Methods and compositions for treating diabetic retinopathy

Abstract

This invention provides a method for treating diabetic retinopathy and inflammatory eye diseases using anti-ceramide antibodies and antibody fragments. [Solution] A method is provided for treating or preventing diabetic retinopathy in a subject requiring such treatment, comprising administering an anti-ceramide antibody or an antigen-binding fragment thereof to the subject via the eye.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 62 / 907,287, filed on 27 September 2019, the contents of which are incorporated herein by reference in their entirety. Statement regarding sequence listings The sequence listing associated with this application is provided in text format instead of paper copy and is incorporated herein by reference. Computer-readable format copy of the sequence listing: File name: CERA-011_01WO_SeqList_ST25.txt, Date recorded: September 28, 2020, File size: 32.9 kilobytes. This disclosure relates to an anti-ceramide composition for treating diabetic retinopathy and a method of using the same. [Background technology]

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

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

Summary of the Invention

[0004] This disclosure relates to a method for treating diabetic retinopathy, comprising administering an anti-ceramide antibody or its antigen-binding fragment into the eye. Methods for treating subjects who have previously received treatment for diabetic retinopathy are also provided. In some embodiments, this disclosure further provides a method for treating diabetic retinopathy with a single dose of an anti-ceramide antibody or its antigen-binding fragment. Furthermore, methods for treating diabetic retinopathy with two or more doses of an anti-ceramide antibody or its antigen-binding fragment, spaced at intervals of at least two weeks to at least one year, are also provided. Another aspect of this disclosure relates to a method for treating inflammatory diseases of the eye using an anti-ceramide antibody or its antigen-binding fragment. In some embodiments, the disclosure provides a method for treating or preventing diabetic retinopathy in a subject requiring such treatment, comprising administering an anti-ceramide antibody or its antigen-binding fragment ocularly to the subject. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv). In some embodiments, ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon's capsule administration, administration into the suprachoroidal space (SCS), administration into the supracial space, and intravitreous administration. In some embodiments, administration is intravitreous.

[0005] In some embodiments, subjects undergo pretreatment for diabetic retinopathy. In some embodiments, subjects did not respond to pretreatment for diabetic retinopathy. In some embodiments, pretreatment is a treatment procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroid and anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, 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 its antigen-binding fragment is administered as a single dose. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least two weeks, at least three weeks, or at least four weeks. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of approximately two weeks to approximately four weeks. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of 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. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of approximately one month to approximately six months. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least one year. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered before the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered after the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered during the non-proliferative phase of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered during the proliferative phase of diabetic retinopathy.

[0006] In some embodiments, the disclosure provides a method for treating or preventing diabetic retinopathy in a subject requiring such treatment, comprising administering an anti-ceramide antibody or its antigen-binding fragment to the subject, the subject having undergone pretreatment for diabetic retinopathy. In some embodiments, the subject did not respond to pretreatment for diabetic retinopathy. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv). In some embodiments, administration is ocular. In some embodiments, ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon's capsule administration, administration into the epichoroidal space (SCS), administration into the epiciliary space, and intravitreous administration. In some embodiments, ocular administration is intravitreous. In some embodiments, pretreatment included vitrectomy, laser surgery, steroids, and / or anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the anti-VEGF therapy was an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

[0007] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered as a single dose. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least two weeks, at least three weeks, or at least four weeks. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of approximately two weeks to approximately four weeks. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of 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. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of approximately one month to approximately six months. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least one year. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered before the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered after the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered during the non-proliferative phase of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered during the proliferative phase of diabetic retinopathy.

[0008] In some embodiments, the disclosure provides a method for treating or preventing diabetic retinopathy in a subject, comprising administering a single dose of an anti-ceramide antibody or an antigen-binding fragment thereof to the subject. In some embodiments, the 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 its antigen-binding fragment to the subject, with at least two weeks between doses. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses with at least two weeks, at least three weeks, or at least four weeks between doses. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses with intervals of about two weeks to about four weeks. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses with intervals of 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. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses with intervals of about one month to about six months. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses with an interval of at least one year between doses.

[0009] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is a single-strand variable fragment (scFv). In some embodiments, administration is ocular. In some embodiments, ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon's capsule administration, administration into the suprachoroidal space (SCS), administration into the supracial space, and intravitreous administration. In some embodiments, ocular administration is intravitreous. In some embodiments, subjects have undergone pretreatment for diabetic retinopathy. In some embodiments, subjects did not respond to pretreatment for diabetic retinopathy. In some embodiments, pretreatment was vitrectomy, laser surgery, steroids, and / or anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, 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 its antigen-binding fragment is administered before the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered after the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered during the non-proliferative phase of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered during the proliferative phase of diabetic retinopathy. In some embodiments, the disclosure provides a method for treating an inflammatory disease of the eye, comprising administering an anti-ceramide antibody or its antigen-binding fragment into the eye. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is a single-strand variable fragment (scFv). In some embodiments, ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon's capsule administration, administration into the epichoroidal space (SCS), administration into the epiciliociliary space, and intravitreous administration. In some embodiments, inflammatory diseases of the eye are 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, cystic macular edema, papilledema, recurrent ischemia, ocular hemorrhage, and proliferative vitreoretinopathy.

[0011] In some embodiments, ocular administration is intravitreous administration. In some embodiments, subjects have undergone pretreatment for diabetic retinopathy. In some embodiments, subjects did not respond to pretreatment for diabetic retinopathy. In some embodiments, pretreatment was a treatment procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroid and anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, 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 its antigen-binding fragment is administered as a single dose. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least two weeks, at least three weeks, or at least four weeks. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of approximately two weeks to approximately four weeks. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of 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. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of approximately one month to approximately six months. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least one year.

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

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

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

[0015] In some embodiments, preventing diabetic retinopathy or inflammatory disease of the eye includes delaying the onset of diabetic retinopathy or inflammatory disease of the eye. In some embodiments, one or more symptoms of diabetic retinopathy or inflammatory disease of the eye are reduced in subjects compared to control subjects or compared to subjects before treatment with anti-ceramide antibodies or their antigen-binding fragments. In some embodiments, one or more symptoms of diabetic retinopathy or inflammatory disease of the eye are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemic-reperfusion injury, retinal leakage areas, and occlusion disruption. In some embodiments, one or more symptoms of diabetic retinopathy or inflammatory disease of the eye are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to control subjects or compared to subjects before treatment with anti-ceramide antibodies or their antigen-binding fragments.

[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 eye or compared to the expression level in the target eye before treatment with an anti-ceramide antibody or its antigen-binding fragment. In some embodiments, the one or more inflammatory markers are selected from cytokines, growth factors, and adhesion molecules. 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 target 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 eye or compared to the expression level in the target eye before treatment with an anti-ceramide antibody or its antigen-binding fragment.

[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 an anti-ceramide antibody or its antigen-binding fragment. In some embodiments, 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 target 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 target eye, or compared to the expression level in the target eye before treatment with an anti-ceramide antibody or its antigen-binding fragment. In some embodiments, the mean early-treatment diabetic retinopathy study (ETDRS) grade score in the control 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 the control group, or compared to the mean ETDRS grade score in the control group before treatment with an anti-ceramide antibody or its antigen-binding fragment. [Modes for carrying out the invention]

[0018] Overview This 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), as well as methods for their use in the treatment or prevention of diabetic retinopathy, are provided. Such compositions and methods can be used for other treatments for diabetic retinopathy, for example, for the treatment of diabetic retinopathy in patients who have previously failed anti-VEGF antibody therapy. Furthermore, a method for administering anti-ceramide scFv intravitreously is provided. definition As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the content expressly indicates otherwise. Where 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 otherwise understood in context, the terms “comprise,” or variations such as “comprises,” or “comprising,” are understood to mean encompassing the element or integer, or group of elements or integers, described, but not to mean excluding other elements or integers, or groups of elements or integers. Where used in this application, the terms “about” and “approximately” are used interchangeably. Where used in this application, the presence or absence of “about” or “approximately” in any number means that any normal variation as understood by those skilled in the art is covered. In certain embodiments, unless otherwise stated or evident from the context, the terms “approximately” or “about” refer to a range of values ​​that fall within 10% (greater than or less than) of a given reference value in either direction (except where such a number is greater than 100% of the possible value or less than 0% of the possible value). The term “sample” refers to a biological composition (e.g., a portion of cells or tissue) subjected to analysis and / or modification. In some embodiments, the sample is a “primary sample” in that it is obtained directly from the subject; in some embodiments, the “sample” is the result of processing a primary sample, for example, to remove certain components and / or isolate or purify certain components of interest.

[0020] The term "subject" includes animals, such as mammals. In some embodiments, mammals are primates. In some embodiments, mammals are humans. In some embodiments, subjects are livestock such as cattle, sheep, goats, cows, and pigs; or domesticated animals such as dogs and cats. In some embodiments (e.g., particularly in research contexts), subjects are rodents (e.g., mice, rats, hamsters), rabbits, primates, or pigs, such as inbred pigs. The terms "subject" and "patient" are used interchangeably herein. In some embodiments, subjects may be newborns, infants, or adults. Mammalian subjects are of particular interest. Mammalian species that can be treated by this method include dogs and cats; horses; cows; sheep, etc., and primates, especially 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,” “to treat,” or “to improve” refer to either a therapeutic treatment or a prophylactic / preventive treatment. A treatment is one in which at least one symptom of a disease is improved in the individual being treated, or the treatment can slow the progression of a progressive disease in the individual, or prevent the onset of additional related diseases.

[0021] As used herein, the term “effective dose” refers to the minimum amount of a drug or composition required to produce a particular physiological effect. The effective dose of a particular drug can be expressed in various ways based on the properties of the drug, such as mass / volume, cell count / volume, particle / volume, (mass of drug) / (mass of target), cell count / (mass of target), or particle / (mass of target). Furthermore, the effective dose of a particular active substance refers to the half-maximum effective concentration (EC2), which is the concentration of the active substance that produces a particular physiological response that is half the level between the baseline and the maximum response level. 50 It can also be expressed as ). The term “antibody” refers to an immunoglobulin (Ig) molecule capable of binding to specific targets such as carbohydrates, polynucleotides, lipids, or polypeptides via at least one epitope recognition site located in the variable region of the immunoglobulin (Ig) molecule. As used herein, the term encompasses intact polyclonal or monoclonal antibodies and their antigen-binding fragments. For example, a natural immunoglobulin molecule is composed of two heavy-chain polypeptides and two light-chain polypeptides. Each heavy-chain polypeptide is bound to a light-chain polypeptide by an interchain disulfide bond between the heavy-chain and light-chain polypeptides to form two heterodimeric proteins or polypeptides (i.e., proteins composed of two different polypeptide chains). The two heterodimeric proteins are then bound by further interchain disulfide bonds 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 comprising at least one complementarity-determining region (CDR) of an immunoglobulin heavy chain and / or light chain that binds to at least one epitope of the antigen of interest. In this regard, the antigen-binding fragments of antibodies described herein may comprise 1, 2, 3, 4, 5, or all 6 CDRs of variable heavy chain (VH) and variable light chain (VL) sequences from an antibody that specifically binds to ceramide. Antigen-binding fragments include proteins containing a portion of a full-length antibody, generally their antigen-binding or variable regions, e.g., Fab, F(ab')2, Fab', Fv fragments, minibodies, diabodies, single-chain antibodies (dAb), single-chain variable fragments (scFv), multispecific antibodies formed from antibody fragments, and other modified configurations of immunoglobulin molecules containing antigen-binding sites or fragments of the desired specificity. In certain embodiments of this disclosure, antigen-binding fragments, rather than intact antibodies, are used to increase tissue penetration or tumor penetration. In other embodiments, the antigen-binding fragment is further modified to increase its serum half-life.

[0023] The term "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from a portion of an antibody that can bind to the Fc receptor and / or the C1q component of complement on a cell, thereby mediating the effector function of the antibody. Fc signifies "fragment crystalline," meaning a fragment of the antibody that readily forms protein crystals. Distinguishable protein fragments, first described by proteolytic digestion, can define the overall structure of an immunoglobulin protein. As first defined in the literature, an Fc region is a homodimeric protein comprising two polypeptides linked by a disulfide bond, each containing a hinge region, a CH2 domain, and a CH3 domain. More recently, however, the term has been applied to a single-chain monomer component consisting of at least a portion of the CH3, CH2, and hinge, sufficient to form a disulfide-bonded dimer with a second such chain. Therefore, depending on the context, the use of the term "Fc region" or "Fc domain" herein refers to either the dimeric form or the individual monomers that bind to form a dimeric protein. For an overview of the structure and function of immunoglobulins, 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 that corresponds to or is derived from one or more constant domains (e.g., CH1, CH2, CH3) of an immunoglobulin. In certain embodiments, the constant region does not include the CH1 domain. In certain embodiments, the constant domains constituting 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 the antibody light chain and heavy chain, respectively. The variable binding region consists of discrete, well-defined subregions known as "complementarity-determining regions" (CDRs) and "framework regions" (FRs). The term "immunoglobulin light chain constant region" (also called "light chain constant region" or "CL") refers to the constant region derived from the antibody light chain. The term "immunoglobulin heavy chain constant region" (also called "heavy chain constant region" or "CH") refers to the constant region derived from the antibody heavy chain. Depending on the antibody isotype, the CH can be further divided into CH1, CH2, and CH3 (IgA, IgD, IgG) or CH1, CH2, CH3, and CH4 domains (IgE, IgM).

[0025] The term "F(ab)" refers to two protein fragments resulting from the proteolytic cleavage of an IgG molecule by the enzyme papain. Each F(ab) contains a covalent heterodimer of the VH and VL chains and 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 small portion of Fc. Each Fab' fragment is a monovalent antigen-binding fragment. The term "F(ab')2" refers to a protein fragment of IgG produced by proteolytic cleavage by the enzyme pepsin. Each F(ab')2 fragment contains two F(ab') fragments and is therefore a bivalent antigen-binding fragment. The "Fd fragment" contains the VH and CH1 domains. "Fv fragment" refers to a non-covalent VH::VL heterodimer that retains most of the antigen recognition and binding ability of an undenatured antibody molecule but contains an antigen-binding site lacking 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 induced using recombinant techniques known in the art.

[0026] The "dAb fragment" (Ward et al., Nature 341:544 546, 1989) contains a VH domain. A "single-chain antibody" or "scFv" is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, linked to a short linker peptide of 10 to approximately 25 amino acids. The linker may be glycine-rich for solubility, serine or threonine-rich for solubility, and may link the N-terminus of the VH to the C-terminus of the VL, or vice versa. The scFv retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. In this disclosure, any description of an antibody or antibody fragment or its use is intended to include the scFv molecule and its use. A "minibody" refers to a fusion protein containing an scFv connected to a CH3 domain, and is included in this specification (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). For example, Ward, ES 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. (See S. Hu et al., Cancer Res., 56, 3055-3061, 1996).

[0027] The term "diabody" refers to a bispecific antibody in which the VH and VL domains are expressed in a single polypeptide chain using a linker that is too short to allow pairing between the two domains on the same chain, thereby pairing the domains with complementary domains on another chain and creating two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993) and Poljak et al., Structure 2:1121-23 (1994)). The terms "nanobody" or "single-domain antibody" refer to antigen-binding fragments consisting of a single monomeric variable antibody domain. Nanocloning is a method for generating nanobodies against a desired target based on automated, high-throughput selection of B cells. (See International Publication No. 2006 / 079372). As used herein, the term "monoclonal antibody" means an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies within the population are identical except for any naturally occurring mutations that may be present in small amounts.

[0028] As used herein, the term "chimeric antibody" refers to a monoclonal antibody in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody originating from a particular species or belonging to a particular antibody class or subclass, while the remaining chain is identical or homologous to a corresponding sequence in an antibody originating from another species or belonging to another antibody class or subclass, and in a fragment of such antibody, insofar as it exhibits the desired biological activity. The term “single-stranded variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, linked to 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 the VH to the C-terminus of the VL, or vice versa. Numerous methods have been described for identifying the chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from the antibody V region into scFv molecules, which fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patents 5,091,513 and 5,132,405 by Huston et al., and U.S. Patent 4,946,778 by Ladner et al.

[0029] As used herein, the term “CDR” refers to the “complementarity-determining region” of an immunoglobulin (antibody) molecule. A CDR is a portion of the variable domain in an antibody that binds to its specific antigen. There are three CDRs per variable domain (i.e., CDR1, CDR2, and CDR3 in the variable domain of the light chain, and CDR1, CDR2, and CDR3 in the variable domain of the heavy chain). Within the variable domain, CDR1 and CDR2 are found in the variable (V) region of the polypeptide chain, while CDR3 exhibits the greatest variability, as it is encoded by VJ recombination when it is in the light chain region and by VDJ when it is in the heavy chain region. "Isolated antibodies" are antibodies that (1) are not naturally related components, including other naturally related antibodies that are associated with them in nature, (2) do not contain other proteins from the same species, (3) are expressed by heterologous cells, or (4) do not occur in nature. The term "human antibody" includes all antibodies having one or more variable and constant regions derived from a human immunoglobulin sequence. In preferred embodiments, all variable and constant domains are derived from a human immunoglobulin sequence (a fully human antibody). These antibodies can be prepared by various methods, as described below.

[0030] As used herein, the term “humanized” refers to an antibody or its antigen-binding fragment derived from a non-human species that retains the antigen-binding properties of the original non-human antibody. In some embodiments, the antibody-binding fragment (e.g., light chain and heavy chain variable regions, Fab, scFv) is humanized. Non-human antigen-binding fragments undergo CDR grafting (Jones et al., Nature 321:522 (1986)) and its modifications, such as "reformation" (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), and 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). Humanization can be performed using techniques known as 148:1149-1154) and “veneering” (Mark, et al., “Derivation of therapeutically active humanized and veneered anti-CD18 antibody.” In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312). If derived from a non-human source, other regions of the antibody, such as the hinge region and constant region domain, can also be humanized.

[0031] As used herein, the term “pharmaceutically acceptable” means a molecular entity and composition that, when administered by a route known in the art, does not generally cause allergic or other serious adverse reactions. Molecular entities and compositions for use in animals, more specifically in humans, that are approved by federal or state regulatory authorities or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias are considered “pharmaceutically acceptable.” The terms "prevent," "prophylaxis," and "prophylactically" refer to the administration of a compound, such as an anti-ceramide antibody or its antigen-binding fragment, before the onset of a disease (for example, before the onset of certain symptoms of the disease). Preventing a disease includes reducing the likelihood of the disease occurring, delaying the onset of the disease, improving long-term symptoms, or slowing the eventual progression of the disease. In this specification, the term "specifically binds" means at least 10 5 M -1 This refers to the ability of an antibody or its antigen-binding fragment to bind to a target antigen with a binding affinity (Ka) of 1, while not significantly binding to other components or antigens present in the mixture. In this specification, references to anti-ceramide antibodies refer to antibodies or their antigen-binding fragments 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. Sequences are said to be “identical” at a position if the position of one sequence is occupied by the same nucleic acid base or amino acid residue at the corresponding position in the comparison sequence. The sequence identity percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue exists, thereby obtaining the number of identical positions. The number of identical positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the sequence identity percentage. The sequence identity percentage is determined by comparing two optimally aligned sequences on the comparison window. The comparison window for polynucleotide sequences may be, for example, nucleic acids with lengths of 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. The comparison window for polypeptide sequences may be, for example, amino acids with lengths of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more. To optimally align sequences for comparison, portions of a polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions called gaps, while the reference sequence remains constant. Optimal alignment is the alignment that, even with gaps, produces the maximum possible number of "identical" positions between the reference and comparison sequences.The "sequence identity" percentage between two sequences can be determined using the version of the "BLAST 2 Sequences" program available from the National Center for Biotechnology Information as of September 1, 2004. This program incorporates BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When using "BLAST 2 Sequences," as of September 1, 2004, default parameters include word size (3), open gap penalty (11), extended gap penalty (1), gap drop-off (50), expected value (10), and other necessary parameters, including but not limited to matrix options. Two nucleotide sequences 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 with respect to each other.

[0033] "Angiogenesis" refers to the formation of new blood vessels. As used herein, the terms “eye” or “into the eye” refer to the administration of a drug to the eye and surrounding tissues. For example, in some embodiments, ocular administration includes topical administration (e.g., administration to the surface of the eye, such as the sclera), intraocular administration, subconjunctival administration (such as under the conjunctiva or under the inner conjunctival layer in the case of eyelid administration), anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon’s capsule administration, administration into the suprachoroidal space (SCS), administration into the supracial space, or intravitreal administration.

[0034] Anti-ceramide antibodies, antibody fragments, and derivatives This disclosure relates to an anti-ceramide antibody and its antigen-binding fragment for treating diabetic retinopathy. Ceramides are a family of waxy lipid molecules. Ceramides are composed of sphingosine and fatty acids. Because ceramides are component lipids that make up sphingomyelin, one of the major lipids in the lipid bilayer, they are found in high concentrations within the cell membrane of eukaryotic cells. Ceramides are involved in various cellular signaling pathways, including the regulation of cell differentiation, proliferation, and programmed cell death (PCD). As bioactive lipids, ceramides are involved in various physiological functions, including apoptosis, cell proliferation arrest, differentiation, cellular senescence, cell migration, and adhesion. The roles of ceramides and their downstream metabolites have 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 Publications 2010 / 0239572 and 2017 / 0335014, respectively, which are incorporated herein by reference. The sequences of exemplary anti-ceramide antibodies are shown in Table 1. However, any anti-ceramide antibody or its antigen-binding fragment can be used according to the disclosed methods and uses.

[0036] [Table 1] TIFF2026102575000003.tif252170 TIFF2026102575000004.tif120170

[0037] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is humanized 6B5 (h6B5). In some embodiments, the h6B5 antibody or its antigen-binding fragment includes 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 whole for all purposes. The sequences of each CDR of the h6B5 antibody or its antigen-binding fragment 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 monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, recombinant antibodies, or synthetic antibodies. In some embodiments, the anti-ceramide antigen-binding antibody fragment is one of the above antigen-binding fragments. In some embodiments, the anti-ceramide antigen-binding antibody fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, an Fd fragment, a dAb fragment, a diabody, scFv, etc. In some embodiments, the anti-ceramide antibody and its antigen-binding fragment are produced using recombinant DNA technology. Procedures for the expression and purification of recombinant proteins are well established in the art. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv). In some embodiments, the scFv comprises a CDR sequence and / or variable chain sequence of a 2A2, h2A2, 6C8, 7B10, 9H10, h6B5, or 6B5 antibody.

[0040] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is the 2A2 antibody or its antigen-binding fragment as described in U.S. Patent Application Publication 2010 / 0239572. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is the 6B5 antibody or its antigen-binding fragment as described in U.S. Patent Application Publication 2017 / 0335014. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is the h6B5 antibody or its antigen-binding fragment as disclosed herein. In some embodiments, the anti-ceramide antibody or its antigen-binding fragment is scFv. In some embodiments, scFV comprises the CDR sequence of any of the antibodies disclosed in Table 1. In some embodiments, scFv comprises the CDR sequence of 2A2. In some embodiments, scFv comprises the CDR sequence of 6B5. In some embodiments, scFv comprises the CDR sequence of h6B5. In some embodiments, scFv comprises the variable heavy and light chain sequences of h2A2. In some embodiments, scFv includes a variable heavy chain and light chain sequence of 6B5. In some embodiments, scFv includes a variable heavy chain and light chain sequence of h6B5.

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

[0042] In some embodiments, the anti-ceramide antibody or its antigen-binding fragment includes 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 detection method is determined by the selected label. 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 a spectrophotometer, luminometer, or fluorometer, or by ELISA or Western blotting.

[0043] diabetic retinopathy In some embodiments, this 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 due to diabetes is associated with damage to the small blood vessels in the retina, leading to diabetic retinopathy. In diabetic retinopathy, leakage of fluid or bleeding from the blood vessels in the retina can lead to decreased vision. In the most advanced stages, new abnormal blood vessels proliferate on the surface of the retina, causing scarring and cell loss in the retina. In some embodiments, the disclosed methods and compositions may be used to treat different stages of diabetic retinopathy. Diabetic retinopathy may progress through a non-proliferative phase, also called the early stage, and a proliferative phase, also called the late stage.

[0044] In some embodiments, this disclosure provides methods and compositions that may be used to treat diabetic retinopathy during the non-proliferative stage. Nonproliferative diabetic retinopathy (NPDR) can be mild, moderate, or severe. Mild NPDR is characterized by microaneurysms of the retinal blood vessels. These microaneurysms can leak fluid into the retina. As the disease progresses to the moderate NPDR stage, the retinal blood vessels become distorted and lose their ability to carry blood, leading to characteristic changes in the appearance of the retina and contributing to diabetic macular edema. In the severe NPDR stage, even more blood vessels become occluded, cutting off the blood supply to areas of the retina. These areas then secrete pro-angiogenic growth factors. Ocular angiogenesis molecules include VEGF, FGF, PIGF, TGF-alpha, TGF-beta, IGF, PDGF, MMP, HGF / SF, TNF-alpha, CTGF, IL-1, IL-8, MCP-1, leptin, integrin, 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 decrease levels of retinal microaneurysms, retinal fluid leakage, diabetic macular edema, and / or pro-angiogenic growth factors. In some embodiments, one or more of VEGF, FGF, P1GF, TGF-alpha, TGF-beta, IGF, PDGF, MMP, HGF / SF, TNF-alpha, CTGF, IL-1, IL-8, MCP-1, integrin, and angiogenin are reduced by the method. In some embodiments, this disclosure provides methods and compositions that may be used to treat diabetic retinopathy during the proliferative stage. Proliferative diabetic retinopathy (PDR) is the progressive stage of the disease. At this stage, pro-angiogenic growth factors secreted from the retina proliferate along the inner surface of the retina, causing the proliferation of new blood vessels that enter the vitreous gel. These neovascularizations are fragile and prone to leakage and bleeding. The accompanying scar tissue may contract, leading to retinal detachment (i.e., separation of the retina from the underlying tissue) and potentially resulting in permanent vision loss. In some embodiments, the methods may prevent, reduce, suppress, or decrease the levels of retinal neovascularization, retinal hemorrhage, retinal scarring, retinal detachment, and vision loss.

[0045] In some embodiments, the disclosure provides methods and compositions that can be used to prevent diabetic retinopathy by administering them to a diabetic subject before the onset of diabetic retinopathy, for example, before the onset of one or more of its symptoms. 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 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, by more than 3, 5, 10, or 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 having 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 degree of diabetes severity (as indicated by comparison between insulin and oral antidiabetic drugs alone or tablets alone and no treatment), having a high mean systolic blood pressure, and having a high hemoglobin A1c are additional factors to consider.

[0046] Symptoms and detection In some embodiments, the method may affect one or more symptoms and / or detectable markers of diabetic retinopathy. Symptoms are usually absent in the early, non-proliferative phase of diabetic retinopathy. In some embodiments, the method and composition are intended for use in diabetic patients before the onset of symptoms of diabetic retinopathy. The disease often progresses unnoticed until it affects vision. Bleeding from retinal blood vessels in the early stages of the disease can cause "floating" spots that may disappear 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 can reduce the incidence, severity, or level of floating spots, retinal hemorrhages, vision loss, and / or blurred vision. In some embodiments, but not limited to, the method can improve one or more parameters of visual acuity, including overall visual acuity, peripheral visual acuity, night vision, color vision, distance vision, near vision, and visual clarity.

[0047] Diabetic retinopathy and diabetic macular edema may be detected during a comprehensive eye examination that includes visual acuity testing (fundus examination to measure visual acuity at various distances), tomometry (measurement of intraocular pressure), pupillary dilation, and optical coherence tomography (OCT). During such an examination, a doctor may check for one or more of the following: changes in retinal blood vessels such as the formation of new blood vessels, swelling, and bleeding; leakage of retinal blood vessels, or warning signs of leaking vessels such as fat deposits, weakening of vessel walls, and bulging of 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 composition and method may be used to treat a subject using any one of these symptoms of diabetic retinopathy. In some embodiments, the composition and method may prevent, treat, or improve any one of these symptoms. If diabetic macular edema or severe diabetic retinopathy is suspected, fluorescein angiography can be used to examine vascular damage or leakage. In this test, a fluorescent dye is injected into the bloodstream and then into a vein in the arm. Once the dye reaches the eyeball, an image of the retinal blood vessels is taken. In some embodiments, the composition and method may be used to prevent or reduce the incidence, prevalence, or severity of damaged and / or leaking blood vessels of the eye. Existing treatments for diabetic retinopathy include anti-VEGF therapy, steroids, laser surgery, and vitrectomy.

[0048] Anti-VEGF injection therapy involves injecting anti-VEGF drugs into a vitreous gel to block the action of pro-angiogenic growth factor (VEGF). Blocking VEGF can reverse abnormal vascular proliferation in the retina and reduce fluid. 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 to treat cancer, but is commonly used to treat eye conditions such as diabetic macular edema. Most people require monthly anti-VEGF injections for the first six months of treatment. Subsequently, the need for injections is not very frequent: typically, 3-4 times in the first 6 months after treatment, about 4 times in the second year, 2 times in the third year, 1 time in the fourth year, and none in the fifth year. Recent studies have shown that anti-VEGF treatment is effective in treating diabetic macular edema and delaying the progression of diabetic retinopathy, including PDR, and anti-VEGF is increasingly being used as a first-line treatment for PDR. However, several large clinical studies have revealed that about 40% of patients do not respond to anti-VEGF therapy. Furthermore, anti-VEGF treatment is directed towards very late stages of the disease when complete recovery of retinal damage is difficult.

[0049] Laser surgery. In localized / lattice macular laser surgery, several to hundreds of tiny laser burns are made to release 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 one session, but some patients may require more than one treatment. Localized / lattice laser treatment can be used in combination with anti-VEGF therapy. For example, localized / lattice laser treatment is sometimes applied before anti-VEGF injections, sometimes on the same day or a few days after anti-VEGF injections, and sometimes only when diabetic macular edema has not adequately improved after 6 months of anti-VEGF therapy. PDR can also be treated with a scattering laser surgery called panretinal laser surgery or panretinal photocoagulation. The procedure involves creating 1,000 to 2,000 small laser burns in an area of ​​the retina away from the macula. These laser burns are intended to constrict abnormal blood vessels. The procedure can be completed in one session, but may require two or more sessions. Central vision can be preserved, but scattering laser surgery may result in a slight decrease in lateral (peripheral), color, and night vision. Scatter laser surgery is most effective before fragile neovascularization begins to bleed.

[0050] Corticosteroids. Corticosteroids may be used alone, in combination with other medications, or via laser surgery to treat diabetic macular edema. Corticosteroids can be injected into the eye or implanted. Ozurdex® (dexamethasone) implants are used for short periods, while Iluvien® (fluocinolone acetonide) implants are used for longer periods. Both are biodegradable and release a sustained dose of corticosteroids to suppress diabetic macular edema. Vitrectomy. Vitrectomy is a surgical procedure to remove the vitreous gel located in the center of the eye. This procedure is used to treat severe bleeding into the vitreous humor and is performed under local or general anesthesia. A clear saline solution is gently pumped into the eye through one or more ports to maintain intraocular pressure during the surgery and to replace the removed vitreous humor. Scar tissue can also be removed or a detached retina repaired using the same instruments as in vitrectomy.

[0051] The role of ceramide and ASM in diabetic retinopathy While we do not wish to be bound by any one theory, this treatment is thought to be able to downregulate retinal inflammation signaling by selectively inhibiting ceramide in the retina through treatment of the eye with anti-ceramide antibodies or their antigen-binding fragments. Sphingolipids are major components of membrane microdomains, and ceramide-rich microdomains are considered prerequisites for inflammatory cytokine signaling. Acid sphingomyelinase (ASM) and neutral sphingomyelinase (NSM) are important regulatory enzymes of sphingolipid metabolism, promoting sphingomyelin hydrolysis to inflammatory ceramides. ASM is an important early responder in inflammatory cytokine signaling. The sphingomyelinase pathway is crucial for inflammatory signaling in human retinal endothelial cells (HRECs), a commensal vascular system affected by diabetic retinopathy. Inflammatory cytokines TNFα and IL-1β induce rapid increases in cell adhesion molecule (CAM) expression and ASM and NSM activity in HRECs.

[0052] Previous studies have demonstrated 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 administration of anti-ASM inhibitors reduced retinal inflammation 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 incorporated collectively by reference in this specification. However, ASM is an essential enzyme, and its deficiency or inhibition can cause Niemann-Pick disease, which can be fatal. Thus, ASM gene knockout and direct inhibition of the enzyme are not viable therapeutic strategies for treating diabetic retinopathy. In contrast, the administration of anti-ceramide antibodies and antigen-binding fragments described herein offers the remarkable result of selectively inhibiting 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 diseases In some embodiments, the composition and method are used to treat diabetic retinopathy. In some embodiments, the composition and method are used to treat diabetic macular edema. In some embodiments, the composition and method are used to treat macular edema after retinal vein occlusion (RVO). In some embodiments, the composition and method are used to treat myopic choroidal neovascularization (mCNV). This disclosure provides methods for treating diseases or disorders involving underlying neovascularization. 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, cystic macular edema, papilledema, recurrent ischemia, ocular hemorrhage, and proliferative vitreoretinopathy. Pharmaceutical composition, route of administration, dosage, and administration schedule In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-ceramide antibody or an antigen-binding fragment thereof for the treatment of diabetic retinopathy.

[0054] For administration, the antibodies or fragments of this disclosure (e.g., anti-ceramide antibodies and their antigen-binding fragments) can be formulated as pharmaceutical compositions. A pharmaceutical composition may comprise (i) an anti-ceramide antibody or its antigen-binding fragment; and (ii) a pharmaceutically acceptable carrier, diluent, or excipient. A pharmaceutical composition 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, thereby combining the therapeutic molecule with a pharmaceutically acceptable carrier, diluent, or excipient in a mixture. 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 include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, etc. In some embodiments, the pharmaceutical composition may be formulated in dosage forms selected from the group consisting of oral, intravenous, intranasal, suppository, intradermal, intramuscular, intraperitoneal, subcutaneous, subdural, sublingual, intracerebral, anterior chamber, subconjunctival, sub-Tenon's capsule, posterior bulbar, posterior scleral, and intravitreous forms. In some embodiments, the pharmaceutical composition may be formulated in an intravitreous administration form. Since the anti-ceramide antibody or its antigen-binding fragment is not expected to cross the blood-brain barrier, the systemic administration route is not expected to deliver the anti-ceramide antibody or its antigen-binding fragment to the eye.

[0055] The anti-ceramide antibodies and their antigen-binding fragments described herein can be administered to subjects by various modes of administration, for example, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, oral, topical, intraocular, anterior chamber, subconjunctival, subtenon's capsule, posterior ocular, suprascleroscopic, suprachoroidal (SCS), supracappillary, and intravitreous routes. In some embodiments, the anti-ceramide antibodies and their antigen-binding fragments disclosed herein, as well as the pharmaceutical compositions thereof, may be administered directly to the eye by any known route of administration, including topical administration (e.g., eye drops), topical administration to the eye, intravitreous administration, intrachorally, subconjunctival administration, subtenon's capsule administration, posterior ocular administration, and posterior sclera administration. A number of possible delivery modes can be used, including, but are not limited to, intraocular or topical application. In one embodiment, the application is intraocular and is not limited to, subconjunctival injection, intracapillary injection, injection into the anterior space via the temporal limb, intrastitium injection, intracorneal injection, subretinal injection, aqueous fluid injection, sub-Tenon's capsule injection or continuous delivery device, or intravitreal injection (e.g., anterior, mid, or posterior intravitreal injection). In one embodiment, the application is topical and is not limited to, eye drops to the cornea. In some embodiments, the disclosed antibodies, antigen-binding fragments, and compositions are prepared for intravitreous administration.

[0056] For preventive and therapeutic purposes, anti-ceramide antibodies and their antigen-binding fragments can be administered to subjects via long-term continuous delivery (e.g., continuous transdermal delivery) or in a single bolus delivery using a repeated-dose protocol (e.g., every hour, daily, weekly, monthly, or yearly). In some embodiments, the methods provided herein involve administering a therapeutically effective dose of an anti-ceramide antibody or its antigen-binding fragment. A therapeutically effective dose, dosage, or quantity refers to the minimum amount of an anti-ceramide antibody or its antigen-binding fragment required to produce a specific physiological effect, e.g., prevention or improvement of one or more symptoms of diabetic retinopathy, as defined above. Determining a therapeutically effective dosage 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 the model subjects. The effective dose of the compositions of this disclosure varies depending on a number of different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs administered, whether the treatment is prophylactic or therapeutic, and the specific activity of the composition itself and its ability to elicit a desired response in an individual. Typically, the dosage regimen is adjusted to provide an optimal therapeutic response, i.e., to optimize safety and efficacy.

[0057] In some embodiments, the dose of the anti-ceramide antibody or its antigen-binding fragment 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, the effective dose of the anti-ceramide antibody or its antigen-binding fragment is between about 1 μg / kg to about 20 mg / kg, about 10 μg / kg to about 10 mg / kg, or about 0.1 to about 5 mg / kg. The anti-ceramide antibody and its antigen-binding fragment described herein can also be administered in doses of about 0.001 to about 10 milligrams (mg) per kg of body weight, and can be administered as a single dose or in two or more doses. For administration to adult human patients, the therapeutically effective dose is not limited, but can be administered in doses ranging from 0.2 mg to 800 mg / dose, including 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 may be administered during the course of treatment. In some embodiments, the total daily dose of the anti-ceramide antibody and its antigen-binding fragment described herein may be in the range of 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, anti-ceramide antibodies, their antigen-binding fragments, or compositions containing the same may be formulated at concentrations of approximately 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 concentrations may be between 0.1 and 1 mg / mL, 1 and 5 mg / mL, 5 and 10 mg / mL, or 10 and 50 mg / mL. In some embodiments, the disclosed antibodies, fragments, or compositions may be administered intravitreally at doses of approximately 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 volume of the dose may be approximately 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 their antigen-binding fragments described herein can be administered at different times of day. In some embodiments, doses can be administered in the evening. In other embodiments, doses can be administered in the morning. Dosage can be in single or multiple doses, including, for example, multiple times a week, once every two weeks, once a month, or once a year. In some embodiments, a single dose of the anti-ceramide antibody or antibody fragment is administered to a subject who needs it. In some embodiments, a patient can receive two or more doses of anti-ceramide antibody treatment at intervals of at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, 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, at least eleven months, or at least one year. In some embodiments, two or more doses may be administered to patients in need at intervals of approximately 1 to 2 weeks, 2 to 4 weeks, 1 to 2 months, 2 to 4 months, 1 to 6 months, 6 months to 1 year, or 1 to 2 years. In some embodiments, administration may be irregular, as indicated by monitoring the clinical symptoms of the disorder.

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

[0061] Treatment method and use In some embodiments, the Disclosure provides a method for treating, preventing, or improving symptoms of inflammatory eye disease in a subject, comprising administering a therapeutically effective amount of an anti-ceramide antibody or an antigen-binding fragment thereof to the subject. In some embodiments, the disclosure provides a method for preventing and / or treating diabetic retinopathy in a subject requiring such treatment, comprising administering a therapeutically effective amount of an anti-ceramide antibody or anti-ceramide antibody fragment to the subject. Previous studies have indicated that the initial metabolic dysfunction leading to inflammation and increased retinal vascular permeability involves the 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 ischemic-reperfusion model, and chimeras with ASM- / - bone marrow transplanted into wild-type mice were also protected from diabetes-induced vascular injury. Several strategies have been employed to inhibit ASM activity, including the administration of docosahexaenoic acid (DHA)-rich fish oil and the tricyclic antidepressant desipramine. Both strategies show some efficacy in cell culture models. However, although desipramine improved retinal health in diabetic animals, it was not well tolerated in animals in long-term studies (see Example 8) and long-term high-dose DHA treatment, and caused retinal pathology in control animals (see Example 9). Furthermore, ASM is an essential enzyme, and ASM deficiency causes neurodegenerative diseases (Niemann-Pick). Therefore, direct inhibition of ASM is not a viable clinical strategy in the treatment of DR. Anti-ceramide antibodies and their antigen-binding fragments are highly effective in binding monomeric ceramides produced on the surface of endothelium and other cells, thereby interfering with ASM-induced ceramide-rich platform formation, resulting in pro-inflammatory and apoptotic signaling, without affecting essential lysosomal ASM function.

[0062] subject The treatments disclosed herein include subjects who have developed or are at risk of developing diabetic retinopathy. Subjects with diabetic retinopathy may have early or late-stage disease. In some embodiments, subjects have previously received one or more treatments for diabetic retinopathy but have not responded to those treatments. In such embodiments, “failure to respond” indicates that the previous treatment did not improve and / or alleviate one or more symptoms of diabetic retinopathy. In some embodiments, the previous treatment may not have resulted in any improvement in visual acuity 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 be effective after a certain period of time. Existing treatments for diabetic retinopathy include therapeutic procedures such as vitrectomy and laser surgery, as well as therapeutic agents such as steroids and anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the subjects may have partially responded to previous treatments for diabetic retinopathy: that is, one or more symptoms of diabetic retinopathy were not sufficiently improved, and / or the effects of the previous treatment were not sufficiently sustained. In some embodiments, the subjects had previously received anti-VEGF treatment and had not responded. In some embodiments, the anti-VEGF treatment was selected from Eylea® (aflibercept), Avastin® (bevacizumab), or Lucentis® treatment (ranibizumab).

[0063] Treatment readout Treatment and / or prevention of diabetic retinopathy can be measured by various means. In some embodiments, treatment or prevention includes treating or preventing one or more of the following in diabetic retinopathy in a subject requiring treatment or prevention: apoptosis, inflammation, acellular capillary formation, neovascularization, retinal endothelial cell death, retinal vascular permeability, ischemic reperfusion injury, and occlusion disruption. The method includes 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. A method is provided for treating diabetic retinopathy with an anti-ceramide antibody or its antigen-binding fragment. The effectiveness of such treatment can be characterized, evaluated, measured, and / or monitored based on several parameters.

[0064] In some embodiments, the methods provided herein result in a reduction of 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 the eye sample undergoing 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 eye 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 ischemic-reperfusion (IR) injury. Further methods for detecting IR injury include fluorescein analysis, fluorescent zinc 2,2'-dipicolylamine coordination complex PSVue® 794, 99mTc glucarate, and electroretinography. In some embodiments, the methods provided herein reduce the levels 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. The inflammatory marker levels can be monitored via enzyme-linked immunosorbent assay (ELISA), Luminex, cytokine bead arrays, Proteo Plex, FAST Quant, etc. In some embodiments, the methods provided herein reduce the levels of one or more inflammatory markers in the eye 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 eye sample.

[0066] In some embodiments, the method provided herein reduces the level of TNFα in the eye 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 eye sample. In some embodiments, the method provided herein reduces the level of TNFα by at least 10%. In some embodiments, the method provided herein reduces the level of TNFα by at least 20%. In some embodiments, the method provided herein reduces the level of TNFα by at least 30%. In some embodiments, the method provided herein reduces the level of TNFα by at least 50%. In some embodiments, the method provided herein reduces the level of TNFα by at least 70%.

[0067] In some embodiments, the method provided herein reduces the level of IL-1β in the eye 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 eye sample. In some embodiments, the method provided herein reduces the level of IL-1β by at least 10%. In some embodiments, the method provided herein reduces the level of IL-1β by at least 20%. In some embodiments, the method provided herein reduces the level of IL-1β by at least 30%. In some embodiments, the method provided herein reduces the level of IL-1β by at least 50%. In some embodiments, the method provided herein reduces the level of IL-1β by at least 70%.

[0068] In some embodiments, the method provided herein reduces the level of IL-6 in the eye 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 eye sample. In some embodiments, the method provided herein reduces the level of IL-6 by at least 10%. In some embodiments, the method provided herein reduces the level of IL-6 by at least 20%. In some embodiments, the method provided herein reduces the level of IL-6 by at least 30%. In some embodiments, the method provided herein reduces the level of IL-6 by at least 50%. In some embodiments, the method provided herein reduces the level of IL-6 by at least 70%.

[0069] In some embodiments, the method provided herein reduces the level of VEGF in the eye 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 eye sample. In some embodiments, the method provided herein reduces the level of VEGF by at least 10%. In some embodiments, the method provided herein reduces the level of VEGF by at least 20%. In some embodiments, the method provided herein reduces the level of VEGF by at least 30%. In some embodiments, the method provided herein reduces the level of VEGF by at least 50%. In some embodiments, the method provided herein reduces the level of VEGF by at least 70%. In some embodiments, the method provided herein reduces the level of ICAM-1 in the eye 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 eye sample. In some embodiments, the method provided herein reduces the level of ICAM-1 by at least 10%. In some embodiments, the method provided herein reduces the level of ICAM-1 by at least 20%. In some embodiments, the method provided herein reduces the level of ICAM-1 by at least 30%. In some embodiments, the method provided herein reduces the level of ICAM-1 by at least 50%. In some embodiments, the method provided herein reduces the level of ICAM-1 by at least 70%.

[0070] In some embodiments, the method provided herein reduces the level of VCAM-1 in the eye 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 eye sample. In some embodiments, the method provided herein reduces the level of VCAM-1 by at least 10%. In some embodiments, the method provided herein reduces the level of VCAM-1 by at least 20%. In some embodiments, the method provided herein reduces the level of VCAM-1 by at least 30%. In some embodiments, the method provided herein reduces the level of VCAM-1 by at least 50%. In some embodiments, the method provided herein reduces the level of VCAM-1 by at least 70%.

[0071] In some embodiments, the method provided herein reduces the level of MCP1 in the eye 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 eye sample. In some embodiments, the method provided herein reduces the level of MCP1 by at least 10%. In some embodiments, the method provided herein reduces the level of MCP1 by at least 20%. In some embodiments, the method provided herein reduces the level of MCP1 by at least 30%. In some embodiments, the method provided herein reduces the level of MCP1 by at least 50%. In some embodiments, the method provided herein reduces 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 or prevent further deterioration of typical symptoms of diabetic retinopathy in the retinal vascular system. Other measures of vascular permeability and 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 a subject before receiving 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 decreased night vision, blurred vision, floating spots, black spots or flashing lights in the field of vision, fluctuating visual acuity, color vision impairment, dark or empty areas of vision, vision loss, and sudden, severe, painless vision loss. In some embodiments, subjects treated according to the methods provided herein may experience one or more of the following effects: improved visual acuity, reduced vision loss, improved night vision, improved low-light vision, improved reading ability, improved peripheral vision, reduced spots in the field of vision, reduced flashing lights in the field of vision, 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 subjects are human. The ETDRS grading scale uses three standard photographs. Standard Photograph 1 shows mild microaneurysms and hemorrhages, Standard Photograph 2A shows moderate microaneurysms and hemorrhages, and Standard Photograph 2B shows severe microaneurysms and hemorrhages. In ETDRS, microaneurysms are defined as red spots with sharp margins and a longest dimension of 125 μm. This is distinguished from hemorrhages, which are defined as red spots with irregular margins and a dimension greater than 125 μm. To compare non-proliferative diabetic retinopathy (NPDR) with standard fundus photographs, the grade scores for describing non-proliferative retinopathy (NPDR), using the extent of retina covered with microaneurysms and / or hemorrhages, 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 of a subject 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 a control subject or a subject before receiving the method disclosed. In some embodiments, the methods provided herein reduce the average ETDRS grade score of a subject with DR by at least 0.2. In some embodiments, the methods provided herein reduce the average ETDRS grade score of a subject with DR by at least 0.5.In some embodiments, the method provided herein reduces the average ETDRS grade score in subjects with DR by at least 1. In some embodiments, the method provided herein reduces the average ETDRS grade score in subjects with DR by at least 1.5. In some embodiments, the method provided herein reduces the average ETDRS grade score in subjects with DR by at least 2. In some embodiments, the method provided herein reduces the average ETDRS grade score in subjects with DR by at least 2.5. In some embodiments, the subjects are human.

[0075] In some embodiments, the methods provided herein prevent diabetic retinopathy. The methods can be administered to patients at risk of developing diabetic retinopathy. In such subjects, prevention of diabetic retinopathy can be monitored by maintenance of visual acuity or absence of typical features of diabetic retinopathy. For example, subjects prophylactically administered with an 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, cotyledonous spots, neovascularization (new blood vessel formation 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 scar tissue formation, glaucoma, decreased night vision, blurred vision, floating spots, black spots or flashing lights in the visual field, fluctuating visual acuity, color vision impairment, dark or empty areas of vision, vision loss, sudden severe painless vision loss, tractional retinal detachment, macular edema, venous dilation, and intraretinal microvascular abnormalities. In some embodiments, the methods provided herein prevent or reduce macular edema. Macular edema can be seen as a bulge and blurring of the retinal layer on slit-lamp microscopy.

[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, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years after the initial diagnosis of diabetes. In some embodiments, the disclosed methods can be used to reduce, improve, lessen the severity of, or reverse one or more symptoms of diabetic retinopathy. In some embodiments, a method of treating diabetic retinopathy using an anti-ceramide antibody or its antigen-binding fragment can reduce, improve, lessen the severity of, or reverse one or more of the following symptoms: microaneurysms, hemorrhage, intraretinal microvascular abnormalities, venous ligation, cotyledonous spots, neovascularization (new blood vessel formation in other locations and the optic nerve), fibrous proliferation in other locations and the optic nerve, preretinal and vitreous hemorrhage, retinal detachment due to scar tissue formation, visual impairment, glaucoma, decreased night vision, blurred vision, floating spots, black spots or flashing lights in the visual field, sudden severe painless visual impairment, tractional 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 vascular system, including, but not limited to, permeability, NFκB levels, inflammatory marker levels, apoptosis incidence, sphingolipid metabolism, and reendothelialization. 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 examinations, etc. In some embodiments, the methods for treating diabetic retinopathy disclosed herein reduce NFκB and / or other inflammatory marker levels in the retinal vascular system. 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 incidence of apoptosis in the retinal vascular system. As discussed above, apoptosis can be measured by conventional means, e.g., 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 for treating diabetic retinopathy disclosed herein can increase the re-endothelialization of healthy tissue. [Examples]

[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, the damaging effects of diabetic retinopathy on the retinal vascular system were simulated using a mouse ischemic-reperfusion (I / R) model of diabetic retinopathy. Retinal ischemic-reperfusion (I / R): Each mouse had one I / R eye and one undamaged control eye. Retinal I / R was created by temporarily increasing intraocular pressure (IOP) to 90 mmHg as follows: Male C57BL / 6J mice weighing 25-30 g were anesthetized. A 30-gauge needle attached to a saline injection line was cannulated into the anterior chamber of one eye. IOP was measured in the mouse eye using a handheld tonometer (TONO Pen; Medtronic Solan, Jacksonville, FL) and adjusted to 80-90 mmHg using a pressure injector (Infu-surg; Ethox Corp., Buffalo, NY). The other eye of the same animal was set as the control. The duration of ischemia was 90 minutes in the mice. After ischemia, the needle was removed, IOP was normalized, and retinal circulation reflow was visually recorded. Animals were euthanized at different times after I / R injury. The retina was isolated 2 or 7 days after retinal I / R.

[0079] Vehicle control or anti-ceramide scFv administration: 24 hours prior to I / R, control mice were injected with a vehicle (phosphate-buffered saline), and test mice were intravitreously injected with 1 μL of anti-ceramide scFv 6B5 at a concentration of 1.73 mg / mL. 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. Possible further measurements: In comparative experiments using this model, permeability, inflammatory markers, and sphingolipid metabolism will be measured after 48 hours, and apoptosis, cell-free capillaries, and sphingolipid metabolism will be measured after 7 days, as shown in Figures 2A and 2B.

[0080] Retinal vascular permeability: The retina was 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). After 2 hours, blood was collected from each mouse and centrifuged to obtain plasma; the animals were perfused with 1% formaldehyde and the nuclei were removed. The retina was removed, flat-mounted with four slits, and placed on a glass slide 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 field regions were selected to retrieve images from each sample. The retina was mechanically destroyed 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 in untreated I / R eyes for each of the tested markers (TNFα, IL-1β, IL6, ICAM-1, VCAM-1, and MCP1). The decreases observed for TNFα, IL6, ICAM-1, and MCP1 were statistically significant. Figure 2B shows clear differences in retinal vascular permeability, as indicated by fluorescence microscopy images and quantitative analysis. Permeability was significantly higher in untreated I / R eyes than in either control eyes or anti-ceramide-treated I / R eyes. This example demonstrates that a single intravitreous 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 We used an STZ-induced model of diabetic retinopathy in rats to simulate the damaging effects of diabetic retinopathy on the retinal vascular system, for example, as shown in Figure 3. Induction of diabetes in animals and STZ-induced diabetes: Male Sprague-Dawley rats weighing 237-283 g were induced with diabetes by a single intraperitoneal injection of 65 mg of streptozotocin (STZ) per kg of body weight. The rats were fed Harlan-Teklad experimental feed (number 8,640) and water ad libitum. Body weight gain and blood glucose levels were monitored every two weeks in both the control group and the STZ-induced diabetes group. Vehicle control or anti-ceramide scFv administration: After confirmation of diabetes (hyperglycemia of ≥250 mg / dL, 7-10 days after STZ injection), diabetic control rats received vehicle PBS injection, and diabetic test rats received intravitreal injection of 2 μL of anti-ceramide scFv6B5 at 1.73 mg / mL. Non-diabetic control rats also received either vehicle control or anti-ceramide scFv injection.

[0083] Retinal vascular permeability analysis: The retina was 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). After 2 hours, blood was collected from each mouse and centrifuged to obtain plasma; the animals were perfused with 1% formaldehyde and the nuclei were removed. The retina was removed, flat-mounted with four slits, and placed on a glass slide 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 field regions were selected to retrieve images from each sample. The retina was mechanically destroyed 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, the experiments are performed in a mouse model or a diabetes model with db / db leptin deficiency, as shown in Figure 3. Furthermore, 6–8 weeks after diabetes induction in the STZ-induced model, the retinal vascular system of each animal is evaluated for permeability, NFκB and inflammatory markers, apoptosis, and sphingolipid metabolism, as shown in Figure 3. result The results shown in Figure 4 demonstrate that diabetic rats treated with 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 this rat model.

[0085] (Example 3) Intravitreal vs. systemic administration of anti-ceramide scFv to treat diabetes-induced pro-inflammatory changes in DR. This study will compare the effects of intravitreous anti-ceramide treatment versus systemic anti-ceramide treatment on diabetes-induced pro-inflammatory changes in a DR mouse model. STZ-induced type 1 diabetic mice will receive a single intravitreous or systemic (intravenous) anti-ceramide treatment with 6B5 scFv at the onset of diabetes. Control, diabetic, and anti-ceramide-treated diabetic animals will be sacrificed 6–8 weeks after STZ administration. The retinas will be isolated from each animal, and inflammatory cytokines, growth factors, and adhesion molecules will be profiled, and endothelial ASM expression and activity in the retina will be measured. These measurements will be performed using qPCR, Western blotting, ESI-MS / MS, fluorescence microscopy, and immunogold-electron microscopy. 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 conducted to evaluate the ability of single intravitreal administration of anti-ceramide antibodies or their antigen-binding fragments to treat existing DR pathologies.

[0086] (Example 4) Intravitreal vs. systemic administration of anti-ceramide scFv for the treatment of DR vascular dysfunction In a DR mouse model, experiments are conducted to compare the effects of intravitreous versus systemic anti-ceramide treatment on retinal vascular permeability. 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 intravitreous or systemic (intravenous) dose of anti-ceramide scFv at the onset of diabetes. Six to eight weeks after the induction of diabetes, 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 in DR vascular dysfunction. Additional experiments will be conducted to evaluate the ability of single intravitreal administration of anti-ceramide antibodies or their antigen-binding fragments to treat existing DR pathologies.

[0087] (Example 5) Anti-ceramide scFv vs. DHA or desipramine to reduce pro-inflammatory changes in DR In a DR mouse model, experiments were conducted to compare the effects of a single intravitreal or systemic anti-ceramide scFv treatment on diabetes-induced pro-inflammatory changes with those of DHA-rich fish oil or desipramine treatment. STZ-induced type 1 diabetic mice received a single forced oral 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. Animals were sacrificed 6–8 weeks after STZ-induced diabetes, and the retinas were isolated. Inflammatory cytokines, growth factors, and adhesion molecules were profiled. Endothelial ASM expression and activity were measured. These measurements were performed using a series of qPCR, Western blotting, 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 conducted to evaluate the ability of single intravitreal administration of anti-ceramide antibodies or their antigen-binding fragments to treat existing DR pathologies.

[0088] (Example 6) Anti-ceramide scFv administration versus DHA or desipramine to treat DR vascular insufficiency Experiments will be conducted in a DR mouse model to compare the effects of a single intravitreous or systemic anti-ceramide scFv treatment versus a single DHA-rich fish oil or desipramine treatment on retinal vascular permeability. STZ-induced type 1 diabetic mice will receive a single forced oral dose of DHA-rich fish oil, a single intravenous injection of desipramine, a single intravitreous 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 will be evaluated 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 in DR vascular dysfunction. Additional experiments will be conducted to evaluate the ability of single intravitreal administration of anti-ceramide antibodies or their antigen-binding fragments to treat existing DR pathologies.

[0089] (Example 7) Anti-ceramide scFv administration inhibits stress-induced apoptosis. The experiment will be conducted to demonstrate that anti-ceramide scFv is effective against cultured human retinal endothelial cells (HRECs) and inhibits stress-induced apoptosis under conditions in which anti-angiogenic agents are ineffective. The mechanism of DR therapy with anti-ceramide scFv and anti-angiogenic agents is distinct from previously characterized mechanisms. Though not limited to theory, it is thought that there is persistent death and remodeling in diabetes that responds to anti-ceramide but cannot be addressed by anti-VEGF treatment. Therefore, while anti-VEGF can only prevent neoangiogenesis, administration of anti-ceramide antibodies can prevent ongoing endothelial cell death, promote vascular recovery, and prevent hypoxia and subsequent neoangiogenesis. To test whether anti-ceramide scFv works in vitro under conditions in which anti-angiogenic agents are ineffective, HRECs will be subjected to a variety of stress conditions that induce ASM / ceramide-mediated apoptotic death (e.g., exposure to IL-1β and TNF cytokines or H2O2). HREC is treated with either an anti-VEGF inhibitor (anti-VEGFR2 DC101 Ab or the VEGFR TK inhibitor sorafenib) or an anti-ceramide antibody. Administration of anti-ceramide scFv is expected to have a greater therapeutic effect in inhibiting endothelial cell death.

[0090] (Example 8) Desipramine treatment of diabetic mice Diabetes mellitus was induced in male C57BL / 6J mice (20-25g) by intraperitoneal injection of SZT (65mg / 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 collected from the dorsal vein of the foot. Diabetes mellitus was confirmed by blood glucose levels >300mg / dL. Weight loss, polyuria, water content, and food intake were monitored daily. Desipramine was added to water approximately two weeks after the initial SZT injection to initiate treatment on the first day diabetes was confirmed. Desipramine administration had no effect on control animals. However, the diabetic animals became dehydrated, losing more than 25% of their body weight within 2-4 days, at which point the experiment had to be stopped in accordance with IACUC's regulations on the use of humane animals. Because diabetic animals exhibit polydipsia and polyurea, desipramine in water may influence their drinking habits, potentially leading to dehydration. Therefore, this experiment was repeated using IP injections of 2 mg / ml desipramine at a dose of 20 mg / kg. Similar to desipramine in water treatment, control animals showed good tolerance to this drug. However, diabetic animals exhibited abnormal tremors, became debilitated, stopped grooming, eating, and drinking, and lost more than 25% of their body weight within 5-7 days of daily injections. At this point, the experiment had to be terminated in accordance with IACUC's regulations on the humane use of animals.

[0091] (Example 9) DHA treatment of wild-type mice Male Sprague-Dawley rats (237-283) were fed either a control AIN-93M refined rodent diet containing 10% calories of soybean oil containing 50.8% linoleic acid from Dyets Inc. (Bethlehem, PA), or a DHA-rich fish oil diet in which half the amount of soybean oil, or 5% of the calories, was replaced with Menhaden oil containing 10.26% DHA and 14.16% EPA. Nine months after dietary intervention, the rat retinal vascular system was isolated by trypsin digestion, and two independent researchers systematically counted acellular capillaries in the midretina. As shown in Figure 7, DHA-rich fish oil treatment was harmful to the control animals and caused the development of acellular capillaries. While the health benefits of fish oil as a triglyceride-lowering agent are well accepted, the dose required for retinal effects is approximately 5% of calorie intake, which corresponds to about 12g of fish oil per day, three times the recommended amount for humans. High doses of DHA in humans, particularly those with diabetes, are associated with many well-known side effects, including hyperglycemia, excessive bleeding, reduced 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 for treating or preventing diabetic retinopathy in a subject requiring such treatment, comprising administering an anti-ceramide antibody or an antigen-binding fragment thereof to the subject orally. Embodiment 2. The method according to Embodiment 1, wherein the anti-ceramide antibody or its antigen-binding fragment is a single-strand variable fragment (scFv). Embodiment 3. The method according to Embodiment 1 or 2, wherein ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon's capsule administration, administration into the epichoroidal space (SCS), administration into the epiciliociliary space, and intravitreous administration.

[0093] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the administration is intravitreous. Embodiment 5. The method according to any one of Embodiments 1 to 4, wherein the subject has undergone pretreatment for diabetic retinopathy. Embodiment 6. The method according to Embodiment 5, wherein the subject did not respond to pretreatment for diabetic retinopathy. Embodiment 7. The method according to Embodiment 5 or 6, wherein the pretreatment is a treatment 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 according to 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 according to any one of Embodiments 1 to 8, wherein the anti-ceramide antibody or its antigen-binding fragment is administered as a single dose. Embodiment 10. The method according to any one of Embodiments 1 to 8, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least two weeks, at least three weeks, or at least four weeks.

[0094] Embodiment 11. The method according to any one of Embodiments 1 to 8, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of approximately 2 to 4 weeks. Embodiment 12. The method according to any one of Embodiments 1 to 8, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of 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 according to any one of Embodiments 1 to 8, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of approximately 1 to 6 months. Embodiment 14. The method according to any one of Embodiments 1 to 8, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least one year. Embodiment 15. The method according to any one of Embodiments 1 to 14, wherein an anti-ceramide antibody or its antigen-binding fragment is administered before the onset of one or more symptoms of diabetic retinopathy. Embodiment 16. The method according to any one of Embodiments 1 to 14, wherein an anti-ceramide antibody or its antigen-binding fragment is administered after the onset of one or more symptoms of diabetic retinopathy.

[0095] Embodiment 17. The method according to any one of Embodiments 1 to 14, wherein an anti-ceramide antibody or its antigen-binding fragment is administered during the non-proliferative phase of diabetic retinopathy. Embodiment 18. The method according to Embodiment 17, wherein an anti-ceramide antibody or its antigen-binding fragment is administered during the proliferative phase of diabetic retinopathy. Embodiment 19. A method for treating or preventing diabetic retinopathy in a subject requiring such treatment, comprising administering an anti-ceramide antibody or an antigen-binding fragment thereof to the subject, wherein the subject has undergone pretreatment for diabetic retinopathy. Embodiment 20. The method according to Embodiment 19, wherein the subject did not respond to pretreatment for diabetic retinopathy. Embodiment 21. The method according to Embodiment 19 or 20, wherein the anti-ceramide antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv). Embodiment 22. The method according to any one of Embodiments 19 to 21, wherein the administration is by eye administration. Embodiment 23. The method according to Embodiment 22, wherein ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon's capsule administration, administration into the suprachoroidal space (SCS), administration into the supracial space, and intravitreous administration.

[0096] Embodiment 24. The method according to Embodiment 22 or 23, wherein ocular administration is intravitreous administration. Embodiment 25. The method according to any one of Embodiments 19 to 24, wherein the pretreatment was vitrectomy, laser surgery, steroids, and / or anti-vascular endothelial growth factor (VEGF) therapy. Embodiment 26. The method according to 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 according to any one of Embodiments 19 to 26, wherein an anti-ceramide antibody or its antigen-binding fragment is administered as a single dose. Embodiment 28. The method according to any one of Embodiments 19 to 26, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least two weeks, at least three weeks, or at least four weeks. Embodiment 29. The method according to any one of Embodiments 19 to 26, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of approximately 2 to 4 weeks.

[0097] Embodiment 30. The method according to any one of Embodiments 19 to 26, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of 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 according to any one of Embodiments 19 to 26, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of about 1 month to about 6 months. Embodiment 32. The method according to any one of Embodiments 19 to 26, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least one year. Embodiment 33. The method according to any one of Embodiments 19 to 32, wherein an anti-ceramide antibody or its antigen-binding fragment is administered before the onset of one or more symptoms of diabetic retinopathy. Embodiment 34. The method according to any one of Embodiments 19 to 32, wherein an anti-ceramide antibody or its antigen-binding fragment is administered after the onset of one or more symptoms of diabetic retinopathy. Embodiment 35. The method according to any one of Embodiments 19 to 32, wherein an anti-ceramide antibody or its antigen-binding fragment is administered during the non-proliferative phase of diabetic retinopathy.

[0098] Embodiment 36. The method according to any one of Embodiments 19 to 32, wherein an anti-ceramide antibody or its antigen-binding fragment is administered during the proliferative phase of diabetic retinopathy. Embodiment 37. A method comprising administering a single dose of an anti-ceramide antibody or an antigen-binding fragment thereof to a subject, for treating or preventing diabetic retinopathy in a subject. 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 an antigen-binding fragment thereof to the subject, wherein the two or more doses are spaced at least two weeks apart. Embodiment 39. The method according to Embodiment 38, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least two weeks, at least three weeks, or at least four weeks. Embodiment 40. The method according to Embodiment 38, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of approximately 2 to 4 weeks. Embodiment 41. The method according to Embodiment 38, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of 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 according to Embodiment 38, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of approximately 1 to 6 months. Embodiment 43. The method according to Embodiment 38, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses with an interval of at least one year between doses. Embodiment 44. The method according to any one of Embodiments 37 to 43, wherein the anti-ceramide antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv). Embodiment 45. The method according to any one of Embodiments 37 to 44, wherein the administration is by eye administration. Embodiment 46. The method according to Embodiment 45, wherein ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon's capsule administration, administration into the suprachoroidal space (SCS), administration into the supracial space, and intravitreous administration. Embodiment 47. The method according to Embodiment 45 or 46, wherein the ocular administration is intravitreous administration. Embodiment 48. The method according to any one of Embodiments 37 to 47, wherein the subject has undergone pretreatment for diabetic retinopathy.

[0100] Embodiment 49. The method according to Embodiment 48, wherein the subject did not respond to pretreatment for diabetic retinopathy. Embodiment 50. The method according to Embodiment 48 or 49, wherein the pretreatment was vitrectomy, laser surgery, steroids, and / or anti-vascular endothelial growth factor (VEGF) therapy. Embodiment 51. The method according to 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 according to any one of Embodiments 37 to 51, wherein an anti-ceramide antibody or its antigen-binding fragment is administered before the onset of one or more symptoms of diabetic retinopathy. Embodiment 53. The method according to any one of Embodiments 37 to 51, wherein an anti-ceramide antibody or its antigen-binding fragment is administered after the onset of one or more symptoms of diabetic retinopathy. Embodiment 54. The method according to any one of Embodiments 37 to 51, wherein an anti-ceramide antibody or its antigen-binding fragment is administered during the non-proliferative phase of diabetic retinopathy. Embodiment 55. The method according to any one of Embodiments 37 to 51, wherein an anti-ceramide antibody or its antigen-binding fragment is administered during the proliferative phase of diabetic retinopathy.

[0101] Embodiment 56. A method for treating an inflammatory disease of the eye, comprising administering an anti-ceramide antibody or an antigen-binding fragment thereof to the eye. Embodiment 57. The method according to Embodiment 56, wherein the anti-ceramide antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv). Embodiment 58. The method according to Embodiment 56 or 57, wherein ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon's capsule administration, administration into the suprachoroidal space (SCS), administration into the supracial space, and intravitreous administration. Embodiment 59. The method according to any one of Embodiments 56 to 58, wherein the inflammatory disease of the eye 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, cystic macular edema, papilledema, recurrent ischemia, ocular hemorrhage, and proliferative vitreoretinopathy. Embodiment 60. The method according to any one of Embodiments 56 to 59, wherein the ocular administration is intravitreous administration.

[0102] Embodiment 61. The method according to any one of Embodiments 56 to 60, wherein the subject has undergone pretreatment for diabetic retinopathy. Embodiment 62. The method according to Embodiment 61, wherein the subject did not respond to pretreatment for diabetic retinopathy. Embodiment 63. The method according to Embodiment 61 or 62, wherein the pretreatment is a treatment 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 according to 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 according to any one of Embodiments 56 to 64, wherein an anti-ceramide antibody or its antigen-binding fragment is administered as a single dose. Embodiment 66. The method according to any one of Embodiments 56 to 64, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least two weeks, at least three weeks, or at least four weeks. Embodiment 67. The method according to any one of Embodiments 56 to 64, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of about two weeks to about four weeks.

[0103] Embodiment 68. The method according to any one of Embodiments 56 to 64, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of 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 according to any one of Embodiments 56 to 64, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of about 1 month to about 6 months. Embodiment 70. The method according to any one of Embodiments 56 to 64, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses with an interval of at least one year between doses. Embodiment 71. The method according to any one of Embodiments 56 to 70, wherein an anti-ceramide antibody or its antigen-binding fragment is administered before the onset of one or more symptoms of an inflammatory disease of the eye. Embodiment 72. The method according to any one of Embodiments 56 to 70, wherein an anti-ceramide antibody or its antigen-binding fragment is administered after the onset of one or more symptoms of an inflammatory disease of the eye.

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

[0105] Embodiment 77. An anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V HThe method according to any one of Embodiments 1 to 72, wherein the method comprises a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of GYTFTNYWMH (SEQ ID NO: 33), an HCDR2 containing the amino acid sequence of AIYPGDSDTSYNQKFKG (SEQ ID NO: 34), and an HCDR3 containing the amino acid sequence of GLYYGYD (SEQ ID NO: 35), and VL comprises a light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence of KSSQSLIDSDGKTFLN (SEQ ID NO: 36), an LCDR2 containing the amino acid sequence of LVSKLDS (SEQ ID NO: 37), and an LCDR3 containing the amino acid sequence of WQGTHFPYT (SEQ ID NO: 38). Embodiment 78.V H It contains the amino acid sequence of SEQ ID NO: 39, V L The method according to any one of Embodiments 1 to 72, wherein the amino acid sequence of Sequence ID No. 40 is included. Embodiment 79. The method according to any one of Embodiments 1 to 72 and 77 to 78, wherein the anti-ceramide antibody or its antigen-binding fragment is a 2A2 antibody. Embodiment 80. The method according to any one of Embodiments 1 to 72 and 77 to 78, wherein the anti-ceramide antibody or its antigen-binding fragment is 2A2 scFv.

[0106] Embodiment 81. An anti-ceramide antibody or its antigen-binding fragment comprises a variable heavy chain (VH) and a variable light chain (VL), a) VH includes a heavy chain complementarity determination region 1 (HCDR1) containing or consisting of amino acid sequences selected from SEQ ID NOs: 1 and 43, an HCDR2 containing or consisting of amino acid sequences selected from SEQ ID NOs: 44-47, and an HCDR3 containing or consisting of the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3), and b) The method according to any one of Embodiments 1 to 72, wherein 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 according to Embodiment 81, wherein HCDR1 contains or consists of the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 contains or consists of the amino acid sequence YNYPRDGSTKYNEKFQG (SEQ ID NO: 44). Embodiment 83. The method according to Embodiment 81, wherein HCDR1 contains or consists of the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 contains or consists of the amino acid sequence YNYPREGSTKYNEKFQG (SEQ ID NO: 45). Embodiment 84. The method according to Embodiment 81, wherein HCDR1 contains or consists of the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 contains or consists of the amino acid sequence YNYPRDVSTKYNEKFQG (SEQ ID NO: 46).

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

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

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

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

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

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

[0113] Embodiment 106. The method according to any one of Embodiments 81 to 104, wherein the anti-ceramide antibody or its antigen-binding fragment is h6B5 scFv. Embodiment 107. An anti-ceramide antibody or its antigen-binding fragment has a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of sequence number 48, V L The method according to any one of Embodiments 1 to 72, wherein the amino acid sequence of Sequence ID No. 53 is included. Embodiment 108. Anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of sequence number 48, V L The method according to any one of Embodiments 1 to 72, wherein the amino acid sequence of Sequence ID No. 55 is included. Embodiment 109. Anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of sequence number 49, V L The method according to any one of Embodiments 1 to 72, wherein the amino acid sequence of Sequence ID No. 53 is included.

[0114] Embodiment 110. An anti-ceramide antibody or its antigen-binding fragment has a variable heavy chain (V H ) and variable light chain (V L ) including V HIt contains the amino acid sequence of sequence number 49, V L The method according to any one of Embodiments 1 to 72, wherein the amino acid sequence of Sequence ID No. 54 is included. Embodiment 111. An anti-ceramide antibody or its antigen-binding fragment has a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of SEQ ID NO: 50, V L The method according to any one of Embodiments 1 to 72, wherein the amino acid sequence of Sequence ID No. 53 is included. Embodiment 112. Anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of SEQ ID NO: 50, V L The method according to any one of Embodiments 1 to 72, wherein the amino acid sequence of Sequence ID No. 54 is included. Embodiment 113. Anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of sequence number 51, V L The method according to any one of Embodiments 1 to 72, wherein the amino acid sequence of Sequence ID No. 53 is included. Embodiment 114. An anti-ceramide antibody or its antigen-binding fragment has a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of SEQ ID NO: 52, V L The method according to any one of Embodiments 1 to 72, wherein the amino acid sequence of Sequence ID No. 53 is included.

[0115] Embodiment 115. The method according to any one of Embodiments 107 to 114, wherein the anti-ceramide antibody or its antigen-binding fragment is a humanized antibody. Embodiment 116. The method according to any one of Embodiments 107 to 114, wherein the anti-ceramide antibody or its antigen-binding fragment is a humanized scFv. Embodiment 117. The method according to any one of Embodiments 1 to 116, wherein preventing diabetic retinopathy or inflammatory eye disease is a method comprising delaying the onset of diabetic retinopathy or inflammatory eye disease. Embodiment 118. The method according to any one of Embodiments 1 to 117, wherein one or more symptoms of diabetic retinopathy or inflammatory disease of the eye are reduced in a subject compared to a control subject or compared to a subject before treatment with an anti-ceramide antibody or its antigen-binding fragment. Embodiment 119. The method according to Embodiment 118, wherein one or more symptoms of diabetic retinopathy or inflammatory disease of the eye are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemic-reperfusion injury, retinal leakage area, and occlusion disruption. Embodiment 120. The method according to Embodiment 118 or 119, wherein one or more symptoms of diabetic retinopathy or inflammatory disease of the eye 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 to a subject before treatment with an anti-ceramide antibody or its antigen-binding fragment.

[0116] Embodiment 121. The method according to 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 a control eye or compared to the expression level in the eye of a subject before treatment with an anti-ceramide antibody or its antigen-binding fragment. Embodiment 122. The method according to Embodiment 121, wherein one or more inflammatory markers are selected from cytokines, growth factors, and adhesion molecules. Embodiment 123. The method according to Embodiment 122, wherein the cytokine is selected from TNFα, IL-1β, IL-6, or MCP1. Embodiment 124. The method according to Embodiment 122, wherein the growth factor is VEGF. Embodiment 125. The method according to Embodiment 122, wherein the adhesive molecule is ICAM-1 or VCAM-1. Embodiment 126. The method according to any one of Embodiments 121 to 125, wherein the expression level of one or more inflammatory markers in the target 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 the control target eye, or compared to the expression level in the target eye before treatment with an anti-ceramide antibody or its antigen-binding fragment.

[0117] Embodiment 127. The method according to any one of Embodiments 1 to 126, wherein one or more visual parameters are increased in a subject compared to the visual parameters of a control subject, or compared to the visual parameters of a subject before treatment with an anti-ceramide antibody or its antigen-binding fragment. Embodiment 128. The method according to Embodiment 127, wherein 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 according to any one of Embodiments 1 to 128, wherein retinal vascular permeability in the target 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 target eye, or compared to the expression level in the target eye before treatment with an anti-ceramide antibody or its antigen-binding fragment. Embodiment 130. The method according to any one of Embodiments 1 to 129, wherein the mean early-treatment diabetic retinopathy study (ETDRS) grade score in the control 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 the control group, or compared to the mean ETDRS grade score in the control group before treatment with an anti-ceramide antibody or its antigen-binding fragment.

[0118] Embedding by reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. However, nothing in any reference, article, publication, patent, patent publication, or patent application cited herein shall be, and should not be, construed as, an affirmation or in any form of suggestion that they constitute valid prior art or part of common general knowledge in any country of the world.

Claims

1. A method for treating or preventing diabetic retinopathy in a subject requiring such treatment, comprising administering an anti-ceramide antibody or its antigen-binding fragment to the subject's eyes.

2. The method according to claim 1, wherein the anti-ceramide antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv).

3. The method according to claim 1 or 2, wherein the ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon's capsule administration, administration into the suprachoroidal space (SCS), administration into the supracial space, and intravitreous administration.

4. The method according to any one of claims 1 to 3, wherein the administration is intravitreous.

5. The method according to any one of claims 1 to 4, wherein the subject has undergone pretreatment for diabetic retinopathy.

6. The method according to claim 5, wherein the subject did not respond to pretreatment for diabetic retinopathy.

7. The method according to claim 5 or 6, wherein the pretreatment is a treatment procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroid and anti-vascular endothelial growth factor (VEGF) therapy.

8. The method according to claim 7, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

9. The method according to any one of claims 1 to 8, wherein the anti-ceramide antibody or its antigen-binding fragment is administered as a single dose.

10. The method according to any one of claims 1 to 8, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least two weeks, at least three weeks, or at least four weeks.

11. The method according to any one of claims 1 to 8, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of about two weeks to about four weeks.

12. The method according to any one of claims 1 to 8, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of 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.

13. The method according to any one of claims 1 to 8, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of about one month to about six months.

14. The method according to any one of claims 1 to 8, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses with an interval of at least one year between doses.

15. The method according to any one of claims 1 to 14, wherein an anti-ceramide antibody or its antigen-binding fragment is administered before the onset of one or more symptoms of diabetic retinopathy.

16. The method according to any one of claims 1 to 14, wherein an anti-ceramide antibody or its antigen-binding fragment is administered after the onset of one or more symptoms of diabetic retinopathy.

17. The method according to any one of claims 1 to 14, wherein an anti-ceramide antibody or its antigen-binding fragment is administered during the non-proliferative phase of diabetic retinopathy.

18. The method according to claim 17, wherein an anti-ceramide antibody or its antigen-binding fragment is administered during the proliferative phase of diabetic retinopathy.

19. A method for treating or preventing diabetic retinopathy in a subject requiring such treatment, comprising administering an anti-ceramide antibody or an antigen-binding fragment thereof to the subject, wherein the subject has undergone prior treatment for diabetic retinopathy.

20. The method according to claim 19, wherein the subject did not respond to pretreatment for diabetic retinopathy.

21. The method according to claim 19 or 20, wherein the anti-ceramide antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv).

22. The method according to any one of claims 19 to 21, wherein the administration is by eye administration.

23. The method according to claim 22, wherein ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon's capsule administration, administration into the suprachoroidal space (SCS), administration into the supracial space, and intravitreous administration.

24. The method according to claim 22 or 23, wherein the ocular administration is intravitreous administration.

25. The method according to any one of claims 19 to 24, wherein the pretreatment was vitrectomy, laser surgery, steroids, and / or anti-vascular endothelial growth factor (VEGF) therapy.

26. The method according to claim 25, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

27. The method according to any one of claims 19 to 26, wherein an anti-ceramide antibody or its antigen-binding fragment is administered as a single dose.

28. The method according to any one of claims 19 to 26, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least two weeks, at least three weeks, or at least four weeks.

29. The method according to any one of claims 19 to 26, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of about two weeks to about four weeks.

30. The method according to any one of claims 19 to 26, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of 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.

31. The method according to any one of claims 19 to 26, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of about one month to about six months.

32. The method according to any one of claims 19 to 26, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses with an interval of at least one year between doses.

33. The method according to any one of claims 19 to 32, wherein an anti-ceramide antibody or its antigen-binding fragment is administered before the onset of one or more symptoms of diabetic retinopathy.

34. The method according to any one of claims 19 to 32, wherein an anti-ceramide antibody or its antigen-binding fragment is administered after the onset of one or more symptoms of diabetic retinopathy.

35. The method according to any one of claims 19 to 32, wherein an anti-ceramide antibody or its antigen-binding fragment is administered during the non-proliferative phase of diabetic retinopathy.

36. The method according to any one of claims 19 to 32, wherein an anti-ceramide antibody or its antigen-binding fragment is administered during the proliferative phase of diabetic retinopathy.

37. A method for treating or preventing diabetic retinopathy in a subject, comprising administering a single dose of an anti-ceramide antibody or its antigen-binding fragment to the subject.

38. A method for treating or preventing diabetic retinopathy in a subject, comprising administering two or more doses of an anti-ceramide antibody or an antigen-binding fragment thereof to the subject, wherein the two or more doses are administered at least two weeks apart.

39. The method according to claim 38, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least two weeks, at least three weeks, or at least four weeks.

40. The method according to claim 38, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of about two weeks to about four weeks.

41. The method according to claim 38, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of 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.

42. The method according to claim 38, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of about one month to about six months.

43. The method according to claim 38, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses with an interval of at least one year between doses.

44. The method according to any one of claims 37 to 43, wherein the anti-ceramide antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv).

45. The method according to any one of claims 37 to 44, wherein the administration is by eye administration.

46. The method according to claim 45, wherein ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon's capsule administration, administration into the suprachoroidal space (SCS), administration into the supracial space, and intravitreous administration.

47. The method according to claim 45 or 46, wherein the ocular administration is intravitreous administration.

48. The method according to any one of claims 37 to 47, wherein the subject has undergone pretreatment for diabetic retinopathy.

49. The method according to claim 48, wherein the subject did not respond to pretreatment for diabetic retinopathy.

50. The method according to claim 48 or 49, wherein the pretreatment was vitrectomy, laser surgery, steroids, and / or anti-vascular endothelial growth factor (VEGF) therapy.

51. The method according to claim 50, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

52. The method according to any one of claims 37 to 51, wherein an anti-ceramide antibody or its antigen-binding fragment is administered before the onset of one or more symptoms of diabetic retinopathy.

53. The method according to any one of claims 37 to 51, wherein an anti-ceramide antibody or its antigen-binding fragment is administered after the onset of one or more symptoms of diabetic retinopathy.

54. The method according to any one of claims 37 to 51, wherein an anti-ceramide antibody or its antigen-binding fragment is administered during the non-proliferative phase of diabetic retinopathy.

55. The method according to any one of claims 37 to 51, wherein an anti-ceramide antibody or its antigen-binding fragment is administered during the proliferative phase of diabetic retinopathy.

56. A method for treating an inflammatory disease of the eye, comprising administering an anti-ceramide antibody or an antigen-binding fragment thereof to the eye.

57. The method according to claim 56, wherein the anti-ceramide antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv).

58. The method according to claim 56 or 57, wherein ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, anterior chamber administration, injection into the anterior chamber via the limbus, limbal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon's capsule administration, administration into the suprachoroidal space (SCS), administration into the supracial space, and intravitreous administration.

59. The method according to any one of claims 56 to 58, wherein the inflammatory disease of the eye 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, cystic macular edema, papilledema, recurrent ischemia, ocular hemorrhage, and proliferative vitreoretinopathy.

60. The method according to any one of claims 56 to 59, wherein the ocular administration is intravitreous administration.

61. The method according to any one of claims 56 to 60, wherein the subject has undergone pretreatment for diabetic retinopathy.

62. The method according to claim 61, wherein the subject did not respond to pretreatment for diabetic retinopathy.

63. The method according to claim 61 or 62, wherein the pretreatment is a treatment procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroid and anti-vascular endothelial growth factor (VEGF) therapy.

64. The method according to claim 63, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

65. The method according to any one of claims 56 to 64, wherein an anti-ceramide antibody or its antigen-binding fragment is administered as a single dose.

66. The method according to any one of claims 56 to 64, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of at least two weeks, at least three weeks, or at least four weeks.

67. The method according to any one of claims 56 to 64, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of about two weeks to about four weeks.

68. The method according to any one of claims 56 to 64, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of 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.

69. The method according to any one of claims 56 to 64, wherein an anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses at intervals of about one month to about six months.

70. The method according to any one of claims 56 to 64, wherein the anti-ceramide antibody or its antigen-binding fragment is administered in two or more doses with an interval of at least one year between doses.

71. The method according to any one of claims 56 to 70, wherein an anti-ceramide antibody or its antigen-binding fragment is administered before the onset of one or more symptoms of an inflammatory disease of the eye.

72. The method according to any one of claims 56 to 70, wherein an anti-ceramide antibody or an antigen-binding fragment thereof is administered after the onset of one or more symptoms of an inflammatory disease of the eye.

73. Anti-ceramide antibody or its antigen-binding fragment, variable heavy chain (V H ) and variable light chain (V L ) including, a) V H This includes a heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence of GYTFTDHTIH (SEQ ID NO: 1), HCDR2 containing the amino acid sequence of YNYPRDGSTKYNEKFKG (SEQ ID NO: 2), and HCDR3 containing the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3), and b) V L The method according to any one of claims 1 to 72, comprising a light chain complementarity determining region 1 (LCDR1) containing the amino acid sequence of RASKSISKYLA (SEQ ID NO: 4), an LCDR2 containing the amino acid sequence of SGSTLQS (SEQ ID NO: 5), and an LCDR3 containing the amino acid sequence of QQHNEYPWT (SEQ ID NO: 6).

74. V H It contains the amino acid sequence of Sequence ID No. 7, V L The method according to any one of claims 1 to 72, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO:

8.

75. The method according to any one of claims 1 to 74, wherein the anti-ceramide antibody or its antigen-binding fragment is a 6B5 antibody.

76. The method according to any one of claims 1 to 74, wherein the anti-ceramide antibody or its antigen-binding fragment is 6B5 scFv.

77. Anti-ceramide antibody or its antigen-binding fragment, variable heavy chain (V H ) and variable light chain (V L ) including, a) V H 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 AIYPGDDSDTSYNQKFKLG (SEQ ID NO: 34), and an HCDR3 comprising the amino acid sequence of GLYYYGD (SEQ ID NO: 35), and b) V L The method according to any one of claims 1 to 72, comprising a light chain complementarity determining region 1 (LCDR1) containing the amino acid sequence of KSSQSLIDSDGKTFLN (SEQ ID NO: 36), an LCDR2 containing the amino acid sequence of LVSKLDS (SEQ ID NO: 37), and an LCDR3 containing the amino acid sequence of WQGTHFPYT (SEQ ID NO: 38).

78. V H It contains the amino acid sequence of sequence number 39, V L The method according to any one of claims 1 to 72, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO:

40.

79. The method according to any one of claims 1 to 72 and 77 to 78, wherein the anti-ceramide antibody or its antigen-binding fragment is a 2A2 antibody.

80. The method according to any one of claims 1 to 72 and 77 to 78, wherein the anti-ceramide antibody or its antigen-binding fragment is 2A2 scFv.

81. Anti-ceramide antibody or its antigen-binding fragment, variable heavy chain (V H ) and variable light chain (V L ) including, a) V H This includes a heavy chain complementarity determination region 1 (HCDR1) containing or consisting of amino acid sequences selected from SEQ ID NOs: 1 and 43, an HCDR2 containing or consisting of amino acid sequences selected from SEQ ID NOs: 44 to 47, and an HCDR3 containing or consisting of the amino acid sequence of GFITTVVPSAY (SEQ ID NO: 3), and b) V L The method according to any one of claims 1 to 72, comprising 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.

82. The method according to claim 81, wherein HCDR1 contains or comprises the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 contains or comprises the amino acid sequence YNYPRDGSTKYNEKFQG (SEQ ID NO: 44).

83. The method according to claim 81, wherein HCDR1 contains or comprises the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 contains or comprises the amino acid sequence YNYPREGSTKYNEKFQG (SEQ ID NO: 45).

84. The method according to claim 81, wherein HCDR1 comprises or consists of the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1) and HCDR2 comprises or consists of the amino acid sequence YNYPRDVSTKYNEKFQG (SEQ ID NO: 46).

85. The method according to claim 81, wherein HCDR1 contains or consists of the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1), and HCDR2 contains or consists of the amino acid sequence YNYPRDGSTKYAEKFQG (SEQ ID NO: 47).

86. The method according to claim 81, wherein HCDR1 contains or comprises the amino acid sequence GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 contains or comprises the amino acid sequence YNYPRDGSTKYNEKFQG (SEQ ID NO: 44).

87. The method according to claim 81, wherein HCDR1 contains or comprises the amino acid sequence GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 contains or comprises the amino acid sequence YNYPREGSTKYNEKFQG (SEQ ID NO: 45).

88. The method according to claim 81, wherein HCDR1 contains or comprises the amino acid sequence GYTFTDHTMH (SEQ ID NO: 43) and HCDR2 contains or comprises the amino acid sequence YNYPRDVSTKYNEKFQG (SEQ ID NO: 46).

89. The method according to claim 81, wherein HCDR1 contains or comprises the amino acid sequence GYTFTDHTMH (SEQ ID NO: 43), and HCDR2 contains or comprises the amino acid sequence YNYPRDGSTKYAEKFQG (SEQ ID NO: 47).

90. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 48, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO:

53.

91. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 48, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO:

54.

92. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 48, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO:

55.

93. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 49, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO:

53.

94. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 49, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO:

54.

95. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 49, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO:

55.

96. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 50, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO:

53.

97. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 50, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO:

54.

98. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 50, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO:

55.

99. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 51, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO:

53.

100. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 51, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO:

54.

101. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO: 51, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, or 100% identical to SEQ ID NO:

55.

102. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, 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%, or 100% identical to SEQ ID NO:

53.

103. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, 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%, or 100% identical to SEQ ID NO:

54.

104. The method according to claim 81, wherein VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97%, 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%, or 100% identical to SEQ ID NO:

55.

105. The method according to any one of claims 81 to 104, wherein the anti-ceramide antibody or its antigen-binding fragment is a humanized 6B5 (h6B5) antibody.

106. The method according to any one of claims 81 to 104, wherein the anti-ceramide antibody or its antigen-binding fragment is h6B5 scFv.

107. Anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of sequence number 48, V L The method according to any one of claims 1 to 72, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO:

53.

108. Anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of sequence number 48, V L The method according to any one of claims 1 to 72, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO:

55.

109. Anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of sequence number 49, V L The method according to any one of claims 1 to 72, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO:

53.

110. Anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of sequence number 49, V L The method according to any one of claims 1 to 72, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO:

54.

111. Anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of sequence number 50, V L The method according to any one of claims 1 to 72, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO:

53.

112. Anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of sequence number 50, V L The method according to any one of claims 1 to 72, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO:

54.

113. Anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of sequence number 51, V L The method according to any one of claims 1 to 72, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO:

53.

114. Anti-ceramide antibody or its antigen-binding fragment is a variable heavy chain (V H ) and variable light chain (V L ) including V H It contains the amino acid sequence of sequence number 52, V L The method according to any one of claims 1 to 72, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO:

53.

115. The method according to any one of claims 107 to 114, wherein the anti-ceramide antibody or its antigen-binding fragment is a humanized antibody.

116. The method according to any one of claims 107 to 114, wherein the anti-ceramide antibody or its antigen-binding fragment is a humanized scFv.

117. The method according to any one of claims 1 to 116, wherein preventing diabetic retinopathy or inflammatory eye disease is a method that delays the onset of diabetic retinopathy or inflammatory eye disease.

118. The method according to any one of claims 1 to 117, wherein one or more symptoms of diabetic retinopathy or inflammatory disease of the eye are reduced in a subject compared to a control subject or compared to a subject before treatment with an anti-ceramide antibody or its antigen-binding fragment.

119. The method according to claim 118, wherein one or more symptoms of diabetic retinopathy or inflammatory disease of the eye are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemic-reperfusion injury, retinal leakage area, and occlusion disruption.

120. The method according to claim 118 or 119, wherein one or more symptoms of diabetic retinopathy or inflammatory disease of the eye 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 to a subject before treatment with an anti-ceramide antibody or its antigen-binding fragment.

121. The method according to any one of claims 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 a subject before treatment with an anti-ceramide antibody or its antigen-binding fragment.

122. The method according to claim 121, wherein one or more inflammatory markers are selected from cytokines, growth factors, and adhesion molecules.

123. The method according to claim 122, wherein the cytokine is selected from TNFα, IL-1β, IL-6, or MCP1.

124. The method according to claim 122, wherein the growth factor is VEGF.

125. The method according to claim 122, wherein the adhesive molecule is ICAM-1 or VCAM-1.

126. The method according to any one of claims 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 the control subject, or compared to the expression level in the eye of the subject before treatment with an anti-ceramide antibody or its antigen-binding fragment.

127. The method according to any one of claims 1 to 126, wherein 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 an anti-ceramide antibody or its antigen-binding fragment.

128. The method according to claim 127, wherein one or more visual parameters are selected from peripheral vision, night vision, color vision, distance vision, near vision, and visual clarity.

129. The method according to any one of claims 1 to 128, wherein the retinal vascular permeability in the target eye is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the retinal vascular permeability in the control eye of the target eye, or compared to the expression level in the target eye before treatment with an anti-ceramide antibody or its antigen-binding fragment.

130. The method according to any one of claims 1 to 129, wherein the mean early-treatment diabetic retinopathy study (ETDRS) grade score in the control 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 the control group, or compared to the mean ETDRS grade score in the control group before treatment with an anti-ceramide antibody or its antigen-binding fragment.

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