacksonville, FL), and IOP was adjusted to 80–90 mmHg with a pressure injector (Infu-surg; Ethox Corp., Buffalo, NY). The other eye of the same animal served as a control. The duration of ischemia was 90 min in each mouse. After ischemia, the needle was removed, IOP was normalized, and retinal circulation reflow was visually recorded. Animals were sacrificed at different times after I / R injury. Retinas were isolated 2 or 7 days after retinal I / R.

[0079] Vehicle control or anti-ceramide scFv administration: 24 hours before I / R, control mice were injected with vehicle (phosphate-buffered saline) and test mice were injected intravitreally with 1 μL of 1.73 mg / mL anti-ceramide scFv 6B5. Inflammatory cytokine levels: 48 hours after I / R, the expression levels of inflammatory markers TNFα, IL-1β, IL6, ICAM-1, VCAM-1, and MCP1 were measured using quantitative PCR analysis. Further measurements that may be performed: In comparative experiments using this model, measurements of permeability, inflammatory markers, and sphingolipid metabolism will be performed after 48 hours, and after 7 days, measurements of apoptosis, acellular capillaries, and sphingolipid metabolism will be performed, as shown in Figure 2A-Figure 2B.

[0080] Retinal vascular permeability: Retinas were isolated 48 hours after I / R. Briefly, mice were injected with FITC-albumin (0.5 mg in 100 μL PBS) (Sigma-Aldrich, St. Louis, MO). Two hours later, blood was collected from each mouse and centrifuged to obtain plasma; animals were perfused with 1% formaldehyde and denucleated. Retinas were removed, flat-mounted with four slits, and placed on glass slides using Fluoromount mounting medium (Sigma-Aldrich, St. Louis, MO). Images were acquired using an Olympus FluoView 1000 scanning laser confocal microscope, and at least five different fields were selected to collect images for each sample. Retinas were mechanically disrupted and removed by centrifugation. FITC-albumin in the supernatant was quantified using a spectrofluorometer and normalized to plasma fluorescence (Kielczewski et al., 2011).

[0081] result As shown in Figure 2A, cytokine expression in anti-ceramide-treated I / R eyes was lower than that in untreated I / R eyes for each of the markers tested (TNFα, IL-1β, IL6, ICAM-1, VCAM-1, and MCP1). The observed decreases in TNFα, IL6, ICAM-1, and MCP1 were statistically significant. Figure 2B shows clear differences in retinal vascular permeability, as demonstrated by fluorescence microscopy images and quantitative analysis. Permeability was significantly higher in untreated I / R eyes than in either control or anti-ceramide-treated I / R eyes. This example demonstrates that a single intravitreal injection of anti-ceramide scFv at the onset of diabetes improves the ultimate outcome of diabetic retinopathy by preventing endothelial cell loss and subsequent retinal damage.

[0082] Example 2 Efficacy of anti-ceramide scFv administration in a rat model of diabetic retinopathy An STZ-induced model of diabetic retinopathy was employed in rats to simulate the damaging effects of diabetic retinopathy on the retinal vasculature, for example, as shown in Figure 3 . Animals and induction of STZ-induced diabetes: Male Sprague-Dawley rats weighing 237–283 g were made diabetic by a single intraperitoneal injection of 65 mg of streptozotocin (STZ) per kg of body weight. Rats were maintained on Harlan-Teklad laboratory chow (No. 8,640) and water ad libitum. Weight gain and blood glucose levels in control and STZ-induced diabetic groups were monitored biweekly. Vehicle control or anti-ceramide scFv administration: After confirmation of diabetes (hyperglycemia of 250 mg / dL or higher, 7–10 days after STZ injection), diabetic control rats received a vehicle PBS injection, and diabetic test rats received an intravitreal injection of 2 μL of anti-ceramide scFv 6B5 at 1.73 mg / mL. Non-diabetic control rats also received an injection of either vehicle control or anti-ceramide scFv.

[0083] Retinal vascular permeability analysis: Retinas were isolated 48 hours after I / R. Briefly, mice were injected with FITC-albumin (0.5 mg in 100 μL PBS) (Sigma-Aldrich, St. Louis, MO). Two hours later, blood was collected from each mouse and centrifuged to obtain plasma; animals were perfused with 1% formaldehyde and denucleated. Retinas were removed, flat-mounted with four slits, and placed on glass slides using Fluoromount mounting medium (Sigma-Aldrich, St. Louis, MO). Images were acquired using an Olympus FluoView 1000 scanning laser confocal microscope, and at least five different fields were selected to collect images for each sample. Retinas were mechanically disrupted and removed by centrifugation. FITC-albumin in the supernatant was quantified using a spectrofluorometer and normalized to plasma fluorescence (Kielczewski et al., 2011).

[0084] Additional measurements: In comparable experiments, experiments are performed in a mouse model or a db / db leptin-deficient diabetic model, as shown in Figure 3. Additionally, 6-8 weeks after diabetes induction in the STZ-induced model, the retinal vasculature of each animal is evaluated for permeability, NFkB and inflammatory markers, apoptosis, and sphingolipid metabolism, as shown in Figure 3. result The results contained in Figure 4 show that diabetic rats receiving intravitreal administration of anti-ceramide scFv did not develop vascular leakage indicative of diabetic retinopathy, thus indicating that a single intravitreal injection of anti-ceramide scFv is sufficient to eliminate hyperglycemia-induced mp diabetic retinopathy in a rat model of this condition.

[0085] Example 3 Intravitreal versus systemic administration of anti-ceramide scFv to treat diabetes-induced pro-inflammatory changes in DR Experiments are conducted to compare the effects of intravitreal anticeramide treatment versus systemic anticeramide treatment on diabetes-induced inflammation-induced changes in a DR mouse model. STZ-induced type 1 diabetic mice receive a single intravitreal or systemic (intravenous) anticeramide treatment with 6B5 scFv at the onset of diabetes. Six to eight weeks after STZ administration, control, diabetic, and anticeramide-treated diabetic animals are sacrificed. Retinas are isolated from each animal and profiled for inflammatory cytokines, growth factors, and adhesion molecules, and the expression and activity of endothelial ASM in the retina are measured. These measurements are performed using qPCR, Western blot, ESI-MS / MS, fluorescence microscopy, and immunogold electron microscopy experiments. An exemplary experimental protocol is shown in Figure 5. A single intravitreal injection of anti-ceramide scFv is expected to have a greater therapeutic effect in the development of DR. Additional experiments will be performed to evaluate the ability of a single intravitreal administration of anti-ceramide antibodies or their antigen-binding fragments to treat existing DR pathology.

[0086] Example 4 Intravitreal versus systemic administration of anti-ceramide scFv for the treatment of DR vascular dysfunction Experiments are performed to compare the effects of intravitreal versus systemic anti-ceramide treatment on retinal vascular permeability in a DR mouse model. In this example, the effect of 6B5 anti-ceramide scFv administration on diabetes-induced retinal vascular damage is measured. To assess early blood-retinal barrier disruption, mice receive a single intravitreal or systemic (intravenous) anti-ceramide scFv administration at the onset of diabetes. Six to eight weeks after diabetes induction, retinal vascular permeability is assessed using fluorescein as described in Example 2. An example of the experimental protocol is shown in Figure 5. A single intravitreal injection of scFv is expected to have a greater therapeutic effect on DR vascular dysfunction. Additional experiments will be performed to evaluate the ability of a single intravitreal administration of an anti-ceramide antibody or its antigen-binding fragment to treat existing DR pathology.

[0087] Example 5 Anti-ceramide scFv versus DHA or desipramine to reduce inflammation-induced changes in DR Experiments were conducted to compare the effects of a single intravitreal or systemic anti-ceramide scFv treatment with that of DHA-rich fish oil or desipramine treatment on diabetes-induced inflammatory changes in a DR mouse model. STZ-induced type 1 diabetic mice received a single oral gavage dose of DHA-rich fish oil, a single intravenous injection of desipramine, a single intravitreal injection of anti-ceramide scFv, or a systemic injection of anti-ceramide scFv at the onset of diabetes. Six to eight weeks after induction of diabetes with STZ, animals were sacrificed and retinas were isolated. Proinflammatory cytokines, growth factors, and adhesion molecules were profiled. Endothelial ASM expression and activity were measured. These measurements were performed in a series of qPCR, Western blot, ESI-MS / MS, fluorescence microscopy, and immunogold electron microscopy experiments, as described in Example 3. An exemplary experimental protocol is shown in Figure 6. A single intravitreal injection of anti-ceramide scFv is expected to have a greater therapeutic effect in the development of DR. Additional experiments will be performed to evaluate the ability of a single intravitreal administration of anti-ceramide antibodies or their antigen-binding fragments to treat existing DR pathology.

[0088] Example 6 Anti-ceramide scFv administration versus DHA or desipramine for treating DR vascular dysfunction Experiments were conducted to compare the effects of a single intravitreal or systemic anti-ceramide scFv treatment versus a single DHA-rich fish oil or desipramine treatment on retinal vascular permeability in a DR mouse model. Mice with STZ-induced type 1 diabetes were administered a single dose of DHA-rich fish oil by oral gavage, a single intravenous injection of desipramine, a single intravitreal injection of anti-ceramide scFv, or a systemic injection of anti-ceramide scFv at the onset of diabetes. Six to eight weeks after the induction of diabetes, retinal vascular permeability was assessed using fluorescein as described in Example 4. An example of the experimental protocol is shown in Figure 6. A single intravitreal injection of scFv is expected to have a greater therapeutic effect on DR vascular dysfunction. Additional experiments will be performed to evaluate the ability of a single intravitreal administration of an anti-ceramide antibody or its antigen-binding fragment to treat existing DR pathology.

[0089] Example 7 Anti-ceramide scFv administration inhibits stress-induced apoptosis Experiments are performed to demonstrate that anti-ceramide scFv is effective in human retinal endothelial cells (HRECs) in culture and inhibits stress-induced apoptosis under conditions in which anti-angiogenic drugs are ineffective. The mechanism of DR therapy with anti-ceramide scFv and anti-angiogenic drugs is distinct from previously characterized mechanisms. Without being limited by theory, it is believed that there is ongoing death and remodeling in diabetes in response to anti-ceramide that cannot be addressed by anti-VEGF treatment. Thus, while anti-VEGF can only prevent neovascularization, anti-ceramide antibody administration can prevent ongoing endothelial cell death, promote vascular recovery, and prevent hypoxia and subsequent neovascularization. To test whether anti-ceramide scFv works in vitro under conditions in which anti-angiogenic drugs are ineffective, HRECs are subjected to various stress conditions that induce ASM / ceramide-mediated apoptotic death (e.g., exposure to IL-1β and TNF cytokines or HO). HRECs are treated with either anti-VEGF inhibitors (anti-VEGFR2 DC101 Ab or the VEGFR TK inhibitor sorafenib) or anti-ceramide antibodies. Anti-ceramide scFv administration is expected to have a greater therapeutic effect in inhibiting endothelial cell death.

[0090] Example 8 Desipramine treatment of diabetic mice Diabetes was induced in male C57BL / 6J mice (20-25 g) by intraperitoneal injection of SZT (65 mg / kg) for 5 consecutive days. Control animals were injected with citrate vehicle (pH 4.5). Two weeks after the last injection, blood glucose levels were measured from a drop of blood drawn from the dorsalis pedis vein. Diabetes was confirmed by a blood glucose level >300 mg / dL. Weight loss, polyuria, water intake, and food intake were monitored daily. On the first day diabetes was confirmed, desipramine was added to the water approximately two weeks after the first SZT injection to initiate treatment. Desipramine administration had no effect on control animals. However, diabetic animals became dehydrated and lost more than 25% of their body weight within 2-4 days, at which point the experiment had to be discontinued in accordance with IACUC humane animal use regulations. Because diabetic animals have polydipsia and polyuria, it is possible that desipramine in the water may affect drinking, leading to dehydration. Therefore, this experiment was repeated using an IP injection of 2 mg / ml desipramine at a dose of 20 mg / kg. Similar to desipramine in the water treatment, control animals tolerated the drug well. However, diabetic animals exhibited abnormal tremors, became weak, stopped grooming, eating, and drinking, and lost more than 25% of their body weight within 5–7 days of daily injections, at which point the experiment had to be discontinued in accordance with IACUC humane animal use regulations.

[0091] Example 9 DHA treatment of wild-type mice Male Sprague-Dawley rats (237-283) were fed a control AIN-93M purified rodent diet containing 10% of the calorie intake as soybean oil containing 50.8% linoleic acid from Dyets Inc. (Bethlehem, PA), or a DHA-rich fish oil diet, in which half of the soybean oil or 5% of the calorie intake was replaced with Menhaden oil containing 10.26% DHA and 14.16% EPA. After 9 months on the diet, the rat retinal vasculature was isolated by trypsin digestion, and two independent researchers systematically counted acellular capillaries in the middle retina. As shown in Figure 7, DHA-rich fish oil treatment was harmful to control animals and caused the development of acellular capillaries. Although the health benefits of fish oil as a triglyceride-lowering agent are well accepted, the dose required for retinal effects is approximately 5% of caloric intake, corresponding to approximately 12 g of fish oil per day, three times the amount recommended for humans. High doses of DHA in humans, especially those with diabetes, are associated with many well-known side effects, including high blood sugar levels, excessive bleeding, impaired wound healing, frequent infections, gastrointestinal problems, and weight gain. Due to these concerns, DHA is not recommended for diabetic complications.

[0092] Further Numbering Embodiments Further embodiments of the present invention are provided in the following numbered embodiments: Embodiment 1. A method of treating or preventing diabetic retinopathy in a subject in need thereof, comprising ocularly administering to the subject an anti-ceramide antibody or antigen-binding fragment thereof. Embodiment 2. The method of embodiment 1, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). Embodiment 3. The method of embodiment 1 or 2, wherein the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the limbus, intralimbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0093] Embodiment 4. The method of any one of embodiments 1-3, wherein the administration is intravitreal administration. Embodiment 5. The method of any one of embodiments 1-4, wherein the subject has undergone prior treatment for diabetic retinopathy. Embodiment 6 The method of embodiment 5, wherein the subject has failed to respond to prior treatment for diabetic retinopathy. Embodiment 7. The method of embodiment 5 or 6, wherein the pretreatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroid and anti-vascular endothelial growth factor (VEGF) therapy. Embodiment 8. The method of embodiment 7, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept. Embodiment 9. The method of any one of embodiments 1 to 8, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered as a single dose. Embodiment 10. The method of any one of embodiments 1-8, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least two weeks, at least three weeks, or at least four weeks apart.

[0094] Embodiment 11. The method of any one of embodiments 1-8, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced about two weeks to about four weeks apart. Embodiment 12. The method of any one of embodiments 1-8, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, or at least 11 months. Embodiment 13. The method of any one of embodiments 1-8, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced about 1 month to about 6 months apart. Embodiment 14 The method of any one of embodiments 1-8, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least one year apart. Embodiment 15 The method of any one of embodiments 1-14, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of diabetic retinopathy. Embodiment 16 The method of any one of embodiments 1-14, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of diabetic retinopathy.

[0095] Embodiment 17 The method of any one of embodiments 1-14, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative phase of diabetic retinopathy. Embodiment 18 The method of embodiment 17, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative phase of diabetic retinopathy. Embodiment 19. A method for treating or preventing diabetic retinopathy in a subject in need thereof, comprising administering to the subject an anti-ceramide antibody or antigen-binding fragment thereof, wherein the subject has undergone prior treatment for diabetic retinopathy. Embodiment 20 The method of embodiment 19, wherein the subject has failed to respond to prior treatment for diabetic retinopathy. Embodiment 21 The method of embodiment 19 or 20, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). Embodiment 22. The method of any one of embodiments 19 to 21, wherein the administration is ocular administration. Embodiment 23. The method of embodiment 22, wherein the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the limbus, intralimbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0096] Embodiment 24 The method of embodiment 22 or 23, wherein the ocular administration is intravitreal administration. Embodiment 25. The method of any one of embodiments 19-24, wherein the prior treatment was vitrectomy, laser surgery, steroids, and / or anti-vascular endothelial growth factor (VEGF) therapy. Embodiment 26. The method of embodiment 25, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept. Embodiment 27 The method of any one of embodiments 19-26, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered as a single dose. Embodiment 28. The method of any one of embodiments 19-26, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least two weeks, at least three weeks, or at least four weeks apart. Embodiment 29. The method of any one of embodiments 19-26, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced about two weeks to about four weeks apart.

[0097] Embodiment 30. The method of any one of embodiments 19-26, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, or at least 11 months. Embodiment 31. The method of any one of embodiments 19-26, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced about 1 month to about 6 months apart. Embodiment 32 The method of any one of embodiments 19-26, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least one year apart. Embodiment 33 The method of any one of embodiments 19-32, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of diabetic retinopathy. Embodiment 34 The method of any one of embodiments 19-32, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of diabetic retinopathy. Embodiment 35 The method of any one of embodiments 19-32, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative phase of diabetic retinopathy.

[0098] Embodiment 36 The method of any one of embodiments 19-32, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative phase of diabetic retinopathy. Embodiment 37. A method for treating or preventing diabetic retinopathy in a subject, comprising administering to the subject a single dose of an anti-ceramide antibody or antigen-binding fragment thereof. Embodiment 38. A method for treating or preventing diabetic retinopathy in a subject, comprising administering two or more doses of an anti-ceramide antibody or antigen-binding fragment thereof to the subject, wherein the two or more doses are separated by at least two weeks. Embodiment 39. The method of embodiment 38, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least two weeks, at least three weeks, or at least four weeks apart. Embodiment 40. The method of embodiment 38, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced about two weeks to about four weeks apart. Embodiment 41. The method of embodiment 38, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, or at least 11 months.

[0099] Embodiment 42. The method of embodiment 38, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced about 1 month to about 6 months apart. Embodiment 43 The method of embodiment 38, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least one year apart. Embodiment 44. The method of any one of embodiments 37 to 43, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). Embodiment 45. The method of any one of embodiments 37 to 44, wherein the administration is ocular administration. Embodiment 46. The method of embodiment 45, wherein the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the limbus, intralimbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration. Embodiment 47. The method of embodiment 45 or 46, wherein the ocular administration is intravitreal administration. Embodiment 48. The method of any one of embodiments 37-47, wherein the subject has undergone prior treatment for diabetic retinopathy.

[0100] Embodiment 49. The method of embodiment 48, wherein the subject has failed to respond to prior treatment for diabetic retinopathy. Embodiment 50. The method of embodiment 48 or 49, wherein the prior treatment was vitrectomy, laser surgery, steroids, and / or anti-vascular endothelial growth factor (VEGF) therapy. Embodiment 51. The method of embodiment 50, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept. Embodiment 52 The method of any one of embodiments 37-51, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of diabetic retinopathy. Embodiment 53 The method of any one of embodiments 37-51, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of diabetic retinopathy. Embodiment 54 The method of any one of embodiments 37-51, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative phase of diabetic retinopathy. Embodiment 55 The method of any one of embodiments 37-51, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative phase of diabetic retinopathy.

[0101] Embodiment 56. A method of treating an inflammatory disease of the eye, comprising ocular administration of an anti-ceramide antibody or antigen-binding fragment thereof. Embodiment 57. The method of embodiment 56, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). Embodiment 58. The method of embodiment 56 or 57, wherein the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the limbus, intralimbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration. Embodiment 59. The method of any one of embodiments 56-58, wherein the ocular inflammatory disease is selected from the group consisting of retinal neovascularization, choroidal neovascularization, corneal neovascularization, macular degeneration, age-related macular degeneration, diabetic retinopathy, vitreous hemorrhage, retinal hemorrhage, choroiditis, neovascular glaucoma, choroidal disease, telangiectasia, retinal artery occlusion, retinal vein occlusion, chorioretinitis, epiretinal membrane, choroidal neoplasm, retinopathy of prematurity, cystoid macular edema, papilledema, recurrent ischemia, ocular hemorrhage, and proliferative vitreoretinopathy. Embodiment 60. The method of any one of embodiments 56-59, wherein the ocular administration is intravitreal administration.

[0102] Embodiment 61. The method of any one of embodiments 56-60, wherein the subject has undergone prior treatment for diabetic retinopathy. Embodiment 62. The method of embodiment 61, wherein the subject has failed to respond to prior treatment for diabetic retinopathy. Embodiment 63. The method of embodiment 61 or 62, wherein the pretreatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroid and anti-vascular endothelial growth factor (VEGF) therapy. Embodiment 64. The method of embodiment 63, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept. Embodiment 65. The method of any one of embodiments 56-64, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered as a single dose. Embodiment 66. The method of any one of embodiments 56-64, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least two weeks, at least three weeks, or at least four weeks apart. Embodiment 67. The method of any one of embodiments 56-64, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced about two weeks to about four weeks apart.

[0103] Embodiment 68. The method of any one of embodiments 56-64, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, or at least 11 months. Embodiment 69. The method of any one of embodiments 56-64, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced about 1 month to about 6 months apart. Embodiment 70. The method of any one of embodiments 56-64, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least one year apart. Embodiment 71 The method of any one of embodiments 56-70, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of an ocular inflammatory disease. Embodiment 72 The method of any one of embodiments 56-70, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of an ocular inflammatory disease.

[0104] Embodiment 73. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1), an HCDR2 comprising the amino acid sequence of YNYPRDGSTKYNEKFKG (SEQ ID NO: 2), and an HCDR3 comprising the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3), and L 73. The method of any one of embodiments 1 to 72, comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of RASKSISKYLA (SEQ ID NO: 4), an LCDR2 comprising the amino acid sequence of SGSTLQS (SEQ ID NO: 5), and an LCDR3 comprising the amino acid sequence of QQHNEYPWT (SEQ ID NO: 6). Embodiment 74.V H comprises the amino acid sequence of SEQ ID NO: 7, and V L 73. The method of any one of embodiments 1 to 72, wherein said nucleic acid sequence comprises the amino acid sequence of SEQ ID NO: 8. Embodiment 75. The method of any one of embodiments 1 to 74, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a 6B5 antibody. Embodiment 76. The method of any one of embodiments 1 to 74, wherein the anti-ceramide antibody or antigen-binding fragment thereof is 6B5 scFv.

[0105] Embodiment 77. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H73. The method of any one of embodiments 1 to 72, wherein the VL comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of GYTFTNYWMH (SEQ ID NO: 33), an HCDR2 comprising the amino acid sequence of AIYPGDSDTSYNQKFKG (SEQ ID NO: 34), and an HCDR3 comprising the amino acid sequence of GLYYGYD (SEQ ID NO: 35), and the VL comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of KSSQSLIDSDGKTFLN (SEQ ID NO: 36), an LCDR2 comprising the amino acid sequence of LVSKLDS (SEQ ID NO: 37), and an LCDR3 comprising the amino acid sequence of WQGTHFPYT (SEQ ID NO: 38). Embodiment 78.V H comprises the amino acid sequence of SEQ ID NO: 39, and V L 73. The method of any one of embodiments 1 to 72, wherein said vector comprises the amino acid sequence of SEQ ID NO: 40. Embodiment 79. The method of any one of embodiments 1 to 72 and 77 to 78, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a 2A2 antibody. Embodiment 80. The method of any one of embodiments 1-72 and 77-78, wherein the anti-ceramide antibody or antigen-binding fragment thereof is 2A2 scFv.

[0106] Embodiment 81. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), a) VH comprises a heavy chain complementarity determining region 1 (HCDR1) comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 and 43, an HCDR2 comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 44 to 47, and an HCDR3 comprising or consisting of the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3); and b) The method of any one of embodiments 1 to 72, wherein the VL comprises a light chain complementarity determining region 1 (LCDR1) comprising or consisting of the amino acid sequence of RASKSISKYLA (SEQ ID NO: 4), an LCDR2 (SEQ ID NO: 5) comprising or consisting of the amino acid sequence of SGSTLQS, and an LCDR3 (SEQ ID NO: 6) comprising or consisting of the amino acid sequence of QQHNEYPWT. Embodiment 82 The method of embodiment 81, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYNEKFQG (SEQ ID NO: 44). Embodiment 83 The method of embodiment 81, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence of YNYPREGSTKYNEKFQG (SEQ ID NO: 45). Embodiment 84 The method of embodiment 81, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDVSTKYNEKFQG (SEQ ID NO: 46).

[0107] Embodiment 85. The method of embodiment 81, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYAEKFQG (SEQ ID NO: 47). Embodiment 86 The method of embodiment 81, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYNEKFQG (SEQ ID NO: 44). Embodiment 87 The method of embodiment 81, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 comprises or consists of the amino acid sequence of YNYPREGSTKYNEKFQG (SEQ ID NO: 45). Embodiment 88 The method of embodiment 81, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDVSTKYNEKFQG (SEQ ID NO: 46). Embodiment 89. The method of embodiment 81, wherein HCDR1 comprises or consists of the amino acid sequence of GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 comprises or consists of the amino acid sequence of YNYPRDGSTKYAEKFQG (SEQ ID NO: 47).

[0108] Embodiment 90. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53. Embodiment 91. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. Embodiment 92. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55. Embodiment 93. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 49, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53.

[0109] Embodiment 94. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 49, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. Embodiment 95. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 49, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55. Embodiment 96. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 50, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53.

[0110] Embodiment 97. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 50, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. Embodiment 98. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 50, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55.

[0111] Embodiment 99. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 51, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53. Embodiment 100. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 51, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. Embodiment 101. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 51, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55.

[0112] Embodiment 102. The method of embodiment 81, wherein the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 52, and the VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53. Embodiment 103. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 52, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. Embodiment 104. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 52, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55. Embodiment 105. The method of any one of embodiments 81 to 104, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a humanized 6B5 (h6B5) antibody.

[0113] Embodiment 106. The method of any one of embodiments 81 to 104, wherein the anti-ceramide antibody or antigen-binding fragment thereof is h6B5 scFv. Embodiment 107. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO: 48, and V L 73. The method of any one of embodiments 1 to 72, wherein said vector comprises the amino acid sequence of SEQ ID NO: 53. Embodiment 108. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO: 48, and V L 73. The method of any one of embodiments 1 to 72, wherein said vector comprises the amino acid sequence of SEQ ID NO: 55. Embodiment 109. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO: 49, and V L 73. The method of any one of embodiments 1 to 72, wherein said vector comprises the amino acid sequence of SEQ ID NO: 53.

[0114] Embodiment 110. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V Hcomprises the amino acid sequence of SEQ ID NO: 49, and V L 73. The method of any one of embodiments 1 to 72, wherein said VLK vector comprises the amino acid sequence of SEQ ID NO: 54. Embodiment 111. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO: 50, and V L 73. The method of any one of embodiments 1 to 72, wherein said vector comprises the amino acid sequence of SEQ ID NO: 53. Embodiment 112. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO: 50, and V L 73. The method of any one of embodiments 1 to 72, wherein said VLK vector comprises the amino acid sequence of SEQ ID NO: 54. Embodiment 113. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO: 51, and V L 73. The method of any one of embodiments 1 to 72, wherein said vector comprises the amino acid sequence of SEQ ID NO: 53. Embodiment 114. The anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V H ) and variable light chain (V L ), including V H comprises the amino acid sequence of SEQ ID NO: 52, and V L 73. The method of any one of embodiments 1 to 72, wherein said vector comprises the amino acid sequence of SEQ ID NO: 53.

[0115] Embodiment 115. The method of any one of embodiments 107 to 114, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a humanized antibody. Embodiment 116. The method of any one of embodiments 107 to 114, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a humanized scFv. Embodiment 117. The method of any one of embodiments 1 to 116, wherein preventing diabetic retinopathy or ocular inflammatory disease comprises delaying the onset of diabetic retinopathy or ocular inflammatory disease. Embodiment 118. The method of any one of embodiments 1 to 117, wherein one or more symptoms of diabetic retinopathy or ocular inflammatory disease are reduced in the subject compared to a control subject or compared to the subject prior to treatment with the anti-ceramide antibody or antigen-binding fragment thereof. Embodiment 119. The method of embodiment 118, wherein the one or more symptoms of diabetic retinopathy or ocular inflammatory disease are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemia-reperfusion injury, areas of retinal leakage, and occlusive breakdown. Embodiment 120. The method of embodiment 118 or 119, wherein one or more symptoms of diabetic retinopathy or ocular inflammatory disease are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with the anti-ceramide antibody or antigen-binding fragment thereof.

[0116] Embodiment 121. The method of any one of embodiments 1 to 120, wherein the expression level of one or more inflammatory markers in the eye is reduced compared to the expression level in the eye of a control subject or compared to the expression level in the eye of the subject before treatment with the anti-ceramide antibody or antigen-binding fragment thereof. Embodiment 122. The method of embodiment 121, wherein the one or more inflammatory markers are selected from cytokines, growth factors, and adhesion molecules. Embodiment 123. The method of embodiment 122, wherein the cytokine is selected from TNFα, IL-1β, IL-6, or MCP1. Embodiment 124. The method of embodiment 122, wherein the growth factor is VEGF. Embodiment 125. The method of embodiment 122, wherein the adhesion molecule is ICAM-1 or VCAM-1. Embodiment 126. The method of any one of embodiments 121 to 125, wherein the expression level of one or more inflammatory markers in the eye of the subject is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the expression level in the eye of a control subject, or compared to the expression level in the eye of the subject before treatment with the anti-ceramide antibody or antigen-binding fragment thereof.

[0117] Embodiment 127. The method of any one of embodiments 1 to 126, wherein one or more visual parameters are increased in the subject compared to visual parameters of a control subject or compared to visual parameters of the subject before treatment with the anti-ceramide antibody or antigen-binding fragment thereof. Embodiment 128. The method of embodiment 127, wherein the one or more visual parameters are selected from peripheral vision, night vision, color vision, distance vision, near vision, and visual clarity. Embodiment 129. The method of any one of embodiments 1 to 128, wherein retinal vascular permeability in the subject's eye is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to retinal vascular permeability in a control subject's eye or compared to the expression level in the subject's eye before treatment with the anti-ceramide antibody or antigen-binding fragment thereof. Embodiment 130. The method of any one of embodiments 1 to 129, wherein the mean Early Treatment Diabetic Retinopathy Study (ETDRS) grade score in the subject group is reduced by at least 0.2, at least 0.5, at least 1, at least 1.5, at least 2, or at least 2.5 compared to the mean ETDRS grade score in a control subject group, or compared to the mean ETDRS grade score in the subject group before treatment with the anti-ceramide antibody or antigen-binding fragment thereof.

[0118] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not and should not be construed as an acknowledgment or any form of suggestion that it constitutes valid prior art or forms part of the general technical common knowledge in any country in the world.

Claims

1. 1. A composition for treating or preventing diabetic retinopathy in a subject, comprising an anti-ceramide antibody or an antigen-binding fragment thereof, the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL); a) V H comprises heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1), HCDR2 comprising the amino acid sequence of YNYPREGSTKYNEKFQG (SEQ ID NO: 45), and HCDR3 comprising the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3); and b) V L comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of RASKSISKYLA (SEQ ID NO: 4), an LCDR2 comprising the amino acid sequence of SGSTLQS (SEQ ID NO: 5), and an LCDR3 comprising the amino acid sequence of QQHNEYPWT (SEQ ID NO: 6); composition.

2. The composition of claim 1 , wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least two weeks, at least three weeks, or at least four weeks apart.

3. 10. The composition of claim 1, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced two to four weeks apart.

4. The composition of claim 1, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses spaced at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, or at least 11 months apart.

5. The composition of any one of claims 1 to 4, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

6. A composition described in any one of claims 1 to 5, for intraocular administration.

7. A composition described in any one of claims 1 to 5, for intravitreal administration.

8. A composition described in any one of claims 1 to 5, for subcutaneous administration.

9. The composition according to any one of claims 1 to 8, wherein the subject is undergoing prior treatment for diabetic retinopathy.

10. 10. The composition of claim 9, wherein the subject has failed to respond to prior treatment for diabetic retinopathy.

11. The composition of claim 9 or 10, wherein the prior treatment was vitrectomy, laser surgery, steroids, and / or anti-vascular endothelial growth factor (VEGF) therapy.

12. 12. The composition of claim 11, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

13. The composition of any one of claims 1 to 12, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of diabetic retinopathy.

14. The composition of any one of claims 1 to 12, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of diabetic retinopathy.

15. The composition of any one of claims 1 to 12, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative phase of diabetic retinopathy.

16. The composition of any one of claims 1 to 12, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative phase of diabetic retinopathy.

17. V H comprises the amino acid sequence of SEQ ID NO: 51, and V L comprises the amino acid sequence of SEQ ID NO: 53; The composition according to any one of claims 1 to 16.

18. The composition according to any one of claims 1 to 17, wherein preventing diabetic retinopathy comprises delaying the onset of diabetic retinopathy.

19. The composition of any one of claims 1 to 18, wherein one or more symptoms of diabetic retinopathy are reduced in the subject compared to a control subject or compared to the subject before treatment with the anti-ceramide antibody or antigen-binding fragment thereof.

20. 20. The composition of claim 19, wherein the one or more symptoms of diabetic retinopathy are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemia-reperfusion injury, areas of retinal leakage, and occlusive breakdown.

21. The composition of claim 19 or 20, wherein one or more symptoms of diabetic retinopathy are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject before treatment with the anti-ceramide antibody or its antigen-binding fragment.

22. the expression level of one or more inflammatory markers in the eye is reduced compared to the expression level in a control subject's eye or compared to the expression level in the subject's eye before treatment with the anti-ceramide antibody or antigen-binding fragment thereof, and the one or more inflammatory markers are selected from a cytokine, a growth factor, and an adhesion molecule; (i) the cytokine is selected from TNFα, IL-1β, IL-6, or MCP1; (ii) the growth factor is VEGF, and (iii) the adhesion molecule is ICAM-1 or VCAM-1; the expression level of one or more inflammatory markers in the subject's eye is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the expression level in a control subject's eye or compared to the expression level in the subject's eye before treatment with the anti-ceramide antibody or antigen-binding fragment thereof; The composition according to any one of claims 1 to 21.

23. 23. The composition of any one of claims 1 to 22, wherein one or more visual parameters are increased in the subject compared to visual parameters of a control subject or compared to visual parameters of the subject before treatment with the anti-ceramide antibody or antigen-binding fragment thereof.

24. 24. The composition of claim 23, wherein the one or more vision parameters are selected from peripheral vision, night vision, color vision, distance vision, near vision, and visual acuity.

25. 25. The composition of any one of claims 1 to 24, wherein retinal vascular permeability in the subject's eye is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to retinal vascular permeability in a control subject's eye, or compared to retinal vascular permeability in the subject's eye before treatment with the anti-ceramide antibody or antigen-binding fragment thereof.

26. 26. The composition of any one of claims 1 to 25, wherein the mean Early Treatment Diabetic Retinopathy Study (ETDRS) grade score in a subject group is reduced by at least 0.2, at least 0.5, at least 1, at least 1.5, at least 2, or at least 2.5 compared to the mean ETDRS grade score in a control subject group, or compared to the mean ETDRS grade score in a subject group prior to treatment with the anti-ceramide antibody or antigen-binding fragment thereof.

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