RU2843013C1 - Methods and compositions for treating diabetic retinopathy - Google Patents

Abstract

FIELD: medical science.

SUBSTANCE: group of inventions relates to methods of treating or preventing diabetic retinopathy. Disclosed are versions of a method for treating or preventing diabetic retinopathy in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an anti-ceramide antibody or an antigen-binding fragment thereof, wherein said anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL). VH includes HCDR1 containing the amino acid sequence SEQ ID NO: 1, HCDR2 containing the amino acid sequence SEQ ID NO: 45, and HCDR3 containing the amino acid sequence SEQ ID NO: 3, and VL contains LCDR1, including amino acid sequence SEQ ID NO: 4, LCDR2 containing the amino acid sequence SEQ ID NO: 5, and LCDR3 containing the amino acid sequence SEQ ID NO: 6.

EFFECT: administering said anti-ceramide antibodies or antigen-binding fragments provides selective inhibition of inflammatory markers, neovascularisation and other symptoms of diabetic retinopathy in the eye without inducing negative side effects associated with direct inhibition of ASM.

73 cl, 7 dwg, 2 tbl, 9 ex

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/907,287, filed September 27, 2019, the contents of which are incorporated herein by reference in their entirety.

Sequence Listing Statement

[0002] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. Machine-readable copy of the sequence listing: file name: CERA-01l_01WO_SeqList_ST25.txt, date of recording: September 28, 2020, file size 32.9 kilobytes.

Field of technology

[0003] The present invention relates to anti-ceramide compositions and methods of using the same for treating diabetic retinopathy.

State of the art

[0004] Diabetic retinopathy (DR) affects the blood vessels in the light-sensitive tissue called the retina, which lines the back of the eye. It is the most common cause of vision loss among people with diabetes and the leading cause of vision impairment and blindness among working-age adults. Diabetic retinopathy results in swelling in an area of the retina called the macula, called diabetic macular edema (DME).

[0005] Current treatments for DR and DME include anti-VEGF immunotherapy, which has some efficacy in the treatment of both neovascular diabetic retinopathy (DR) and diabetic macular edema (DME). However, several large clinical trials show that approximately 40% of patients do not respond to anti-VEGF therapy. In addition, anti-VEGF therapy is targeted at a very late stage of the disease, when complete restoration of retinal damage is difficult.

[0006] There remains an unmet need for safe and effective treatments for DR and DME.

BRIEF DESCRIPTION OF DRAWINGS

[0007] Fig. 1 shows an example of an experimental design for measuring the effect of intravitreal administration of anti-ceramide scFv in a mouse model of diabetic retinopathy involving ischemia-reperfusion (I/R) injury.

[0008] Fig. 2A and Fig. 2B show the results of intravitreal administration of anti-ceramide scFv in mice with retinal ischemia-reperfusion (I/R) injury in a mouse model of diabetic retinopathy. Fig. 2A shows the change in expression for TNFα, IL-1β, IL6, ICAM-1, VCAM-1, and MCP1 in the following order: control eyes (white bars), I/R eyes (black bars), and I/R eyes with anti-ceramide scFv (gray bars) compared to cyclophilin A expression. Fig. Figure 2B shows retinal vascular permeability demonstrated by fluorescence microscopy images and quantitative histogram analysis in control (left panel, left column), VR (middle panel, middle column) and I/R eyes treated with anti-ceramide scFv (right panel, right column). Scale bars in images are 50 μm.

[0009] Fig. 3 shows an example of an experimental design for measuring the effect of intravitreal administration of anti-ceramide scFv in a mouse model of diabetes.

[0010] Fig. 4 shows the results of administration of anti-ceramide scFv in diabetic rats.

[0011] Fig. 5 shows an example of an experimental design to compare the effects of intravitreal and systemic administration of anti-ceramide scFv in a mouse model of diabetic retinopathy.

[0012] Fig. 6 shows an example of an experimental design to compare the effects of anti-ceramide scFv administration and DHA administration in a mouse model of diabetic retinopathy.

[0013] Fig. 7 shows acellular capillaries in DHA-treated control mice.

Brief description

[0014] The present invention relates to methods of treating diabetic retinopathy comprising ocular administration of an anti-ceramide antibody or antigen-binding fragment thereof. Methods of treating subjects who have received previous treatment for diabetic retinopathy are also provided. In some embodiments, the present invention further relates to methods of treating diabetic retinopathy with a single dose of an anti-ceramide antibody or antigen-binding fragment thereof. Further, a method of treating diabetic retinopathy with two or more doses of an anti-ceramide antibody or antigen-binding fragment thereof separated by a period of at least two weeks to at least one year is provided. Another aspect of the present invention relates to a method of treating an inflammatory disease of the eye with an anti-ceramide antibody or antigen-binding fragment thereof.

[0015] In some embodiments, the present invention relates to a method of treating or preventing diabetic retinopathy in a subject in need thereof, comprising ocularly administering to the subject an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

[0016] In some embodiments, the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, anterior chamber injection through the temporal margin, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, suprachoroidal space (SCS) administration, supraciliary space administration, and intravitreal administration. In some embodiments, the administration is intravitreal administration.

[0017] In some embodiments, the subject has received prior treatment for diabetic retinopathy. In some embodiments, the subject has not responded to prior treatment for diabetic retinopathy. In some embodiments, the prior treatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroid therapy and anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

[0018] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in a single dose. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least two weeks, at least three weeks, or at least four weeks. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about two weeks to about four weeks. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by 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 antigen-binding fragment thereof is administered in two or more doses separated by a period of from about one month to about six months. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least one year. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative stage of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative stage of diabetic retinopathy.

[0019] In some embodiments, a method is provided for treating or preventing diabetic retinopathy in a subject in need thereof, comprising administering an anti-ceramide antibody or antigen-binding fragment thereof to the subject, wherein said subject has received prior treatment for diabetic retinopathy. In some embodiments, said subject has not responded to prior treatment for diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

[0020] In some embodiments, the administration is ocular administration. In some embodiments, the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, transtemporal anterior chamber injection, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, suprachoroidal space (SCS) administration, supraciliary space administration, and intravitreal administration. In some embodiments, the ocular administration is intravitreal administration.

[0021] In some embodiments, the prior treatment was vitrectomy, laser surgery, steroid, and/or anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

[0022] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in a single dose. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least two weeks, at least three weeks, or at least four weeks. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about two weeks to about four weeks. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by 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 antigen-binding fragment thereof is administered in two or more doses separated by a period of from about one month to about six months. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least one year.

[0023] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative stage of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative stage of diabetic retinopathy.

[0024] In some embodiments, a method of treating or preventing diabetic retinopathy in a subject is provided, comprising administering to the subject a single dose of an anti-ceramide antibody or antigen-binding fragment thereof.

[0025] In some embodiments, a method of treating or preventing diabetic retinopathy in a subject is provided, comprising administering to the subject two or more doses of an anti-ceramide antibody or antigen-binding fragment thereof, wherein the two or more doses are separated by at least two weeks. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least two weeks, at least three weeks, or at least four weeks. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of about two weeks to about four weeks. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by 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 antigen-binding fragment thereof is administered in two or more doses separated by a period of from about one month to about six months. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least one year.

[0026] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

[0027] In some embodiments, the administration is ocular administration. In some embodiments, the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, anterior chamber injection through the temporal margin, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, suprachoroidal space (SCS) administration, supraciliary space administration, or intravitreal administration. In some embodiments, the ocular administration is intravitreal administration.

[0028] In some embodiments, the subject has received prior treatment for diabetic retinopathy. In some embodiments, the subject has not responded to prior treatment for diabetic retinopathy. In some embodiments, the prior treatment was vitrectomy, laser surgery, steroid, and/or anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

[0029] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative stage of diabetic retinopathy. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative stage of diabetic retinopathy.

[0030] In some embodiments, the present invention provides a method of treating an inflammatory disease of the eye comprising ocular administration of an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

[0031] In some embodiments, ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, anterior chamber injection through the temporal margin, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, suprachoroidal space (SCS) administration, supraciliary space administration, or intravitreal administration. In some embodiments, said inflammatory eye disease is selected from the group consisting of retinal neovascularization, choroidal neovascularization, corneal neovascularization, macular degeneration, age-related macular degeneration, diabetic retinopathy, vitreous hemorrhage, retinal hemorrhage, choroiditis, neovascular glaucoma, choroidal disease, telangiectasia, retinal artery occlusion, retinal vein occlusion, chorioretinitis, epiretinal membrane, choroidal neoplasm, retinopathy of prematurity, cystoid macular edema, optic disc edema, recurrent ischemia, ocular hemorrhage, and proliferative vitreoretinopathy.

[0032] In some embodiments, ocular administration is intravitreal administration.

[0033] In some embodiments, the subject has received a prior treatment for diabetic retinopathy. In some embodiments, the subject has not responded to a prior treatment for diabetic retinopathy. In some embodiments, the prior treatment was a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroid therapy and anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

[0034] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in a single dose. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least two weeks, at least three weeks, or at least four weeks. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about two weeks to about four weeks. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by 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 antigen-binding fragment thereof is administered in two or more doses separated by a period of from about one month to about six months. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least one year.

[0035] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered prior to the occurrence of one or more symptoms of an inflammatory disease of the eye. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is administered after the occurrence of one or more symptoms of an inflammatory disease of the eye.

[0036] In some embodiments, an anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V _H ) and a variable light chain (V _L ), wherein V _H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1), HCDR2 comprising the amino acid sequence YNYPRDGSTKYNEKFKG (SEQ ID NO: 2), and HCDR3 comprising the amino acid sequence GFITTVVPSAY (SEQ ID NO: 3), and wherein V _L comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence RASKSISKYLA (SEQ ID NO: 4), LCDR2 comprising the amino acid sequence SGSTLQS (SEQ ID NO: 5), and LCDR3 comprising the amino acid sequence QQHNEYPWT (SEQ ID NO: 6). In some embodiments, V _H comprises the amino acid sequence of SEQ ID NO: 7, and wherein V _L comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is antibody 6B5. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is scFv 6B5.

[0037] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a humanized 6B5 (h6B5) antibody. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is an h6B5 scFv. In some embodiments, the h6B5 antibody, scFv, or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises a heavy chain complementarity determining region 1 (HCDR1) comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 and 43, HCDR2 comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 44-47, and HCDR3 comprising or consisting of the amino acid sequence GFITTVVPSAY (SEQ ID NO: 3), and wherein the VL comprises a light chain complementarity determining region 1 (LCDR1) comprising or consisting of the amino acid sequence RASKSISKYLA (SEQ ID NO: 4), LCDR2 comprising or consisting of the amino acid sequence SGSTLQS (SEQ ID NO: 5), and LCDR3 comprising or consisting of the amino acid sequence QQHNEYPWT (SEQ ID NO: 6). In some embodiments, HCDR1 comprises or consists of the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1), 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 GYTFTDHTIH (SEQ ID NO: 1), 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 GYTFTDHTIH (SEQ ID NO: 1), 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 GYTFTDHTIH (SEQ ID NO: 1), and HCDR2 comprises or consists of the amino acid sequence YN YPRDGS TKYAEKFQG (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 YNYPREGSTK YNEKFQG (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 YNYPRDVSTK YNEKFQG (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 comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 48, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 53. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 48, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 54. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 48, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 49, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least at least 97% identical, or 100% identical to SEQ ID NO: 53. In some embodiments, the VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:

49, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 54. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 49, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 55. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, 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% identical, or 100% identical to SEQ ID NO: 53. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO:

50, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 54. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 50, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 55. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, 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% identical, or 100% identical to SEQ ID NO: 53. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 51, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55. is at least 97% identical, 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% identical, or 100% identical to SEQ ID NO: 55. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 52, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 53. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 52, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 54. In some embodiments, VH comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 52, and VL comprises or consists of an amino acid sequence that is at least 90%, at least 95%, at least 97% identical, or 100% identical to SEQ ID NO: 55. In some embodiments, the antibody h6B5 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 wherein 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 wherein 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 wherein 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 wherein VL comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein VH comprises the amino acid sequence of SEQ ID NO: 50, and wherein VL comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein VH comprises the amino acid sequence of SEQ ID NO: 50, and wherein VL comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein VH comprises the amino acid sequence of SEQ ID NO: 51, and wherein VL comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL), wherein VH comprises the amino acid sequence of SEQ ID NO: 52, and wherein VL comprises the amino acid sequence of SEQ ID NO: 53.

[0038] In some embodiments, an anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V _H ) and a variable light chain (V _L ), wherein V _H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence GYTFTNYWMH (SEQ ID NO: 33), HCDR2 comprising the amino acid sequence AIYPGDSDTSYNQKFKG (SEQ ID NO: 34), and HCDR3 comprising the amino acid sequence GLYYGYD (SEQ ID NO: 35), and wherein V _L comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence KSSQSLIDSDGKTFLN (SEQ ID NO: 36), LCDR2 comprising the amino acid sequence LVSKLDS (SEQ ID NO: 37), and LCDR3 comprising the amino acid sequence WQGTHFPYT (SEQ ID NO: 38). In some embodiments, V _H comprises the amino acid sequence of SEQ ID NO: 39, and wherein V _L comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is antibody 2A2. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is scFv 2A2.

[0039] In some embodiments, preventing diabetic retinopathy or an inflammatory eye disease comprises delaying the onset of diabetic retinopathy or an inflammatory eye disease. In some embodiments, one or more symptoms of diabetic retinopathy or an inflammatory eye disease in a subject are reduced compared to a control subject or compared to a subject before treatment with an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the one or more symptoms of diabetic retinopathy or an inflammatory eye disease are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemia-reperfusion injury, retinal leakage area, and occludin abnormality. In some embodiments, one or more symptoms of diabetic retinopathy or inflammatory eye disease are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject before treatment with an anti-ceramide antibody or antigen-binding fragment thereof.

[0040] In some embodiments, the expression level of one or more inflammatory markers in the eye is reduced compared to the expression level in the eye of the subject in a control or compared to the expression level in the eye of the subject before treatment with an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the one or more inflammatory markers are selected from a cytokine, a growth factor, and an adhesion molecule. In some embodiments, the cytokine is selected from TNFα, IL-1β, IL-6, or MCP1. In some embodiments, the growth factor is VEGF. In some embodiments, the adhesion molecule is ICAM-1 or VCAM-1. In some embodiments, the expression level of one or more inflammatory markers in the 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 subject in a control or compared to the expression level in the eye of the subject before treatment with an anti-ceramide antibody or antigen-binding fragment thereof.

[0041] In some embodiments, the subject has an increase in one or more visual parameters compared to the subject's visual parameters in a control or compared to the subject's visual parameters before treatment with an anti-ceramide antibody or antigen-binding fragment thereof. In some embodiments, the one or more visual parameters are selected from peripheral vision; night vision; color vision; distance vision; near vision; and visual clarity.

[0042] In some embodiments, the retinal vascular permeability 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 retinal vascular permeability in the eye of the subject in a control or compared to the expression level in the eye of the subject prior to treatment with an anti-ceramide antibody or antigen-binding fragment thereof.

[0043] In some embodiments, the mean early diabetic retinopathy score (ETDRS) in a group of subjects 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 score in a group of subjects in a control or compared to the mean ETDRS score in a group of subjects before treatment with an anti-ceramide antibody or antigen-binding fragment thereof.

Detailed description

Review

[0044] The present invention relates to compositions and methods for treating diabetic retinopathy. In some embodiments, compositions of anti-ceramide antibodies and antigen-binding fragments thereof (e.g., scFvs) and methods of use for treating or preventing diabetic retinopathy are provided. Such compositions and methods can be used to treat diabetic retinopathy in patients who have not previously received other treatment for diabetic retinopathy, such as anti-VEGF antibody therapy. In addition, a method for intravitreal administration of anti-ceramide scFv is provided.

Definitions

[0045] The use of objects in the singular in this application and the appended claims includes the plural of the said objects, unless the context clearly indicates otherwise.

[0046] As used herein, the term "and/or" is used herein to mean "and" or "or" unless otherwise specified.

[0047] Throughout the description, unless the context clearly requires otherwise, the words "comprise (include)" or variations thereof such as "comprises" or "comprising" shall be construed to include the stated element or integer or group of elements or integers, but not to the exclusion of any other element or integer or group of elements or integers.

[0048] As used in this application, the terms "about" and "approximately" are used as equivalents. Any numbers used in this application, with or without about/approximately, are intended to cover any normal variations understood by one of skill in the art. In some embodiments, the term "about" or "approximately" refers to a range of values that are within 10% or less in either direction (greater or less) of a stated reference value, unless otherwise stated or apparent from context (except where such number exceeds 100% of a possible value or falls below 0% of a possible value).

[0049] The term "sample" refers to a biological composition (e.g., a cell or tissue portion) that is being analyzed and/or modified. In some embodiments, the sample is a "primary sample" because it is obtained directly from the subject; in some embodiments, the "sample" is the result of processing the primary sample, such as to remove certain components and/or isolate or purify certain components of interest.

[0050] The term "subject" includes animals, such as, for example, mammals. In some embodiments, the mammal is a primate. In some embodiments, the mammal is a human. In some embodiments, the subjects are livestock, such as cattle, sheep, goats, cows, pigs, and the like; or companion animals, such as dogs and cats. In some embodiments (e.g., particularly in the context of research), the subjects are rodents (e.g., mice, rats, hamsters), rabbits, primates, or pigs, such as inbred pigs, etc. The terms "subject" and "patient" are used interchangeably herein. In some embodiments, the subject can be a neonate, a juvenile, or an adult. Of particular interest are mammalian subjects. Mammalian species that can be treated using the present methods include dogs and cats; horses; cattle; sheep; etc. and primates, particularly humans. Animal models, especially small mammals (e.g. mice, rats, guinea pigs, hamsters, rabbits, etc.), can be used for experimental research.

[0051] As used herein, the terms "treatment," "treat," or "alleviate" refer to either therapeutic treatment or prophylactic/preventative treatment. A treatment is therapeutic if at least one symptom of a disease in an individual receiving the treatment is improved or the treatment can slow the worsening of a progressive disease in an individual or prevent the occurrence of additional comorbidities.

[0052] In the context of the present application, the term "effective amount" refers to the minimum amount of an agent or composition necessary to achieve a specific physiological effect. An effective amount of a particular agent may be represented in various ways depending on the nature of the agent, such as mass/volume, cell count/volume, particles/volume, (mass of agent)/(mass of subject), cell count/(mass of subject), or particles/(mass of subject). An effective amount of a particular agent may also be expressed as a half-maximal effective concentration (EC ₅₀ ), which refers to the concentration of the agent that results in a magnitude of a specific physiological response that is the midpoint between the reference level and the maximal level of the response.

[0053] The term "antibody" refers to an immunoglobulin (Ig) molecule capable of binding to a specific target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one epitope recognition site located in the variable region of the Ig molecule. As used herein, the term encompasses intact polyclonal or monoclonal antibodies and antigen-binding fragments thereof. For example, a native immunoglobulin molecule is composed of two heavy chain polypeptides and two light chain polypeptides. Each heavy chain polypeptide associates with a light chain polypeptide through interchain disulfide bonds between the heavy and light chain polypeptides to form two heterodimeric proteins or polypeptides (i.e., a protein composed of two heterologous polypeptide chains). The two heterodimeric proteins then associate through additional interchain disulfide bonds between the heavy chain polypeptides to form an immunoglobulin protein or polypeptide.

[0054] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment that comprises at least one complementarity determining region (CDR) of an immunoglobulin heavy and/or light chain that binds to at least one epitope of an antigen of interest. In this regard, the antigen-binding fragment of the antibodies described herein may comprise 1, 2, 3, 4, 5, or all 6 CDRs of the variable heavy chain (VH) and variable light chain (VL) sequences of antibodies that specifically bind ceramide. Antigen-binding fragments include proteins that comprise a portion of a full-length antibody, generally its antigen-binding or variable region, such as Fab, F(ab')2, Fab', Fv fragments, minibodies, diabodies, single-domain antibodies (dAbs), single-chain variable fragments (scFv), multispecific antibodies formed from antibody fragments, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site or fragment with the desired specificity. In certain embodiments, the antigen-binding fragment, rather than the intact antibody, is used to enhance tissue penetration or tumor penetration. In other embodiments, the antigen-binding fragments are further modified to increase serum half-life.

[0055] "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from a portion of an antibody that is capable of binding to Fc receptors on cells and/or complement component C1q, thereby mediating the effector function of the antibody. Fc stands for "crystal fragment," a fragment of an antibody that readily forms a protein crystal. Individual protein fragments, which were originally characterized by proteolytic cleavage, can determine the overall structure of an immunoglobulin protein. As originally defined in the literature, the Fc region is a homodimeric protein comprising two polypeptides that are disulfide-linked, each containing a hinge region, a CH2 domain, and a CH3 domain. However, more recently, the term has been applied to a single-chain monomeric component consisting of CH3, CH2, and at least a portion of the hinge sufficient to form a disulfide-linked dimer with a second such chain. Thus, and depending on the context, use of the terms "Fc region" or "Fc domain" will refer herein to either the dimeric form or to individual monomers that associate to form a dimeric protein. For a review of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol. 31:169-217, 1994. As used herein, the term Fc domain includes variants of naturally occurring sequences.

[0056] The term "immunoglobulin constant region" or "constant region" refers to a peptide or polypeptide sequence that corresponds to or is derived from a portion or all of one or more immunoglobulin constant domains (e.g., CH1, CH2, CH3). In certain embodiments, the constant region does not comprise a CH1 domain. In certain embodiments, the constant domains comprising the constant region are human.

[0057] The terms "light chain variable region" (also referred to as "light chain variable domain" or "VL") and "heavy chain variable region" (also referred to as "heavy chain variable domain" or "VH") refer to the variable binding region of the light and heavy chain of an antibody, respectively. The variable binding regions are comprised of discrete, well-defined subregions known as "complementarity determining regions" (CDRs) and "framework regions" (FRs).

[0058] The term "immunoglobulin light chain constant region" (also referred to as "light chain constant region" or "CL") is a constant region from a light chain of an antibody.

[0059] The term "immunoglobulin heavy chain constant region" (also referred to as "heavy chain constant region" or "CH") refers to the constant region of the heavy chain of an antibody. The CH is further divided, depending on the antibody isotype, into CH1, CH2, and CH3 domains (IgA, IgD, IgG) or CH1, CH2, CH3, and CH4 (IgE, IgM).

[0060] The term "F(ab)" refers to two protein fragments resulting from proteolytic cleavage of IgG molecules by the enzyme papain. Each F(ab) comprises a covalent heterodimer of a VH chain and a VL chain and contains an intact antigen-binding site. Each F(ab) is a monovalent antigen-binding fragment. The term "Fab'" refers to a fragment derived from F(ab')2 and may contain a small Fc portion. Each Fab' fragment is a monovalent antigen-binding fragment.

[0061] The term "F(ab')2" refers to the protein fragment of IgG produced by proteolytic cleavage with the enzyme pepsin. Each E(ab')2 fragment contains two E(ab') fragments and is therefore a bivalent antigen-binding fragment.

[0062] The "Fd fragment" contains the VH and CH1 domains.

[0063] An "Fv fragment" refers to a non-covalent VH::VL heterodimer that includes an antigen-binding site that retains most of the antigen recognition and binding capabilities of a native antibody molecule, but lacks the CH1 and CL domains found in 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, an Fv fragment can be produced by preferential proteolytic cleavage of IgM and, rarely, an IgG or IgA immunoglobulin molecule. Fv fragments, however, are more often produced using recombinant techniques known in the art.

[0064] The "dAb fragment" (Ward et al., Nature 341:544 546, 1989) contains the VH domain.

[0065] A "single chain antibody" or "scFv" is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, linked to a short linker peptide of ten to about 25 amino acids. The linker may be rich in glycine for flexibility and serine or threonine for solubility, and may either connect the N-terminus of the VH to the C-terminus of the VL or vice versa. The scFv retains the specificity of the parent immunoglobulin despite the removal of the constant regions and the introduction of the linker. In the present invention, any reference to antibodies or antibody fragments or their use is intended to include scFv molecules and their use.

[0066] “Minibodies” refer to a fusion protein comprising an scFv fused to a CH3 domain and are also included in the present application (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, e.g., Ward, E. S. et al, Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); PCT/US92/09965; WO94/13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Ni et al., Cancer Res., 56, 3055-3061, 1996.

[0067] The term "diabody" refers to a bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain using a linker that is too short to allow pairing between the two domains on one chain, thereby forcing the domains to pair with the complementary domains of another chain and creating two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993) and Poljak et al., Structure 2:1121-23 (1994)).

[0068] The term "nanobody" or "single-domain antibody" refers to an antigen-binding fragment consisting of a single monomeric variable domain of an antibody. The Nanoclone method is a method for producing nanobodies against a desired target based on automated high-throughput selection of B cells. (See WO 2006/079372).

[0069] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0070] The term "chimeric antibody" as used herein refers to a monoclonal antibody in which a portion of the heavy and/or light chain is identical to or homologous with corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) is identical to or homologous with corresponding sequences in antibodies derived from another species or belonging to a different class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity.

[0071] The term "single chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, linked to a short linker peptide of ten to about 25 amino acids.Huston et al. (1988) Proc. Nat. Acad. Sci. USA S5(16):5879-5883. The linker may join the N-terminus of VH to the C-terminus of VL or vice versa. A number of methods have been described for determining chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from the V region of an antibody into an scFv molecule that will fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Patent No. 4,946,778 to Ladner et al.

[0072] As used herein, the term "CDR" refers to the "complementarity determining region" of an immunoglobulin (antibody) molecule. CDRs are part of the variable domain in an antibody where the antibody 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 located in the variable (V) region of the polypeptide chain, CDR3 exhibiting the greatest variability because it is encoded by VJ recombination in the case of the light chain region and VDJ in the case of the heavy chain regions.

[0073] An "isolated antibody" is an antibody that (1) is not associated with natural components, including other natural antibodies, that accompany it in the native state, (2) does not contain other proteins from the same species, (3) is expressed by a cell of another species, or (4) is not found in nature.

[0074] The term "human antibody" includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In a preferred embodiment, all variable and constant domains are derived from human immunoglobulin sequences (a fully human antibody). These antibodies can be produced by a variety of methods, as described below.

[0075] As used herein, the term "humanized" refers to an antibody or antigen-binding fragment thereof that is derived from a non-human species and retains the antigen-binding properties of the parent non-human antibody. In some embodiments, antibody binding fragments (e.g., light and heavy chain variable regions, Fab, scFv) are humanized. Non-human antigen-binding fragments can be humanized using techniques known as CDR grafting (Jones et al., Nature 321:522 (1986)) and variants thereof, including “shape-shifting” (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al., Bio/Technol 1991 9:266-271), “hyperchimerization” (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol 148:1149-1154), and "veneering" (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD 18 antibodies." In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312). When derived from a non-human source, other regions of the antibody, such as the hinge region and constant region domains, can also be humanized.

[0076] As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not typically produce allergic or other serious adverse reactions when administered using methods well known in the art. Molecular entities and compositions that are approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans, are considered "pharmaceutically acceptable."

[0077] The terms "prevention," "prophylaxis," and "prophylactically" refer to the administration of a compound, such as an antibody against ceramide or an antigen-binding fragment thereof, prior to the onset of a disease (e.g., prior to the onset of certain symptoms of a disease). Preventing a disease may include reducing the likelihood that the disease will occur, delaying the onset of the disease, alleviating long-term symptoms, or slowing the potential progression of the disease.

[0078] As used herein, the term "specifically binds" refers to the ability of an antibody or antigen-binding fragment thereof to bind to a target antigen with a binding affinity (Ka) of at least 10 ⁵ M ^-1 , while not substantially binding to other components or antigens present in the mixture. Reference to an anti-ceramide antibody as used herein refers to an antibody or antigen-binding fragment thereof that specifically binds to ceramide.

[0079] As used herein, the term "sequence identity" refers to the relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. When a position in one sequence is occupied by the same nucleic acid base or amino acid residue at a corresponding position in a comparison sequence, the sequences are said to be "identical" at that position. Percent sequence identity is calculated by determining the number of positions at which an identical nucleic acid base or amino acid residue is found in both sequences to obtain 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 percent sequence identity. Percent sequence identity is determined by comparing two optimally aligned sequences in the comparison window. The comparison window for polynucleotide sequences can be, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more nucleic acids in length. The comparison window for polypeptide sequences can be, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids in length. In order to optimally align the sequences for comparison, a portion of the polynucleotide or polypeptide sequence in the comparison window can contain additions or deletions, called gaps, while the reference sequence remains constant. An optimal alignment is one that, even with gaps, yields the maximum possible number of "identical" positions between the reference and compared sequences. The percentage of "sequence identity" 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, which includes the BLASTN (for comparing nucleotide sequences) and BLASTP (for comparing polypeptide sequences) programs, the programs being based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873–5877, 1993). Using the BLAST 2 Sequences parameters, which were the default parameters as of September 1, 2004, the following can be used for segment size (3), gap opening penalty (11), gap widening penalty (1), gap closing (50), expected value (10), and any other desired parameter, including but not limited to the matrix option. Two nucleotide or amino acid sequences are said to have “substantially similar sequence identity” or “substantially 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.

[0080] "Angiogenesis" refers to the formation of new blood vessels.

[0081] As used herein, the term "ocular" or "ocular" refers to the administration of a drug into the eye and surrounding tissue. For example, in some embodiments, ocular administration includes: topical administration (e.g., administration to the ocular surface, such as the sclera), intraocular administration, subconjunctival administration (e.g., under the bulbar conjunctiva or under the palpebral conjunctiva), intracameral administration, injection into the anterior chamber through the temporal margin, intrastromal administration, intracorneal administration, subretinal administration, injection into the aqueous humor, sub-Tenon's administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, or intravitreal administration.

Anti-ceramide antibodies, antibody fragments and derivatives

[0082] The present invention relates to anti-ceramide antibodies and antigen-binding fragments thereof for the treatment of diabetic retinopathy. Ceramides are a family of waxy lipid molecules. Ceramide consists of sphingosine and a fatty acid. Ceramides are found in high concentrations within the cell membrane of eukaryotic cells, as they are components of the lipids that make up sphingomyelin, one of the major lipids in the lipid bilayer. Ceramide is involved in a variety of cellular signals, including the regulation of differentiation, proliferation, and programmed cell death (PCD). As a bioactive lipid, ceramide has been implicated in a variety of physiological functions, including apoptosis, cell growth arrest, differentiation, cell senescence, cell migration, and adhesion. Roles for ceramide and its downstream metabolites have also been suggested in a number of pathological conditions including cancer, neurodegeneration, diabetes, microbial pathogenesis, obesity and inflammation.

[0083] The sequences and properties of exemplary anti-ceramide antibodies are also disclosed in U.S. Patent Application Publication Nos. 2010/0239572 and 2017/0335014, each of which is incorporated herein by reference. The sequences of exemplary anti-ceramide antibodies are provided in Table 1. However, any anti-ceramide antibody or antigen-binding fragment thereof can be used in accordance with the disclosed methods and uses.

[0084] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a humanized 6B5 antibody (h6B5). In some embodiments, the h6B5 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. In some embodiments, the sequences of the h6B5 antibody are provided in U.S. Provisional Patent Application Ser. No. 62/991,232, filed March 18, 2020, the contents of which are incorporated herein by reference for all purposes. The sequences for each of the CDRs of the h6B5 antibody or antigen-binding fragment thereof are disclosed herein and are summarized in Table 2 below.

[0085] In some embodiments, the anti-ceramide antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a recombinant antibody, or a synthetic antibody. In some embodiments, the antigen-binding fragment of the anti-ceramide antibody is an antigen-binding fragment of any of the above. According to some embodiments of the present invention, the antigen-binding fragment of the anti-ceramide antibody is a Fab fragment, a Fab' fragment, a P(ab')2 fragment, an Fv fragment, an Fd fragment, a dAb fragment, a diabody, an scFv, or the like. In some embodiments, the anti-ceramide antibodies and antigen-binding fragments thereof are produced using recombinant DNA technologies. Methods for expressing and purifying recombinant proteins are well known in the art.

[0086] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). In some embodiments, the scFv comprises CDR sequences and/or variable chain sequences of antibodies 2A2, h2A2, 6C8, 7B10, 9H10, h6B5, or 6B5.

[0087] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the 2A2 antibody or antigen-binding fragment thereof as described in U.S. Patent Publication No. 2010/0239572. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the 6B5 antibody or antigen-binding fragment thereof as described in U.S. Patent Publication No. 2017/0335014. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is the h6B5 antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is an scFv. In some embodiments, the scFV comprises the CDR sequences of any of the antibodies disclosed in Table 1. In some embodiments, the scFv comprises the CDR sequences of 2A2. In some embodiments, the scFv comprises the CDR sequences of 6B5. In some embodiments, the scFv comprises h6B5 CDR sequences. In some embodiments, the scFv comprises h2A2 heavy and light chain variable sequences. In some embodiments, the scFv comprises 6B5 heavy and light chain variable sequences. In some embodiments, the scFv comprises h6B5 heavy and light chain variable sequences.

[0088] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof is of any immunoglobulin isotype. The immunoglobulin may be of any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. The IgG isotype is divided into subclasses in some species: IgG1, IgG2, IgG3, and IgG4 in humans and IgG1, IgG2a, IgG2b, and IgG3 in mice. In some embodiments, the anti-ceramide antibodies or antigen-binding fragments thereof comprise one or more modifications in the Fc region. Some modifications may provide desirable effector functions or serum half-life. In some embodiments, with appropriate Fc regions, a naked antibody bound to the cell surface can induce cytotoxicity, such as through antibody-dependent cellular cytotoxicity (ADCC) or by recruiting complement in complement-dependent cytotoxicity (CDC), or by recruiting non-specific cytotoxic cells that express one or more effector ligands that recognize the bound antibody on a target cell and subsequently cause phagocytosis of the target cell in antibody-dependent cell-mediated phagocytosis (ADCP), or by some other mechanisms. Where it is desirable to eliminate or reduce effector function in order to minimize side effects or therapeutic complications, some other Fc regions can be used. The Fc region of antibodies can be modified to increase binding affinity for FcRn and thus increase serum half-life. Alternatively, the Fc region may be conjugated to PEG or albumin to increase serum half-life or some other conjugation that results in the desired effect.

[0089] In some embodiments, the anti-ceramide antibody or antigen-binding fragment thereof comprises a detectable label or tag. Exemplary detectable labels include fluorescent labels, affinity labels, radioisotopes, luminescent markers, particle labels, chromophores, phosphorescent markers, and enzymatic labels. Exemplary fluorescent labels include GFP, RFP, and YFP. Exemplary enzymatic labels include horseradish peroxidase and alkaline phosphatase. Exemplary peptide tags include a His-tag, MBP, and streptavidin.

[0090] The detection means is determined by the selected label. The appearance of the label or its reaction products can be determined by the naked eye, in the case where the label is particulate and accumulates in appropriate quantities, or by means of instruments such as a spectrophotometer, luminometer, fluorimeter, or by means of ELISA or Western blotting.

Diabetic retinopathy

[0091] According to some embodiments of the present invention, methods and compositions for treating diabetic retinopathy are provided. Diabetic retinopathy refers to a medical condition in which the retina is damaged due to diabetes mellitus. Chronically high blood sugar from diabetes is associated with damage to the finest blood vessels in the retina, which leads to diabetic retinopathy. Diabetic retinopathy can cause fluid leakage or bleeding into the blood vessels of the retina, distorting vision. In the most advanced stage, new abnormal blood vessels proliferate on the surface of the retina, which can lead to scarring and cell loss in the retina.

[0092] In some embodiments, the disclosed methods and compositions can be used to treat various stages of diabetic retinopathy. Diabetic retinopathy can progress through a non-proliferative stage, also referred to as the early stage, and a proliferative stage, also referred to as the late stage.

[0093] In some embodiments, the present invention relates to methods and compositions that can be used to treat non-proliferative stage diabetic retinopathy. Non-proliferative diabetic retinopathy (NPDR) can be mild, moderate, or severe. Mild NPDR is characterized by microaneurysms in the blood vessels of the retina. These microaneurysms can leak fluid into the retina. As the disease progresses to moderate NPDR, the blood vessels of the retina can become distorted and lose their ability to transport blood, which leads to characteristic changes in the appearance of the retina and can contribute to diabetic macular edema. In severe NPDR, many more blood vessels are blocked, which deprives areas of the retina of blood circulation. These areas then secrete pro-angiogenic growth factors. Angiogenic molecules in the eye include VEGF, FGF, P1GF, TGF-alpha, TGF-beta, IGF, PDGF, MMPs, HGF/SF, TNF-alpha, CTGF, IL-1, IL-8, MCP-1, leptin, integrins, and angiogenin. In some embodiments, the methods of the present invention are characterized by preventing or reducing one or more markers of diabetic retinopathy. In some embodiments, the present methods can prevent, reduce, inhibit, or reduce the level of retinal microaneurysm, retinal fluid leakage, diabetic macular edema, and/or proangiogenic retinal growth factors. In some embodiments, one or more of VEGF, FGF, P1GF, TGF-alpha, TGF-beta, IGF, PDGF, MMPs, HGF/SF, TNF-alpha, CTGF, IL-1, IL-8, MCP-1, integrins, and angiogenin are reduced using the present methods.

[0094] In some embodiments, the present invention relates to methods and compositions that can be used to treat diabetic retinopathy in the proliferative stage. Proliferative diabetic retinopathy (PDR) is an advanced stage of the disease. In this stage, proangiogenic growth factors secreted by the retina cause the proliferation of new blood vessels that grow along the inner surface of the retina into the vitreous. The new blood vessels are fragile and are more likely to leak and bleed. The associated scar tissue can contract and cause retinal detachment (i.e., separation of the retina from the underlying tissue), which can lead to permanent vision loss. In some embodiments, the disclosed methods can prevent, reduce, inhibit, or reduce the level of retinal neovascularization, retinal hemorrhage, retinal scarring, retinal detachment, and vision loss.

[0095] In some embodiments, the present invention provides methods and compositions that can be used to prevent diabetic retinopathy by administering to a subject suffering from diabetes before the onset of diabetic retinopathy, such as before the onset of one or more symptoms thereof. Among the most consistent risk factors, the duration of diabetes is a strong predictor of the development and progression of retinopathy. Among young patients with diabetes, the prevalence 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, such as by more than 3 years, more than 5 years, more than 10 years, or more than 15 years after the onset of diabetes.

[0096] In some embodiments, the disclosed methods and compositions can be used to prevent diabetic retinopathy in a subject at risk for developing diabetic retinopathy, such as a subject having one or more risk factors associated with developing diabetic retinopathy. Significant risk factors include hyperglycemia, hypertension, hyperlipidemia, and kidney disease. Additional factors to consider include male gender, higher severity of diabetes (as indicated by use of insulin and oral diabetes therapy versus pill monotherapy or use of pill monotherapy versus no treatment), higher mean systolic blood pressure, and higher hemoglobin A1c.

Symptoms and detection

[0097] In some embodiments, the present methods can affect one or more symptoms and/or detectable markers of diabetic retinopathy. The early, non-proliferative stages of diabetic retinopathy are typically asymptomatic. In some embodiments, the methods and compositions of the present invention are intended for use in a subject with diabetes before symptoms of diabetic retinopathy appear. The disease often progresses undetected until vision is affected. Bleeding from retinal blood vessels in the early stages of the disease can cause floaters that may resolve on their own. Without prompt treatment, the bleeding often recurs, increasing the risk of permanent vision loss. When diabetic macular edema occurs, it can cause blurred vision. In some embodiments, the present compositions and methods can reduce the frequency, severity, or level of floaters, retinal bleeding, vision loss, and/or blurred vision. In some embodiments, one or more parameters of vision may be improved using the present methods, including, but not limited to, overall vision, peripheral vision, night vision, color vision, distance vision, near vision, and visual clarity.

[0098] Diabetic retinopathy and diabetic macular edema may be detected during a comprehensive eye examination that includes visual acuity testing (eye charts to measure a person's ability to see at different distances); tonometry (measurements of the pressure inside the eye); pupil dilation; and optical coherence tomography (OCT). During this examination, a doctor may check for one or more of the following: changes in the blood vessels of the retina, including new vessel formation, swelling, and bleeding; leakage of the blood vessels of the retina or warning signs of leakage of the blood vessels, such as fatty deposits, weakened vessel walls, and bulging vessel walls; swelling of the macular; changes in the lens, including changes in curvature or cataract formation; and damage to nerve tissue.

[0099] In some embodiments, the present compositions and methods can be used to treat a subject with any one or more of these symptoms of diabetic retinopathy. In some embodiments, the compositions and methods of the present invention can prevent, treat, or improve any one or more of these symptoms. If diabetic macular edema or severe diabetic retinopathy is suspected, a fluorescein angiogram can be used to look for damaged or leaky blood vessels. In this test, a fluorescent dye is injected into the bloodstream, often into a vein in the arm. Photographs of the retinal blood vessels are taken as the dye enters the eye. In some embodiments, the compositions of the present invention and the methods of the present invention can be used to prevent or reduce the incidence, prevalence, or severity of damaged and/or leaky blood vessels in the eye.

[0100] Current treatments for diabetic retinopathy include anti-VEGF therapy, steroids, laser surgery, and vitrectomy.

[0101] Anti-VEGF injection therapy. Anti-VEGF drugs are injected into the vitreous to block the action of the pro-angiogenic growth factor VEGF. Blocking VEGF can reverse abnormal blood vessel growth and reduce the amount of fluid in the retina. Available 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) to treat diabetic macular edema. Avastin® (bevacizumab) has been approved by the FDA to treat cancer, but is typically used to treat eye conditions, including diabetic macular edema. Most people need monthly injections of anti-VEGF therapy for the first six months of treatment. Thereafter, injections are required less frequently: typically three to four during the second half of treatment, about four during the second year of treatment, two in the third year, one in the fourth year, and none in the fifth year. Recent studies have shown that anti-VEGF treatment may be effective in treating diabetic macular edema and in slowing the progression of diabetic retinopathy, including PDR, so that anti-VEGF therapy is increasingly used as first-line therapy for PDR. However, several large clinical trials show that approximately 40% of patients do not respond to anti-VEGF therapy. In addition, anti-VEGF treatment is aimed at a very late stage of the disease, when complete restoration of the retinal lesions is difficult.

[0102] Laser surgery. In focal/sectoral macular laser surgery, several to hundreds of small laser burns are made to treat leaking blood vessels in areas of swelling near the center of the macula. Laser burns for diabetic macular edema slow the leakage of fluid, reducing swelling in the retina. The procedure is usually performed in a single session, but some patients may require more than one treatment. Focal/sectoral laser treatment can be used in combination with anti-VEGF therapy. For example, focal/sectoral laser treatment is sometimes used before anti-VEGF injections, sometimes on the same day or a few days after an anti-VEGF injection, and sometimes only when diabetic macular edema has not improved adequately after six months of anti-VEGF therapy.

[0103] PDR can also be treated with scatter laser surgery, sometimes called panretinal laser surgery or panretinal photocoagulation. The treatment involves 1,000 to 2,000 tiny laser burns in areas of the retina away from the macula. These laser burns are designed to cause abnormal constriction of the blood vessels. Although the treatment can be completed in one session, sometimes two or more sessions are required. Although it can preserve central vision, scatter laser surgery can cause some loss of side (peripheral), color, and night vision. Scatter laser surgery works best before the fragile new blood vessels begin to bleed.

[0104] Corticosteroids. Corticosteroids can be used alone or in combination with other medications or laser surgery to treat diabetic macular edema. Corticosteroids can be injected into the eye or implanted into the eye. The Ozurdex® (dexamethasone) implant is intended for short-term use, while the Iluvien® (fluocinolone acetonide) implant is more long-term. Both are biodegradable and release a sustained dose of corticosteroids to suppress diabetic macular edema.

[0105] Vitrectomy. Vitrectomy is the surgical removal of the vitreous gel in the center of the eye. The procedure is used to treat severe bleeding into the vitreous and is performed under local or general anesthesia. A clear saline solution is gently pumped into the eye through one or more holes to maintain pressure in the eye during surgery and to replace the removed vitreous. The same instruments used during vitrectomy can also be used to remove scar tissue or to repair a detached retina.

The role of ceramide and ASM in diabetic retinopathy

[0106] Without being limited by any one theory, it is believed that the present treatments are capable of suppressing inflammatory signaling in the retina by selectively inhibiting ceramide in the retina using ophthalmic treatment with anti-ceramide antibodies or antigen-binding fragments thereof.

[0107] Sphingolipids are a major component of membrane microdomains, and ceramide-rich microdomains appear to be a prerequisite for inflammatory cytokine signaling. Acid sphingomyelinase (ASM) and neutral sphingomyelinase (NSM) are key regulatory enzymes of sphingolipid metabolism, promoting the hydrolysis of sphingomyelin to proinflammatory ceramide. ASM is an important early responder in inflammatory cytokine signaling. The sphingomyelinase pathway is important for inflammatory signaling in human retinal endothelial cells (HREC), the resident vasculature affected by diabetic retinopathy. The inflammatory cytokines TNFa and IL-lp induce cell adhesion molecule (CAM) expression and a rapid upregulation of ASM and NSM in HREC.

[0108] Previous studies have shown the importance of ASM activity in diabetic retinopathy using, for example, ASM-/- mouse models and ASM inhibitors such as DHA and imipramine. Such gene knockout models and administration of an anti-ASM inhibitor reduced retinal inflammatory markers. See Fox et al., Diabetes 2006;55(12):3573-80; Opreanu et al., Diabetes 2011;60(9):2370-8; Opreanu et al., Investigative Ophthalmology & Visual Science 2010;51(6):3253-63; and Chakravarthy et al., Stem Cells 2016;34:972-83, each of which is incorporated herein by reference in its entirety.

[0109] However, ASM is an essential enzyme, the absence or inhibition of which can lead to Niemann-Pick disease, which can be fatal. Thus, knockout of the ASM gene and direct inhibition of the enzyme are not viable therapeutic strategies for the treatment of diabetic retinopathy. In contrast, the administration of anti-ceramide antibodies and antigen-binding fragments in the present application provides the unexpected result of selectively inhibiting inflammatory markers, neovascularization, and other symptoms of diabetic retinopathy in the eye without inducing the negative side effects associated with direct inhibition of ASM.

Additional diseases

[0110] In some embodiments, the compositions and methods of the present invention are used to treat diabetic retinopathy. In some embodiments, the compositions and methods of the present invention are used to treat diabetic macular edema. In some embodiments, the compositions and methods of the present invention are used to treat macular edema following retinal vein occlusion (RVO). In some embodiments, the compositions and methods of the present invention are used to treat myopic choroidal neovascularization (mCNV).

[0111] The present disclosure provides methods for treating diseases or disorders with underlying neovascularization. Typical diseases and disorders of the eye include: retinal neovascularization, choroidal neovascularization, corneal neovascularization, macular degeneration, age-related macular degeneration, diabetic retinopathy, vitreous hemorrhage, retinal hemorrhage, choroiditis, neovascular glaucoma, choroidal disease, telangiectasia, retinal artery occlusion, retinal vein occlusion, chorioretinitis, epiretinal membrane, choroidal neoplasm, retinopathy of prematurity, cystoid macular edema, papilledema, recurrent ischemia, ocular hemorrhage, and proliferative vitreoretinopathy.

Pharmaceutical compositions, routes of administration, dosages and dosing regimens

[0112] In some embodiments, the present invention provides a pharmaceutical composition comprising an anti-ceramide antibody or antigen-binding fragment thereof for the treatment of diabetic retinopathy.

[0113] For administration, an antibody or fragment according to the present disclosure (e.g., anti-ceramide antibodies and antigen-binding fragments thereof) can be formulated as a pharmaceutical composition. The pharmaceutical composition can comprise: (i) an anti-ceramide antibody or antigen-binding fragment thereof; and (ii) a pharmaceutically acceptable carrier, diluent, or excipient. A pharmaceutical composition comprising an anti-ceramide antibody or antigen-binding fragment thereof and/or an scFv can be prepared according to known methods for the preparation of pharmaceutically useful compositions, wherein the therapeutic molecule is combined in admixture with a pharmaceutically acceptable carrier, diluent, or excipient. Suitable carriers, diluents, or excipients are well known to those skilled in the art. (See, e.g., Gennaro (ed), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995).) The formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.

[0114] In some embodiments, the pharmaceutical composition may be prepared in a dosage form selected from the group consisting of: an oral unit dosage form, an intravenous unit dosage form, an intranasal unit dosage form, a suppository unit dosage form, an intradermal unit dosage form, an intramuscular unit dosage form, an intraperitoneal unit dosage form, a subcutaneous unit dosage form, an epidural unit dosage form, a sublingual unit dosage form, an intracerebral unit dosage form, an intracameral unit dosage form, a subconjunctival unit dosage form, a sub-Tenon's unit dosage form, a retrobulbar unit dosage form, a unit dosage form for administration into the posterior parascleral region, and an intravitreal unit dosage form. In some embodiments, the pharmaceutical composition may be prepared in an intravitreal dosage form. Systemic routes of administration do not involve delivery of anti-ceramide antibodies or antigen-binding fragments thereof to the eye, since anti-ceramide antibodies or antigen-binding fragments thereof are not expected to cross the blood-brain barrier.

[0115] The anti-ceramide antibodies and antigen-binding fragments thereof described herein can be administered to subjects by a variety of routes of administration, including, for example, intramuscular, subcutaneous, intravenous, intraatrial, intra-articular, parenteral, intranasal, intrapulmonary, transdermal, intrapleural, intrathecal, oral, topical, intraocular, intracameral, subconjunctival, sub-Tenon, retrobulbar, posterior parascleral, suprachoroidal space (SCS), supraciliary space, and intravitreal routes of administration.

[0116] According to some embodiments of the present invention, the anti-ceramide antibodies and antigen-binding fragments thereof, as well as the pharmaceutical compositions thereof, disclosed herein can be administered directly to the eye by any known route of administration, including topical (e.g., eye drops), topical ocular, intravitreal, intracameral, subconjunctival, sub-Tenon, retrobulbar, and by administration into the posterior periscleral region.

[0117] A variety of possible delivery methods may be used, including, but not limited to, intraocular or topical administration. In one embodiment, administration is intraocular and includes, but is not limited to, subconjunctival injection, intracameral injection, anterior chamber injection through the temporal rim, intrastromal injection, intracorneal injection, subretinal injection, aqueous humor injection, sub-Tenon injection or sustained delivery device, or intravitreal injection (e.g., anterior, middle, or posterior vitreous injection). In one embodiment, administration is topical and includes, but is not limited to, corneal eye drops.

[0118] In some embodiments, the disclosed antibodies, antigen-binding fragments thereof, and compositions thereof are prepared for intravitreal administration. In some embodiments, the disclosed antibodies, antigen-binding fragments, and compositions thereof are administered intravitreally.

[0119] For prevention and treatment purposes, anti-ceramide antibodies and antigen-binding fragments thereof can be administered to a subject as a single bolus delivery, as a continuous delivery (e.g., continuous transdermal delivery) over an extended period of time, or as a repeated administration protocol (e.g., hourly, daily, weekly, monthly, or annually).

[0120] In some embodiments, the methods provided herein comprise administering a therapeutically effective dose of an anti-ceramide antibody or antigen-binding fragment thereof. A therapeutically effective dose, dosage, or amount, as defined above, refers to the minimum amount of an anti-ceramide antibody or antigen-binding fragment thereof necessary to achieve a specific physiological effect, such as preventing or alleviating one or more symptoms of diabetic retinopathy. The determination of therapeutically effective doses in this context is typically based on studies in animal models that are followed by clinical trials in humans, and is based on the determination of effective doses and administration protocols that significantly reduce the incidence or severity of diabetic retinopathy in model subjects. Effective doses of the compositions of the present invention vary depending on many different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other drugs administered, whether the treatment is prophylactic or therapeutic, and the specific activity of the composition itself and its ability to elicit the desired response in the individual. Typically, administration regimens are adjusted to provide an optimal therapeutic response, i.e., to optimize safety and efficacy.

[0121] In some embodiments, the dose of the anti-ceramide antibody or antigen-binding fragment thereof is from about 0.1 μg to 100 mg/kg, or from 1 μg/kg to about 50 mg/kg, or from 10 μg to 5 mg/kg. In some embodiments, the effective amount of the anti-ceramide antibody or antigen-binding fragment thereof is from about 1 μg/kg to about 20 mg/kg, from about 10 μg/kg to about 10 mg/kg, or from about 0.1 mg/kg to about 5 mg/kg. The anti-ceramide antibodies and antigen-binding fragments thereof described herein can also be administered at a dosage of from about 0.001 to about 10 milligrams (mg) per kilogram (mg/kg) of body weight, administered as a single dose or in two or more doses. For administration to an adult human patient, a therapeutically effective amount can be administered in doses of 0.2 mg to 800 mg per dose, including but not limited to 0.2 mg per dose, 0.5 mg per dose, 1 mg per dose, 5 mg per dose, 10 mg per dose, 25 mg per dose, 100 mg per dose, 200 mg per dose, and 400 mg per dose, and one or more doses can be administered over a course of treatment. In some embodiments, the total daily dose of the anti-ceramide antibodies and antigen-binding fragments thereof described herein can be from about 1 mg to about 2 g, from about 100 mg to about 1.5 g, or from about 200 mg to about 1200 mg.

[0122] In some embodiments, anti-ceramide antibodies, antigen-binding fragments thereof, or compositions comprising the same can be prepared at a concentration of about 0.5 mg/mL, 1 mg/mL, 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, or 50 mg/mL. The concentration can be 0.1-1 mg/mL, 1-5 mg/mL, 5-10 mg/mL, or 10-50 mg/mL. In some embodiments, the disclosed antibodies, fragments, or compositions can be administered at a dose of about 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1 mg intravitreally. The dose volume can be about 0.005 ml, 0.01 ml, 0.02 ml, 0.03 ml, 0.04 ml, 0.05 ml, 0.06 ml, 0.07 ml, 0.08 ml, 0.09 ml, or 0.1 ml.

[0123] The anti-ceramide antibodies and antigen-binding fragments thereof described herein may be administered at different times of the day. In one embodiment, the dose may be administered in the evening. In another embodiment, the dose may be administered in the morning. Dosages may be administered once or multiple times, including, for example, multiple weekly, biweekly, monthly, or annual administrations. In some embodiments, a single dose of the anti-ceramide antibody or antibody fragment is administered to a subject in need thereof. In some embodiments, a patient may receive two or more doses of an anti-ceramide antibody separated by a period 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 a patient in need thereof separated by a period of time from about one week to about two weeks, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about one month to about six months, from about six months to about one year, or from about one year to about two years. In some embodiments, administration may be irregular, as indicated by monitoring the clinical symptoms of the disorder.

[0124] The dosage of the pharmaceutical composition comprising the anti-ceramide antibodies and antigen-binding fragments thereof may be adjusted by the treating physician to maintain a desired concentration at the target site. Higher or lower concentrations may be selected based on the type of delivery. The anti-ceramide antibodies or antigen-binding fragments thereof may be administered at any time during the life of the subject. In some embodiments, the administration occurs before the development of symptoms of diabetic retinopathy. In such embodiments, the administration may be used as a prophylactic agent to prevent or delay the onset of diabetic retinopathy. In some embodiments, the administration occurs early in the disease. In some embodiments, the administration occurs late in the disease.

Therapeutic methods and applications

[0125] In some embodiments, the present invention relates to methods of treating, preventing, or ameliorating a symptom of an inflammatory disease of the eye in a subject, comprising administering to the subject a therapeutically effective amount of an anti-ceramide antibody or antigen-binding fragment thereof.

[0126] In some embodiments, the present invention provides methods for preventing and/or treating diabetic retinopathy in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an anti-ceramide antibody or an anti-ceramide antibody fragment.

[0127] Previous studies have suggested that the initial metabolic insult leading to inflammation and increased vascular permeability in the retina involves activation of acid sphingomyelinase (ASM), a central enzyme in sphingolipid signaling that converts sphingomyelin to ceramide. ASM-/- animals were protected from vascular degeneration in a retinal ischemia-reperfusion model, and chimeras with ASM-/- bone marrow transplanted into wild-type mice were also protected from diabetes-induced vascular injury. Several strategies have been implemented to inhibit ASM activity, including administration of docosahexaenoic acid (DHA)-rich fish oil and the tricyclic antidepressant desipramine. Both strategies show some efficacy in cell culture models. However, desipramine was not well tolerated by animals in a long-term study (see Example 8), and chronic treatment with high doses of DHA, although improving retinal health in diabetic animals, caused retinal pathology in control animals (see Example 9). Furthermore, ASM is an essential enzyme, and the absence of ASM results in a neurodegenerative disease (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 ceramide generated on the surface of endothelial and other cells, thereby preventing ASM-induced ceramide-rich platform formation and subsequent proinflammatory and apoptotic signaling, without affecting the essential lysosomal function of ASM.

Subjects

[0128] Subjects for treatment according to the methods described herein include those who have or are at risk of developing diabetic retinopathy. Subjects with diabetic retinopathy may have an early or late stage of the disease.

[0129] In some embodiments, the subjects are those who have received one or more previous treatments for diabetic retinopathy but have not responded to the previous treatment. In such embodiments, "failure to respond" indicates that the previous treatment did not alleviate and/or improve one or more symptoms of diabetic retinopathy. In some embodiments, the previous therapy may not have improved vision or retinal vascular health. In some embodiments, the previous therapy may have demonstrated some results, but may not achieve the desired effectiveness or may cease to demonstrate effectiveness after a period of time.

[0130] Existing treatments for diabetic retinopathy include therapeutic procedures such as vitrectomy and laser surgery, and therapeutic agents such as steroids and anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the subject may have responded partially to a previous treatment for diabetic retinopathy: that is, one or more symptoms of diabetic retinopathy were not sufficiently relieved and/or the effects of the previous treatment were not sufficiently durable.

[0131] In some embodiments, the subject has received prior anti-VEGF treatment and has not responded to the treatment. In some embodiments, the anti-VEGF treatment is selected from Eylea® (aflibercept), Avastin® (bevacizumab), or Lucentis® (ranibizumab).

Determination of treatment indicators

[0132] Treating and/or preventing diabetic retinopathy can be measured in a variety of ways. In some embodiments, treating or preventing comprises treating or preventing one or more of apoptosis, inflammation, acellular capillary formation, neovascularization, retinal endothelial cell death, retinal vascular permeability, ischemia-reperfusion injury, and occludin abnormalities in diabetic retinopathy in a subject in need thereof. The method comprises administering an effective amount of an anti-ceramide antibody or antigen-binding fragment thereof prior to the onset of diabetic retinopathy in a diabetic patient or after the onset of diabetic retinopathy in a diabetic patient.

[0133] Methods for treating diabetic retinopathy with an anti-ceramide antibody or antigen-binding fragment thereof are provided. The effectiveness of such treatment can be characterized, assessed, measured, and/or monitored based on several parameters.

[0134] According to some embodiments of the present invention, methods are provided that reduce apoptosis and/or death of endothelial cells in the eye. Monitoring cell death can be performed in accordance with known methods. Exemplary methods for detecting cell death include nuclear staining techniques such as propidium iodide, Hoechst-33342, 4',6-diamidino-2-phenylindole (DAPI), acridine orange ethidium bromide staining, and the like. Non-nuclear staining techniques include annexin-V staining. In some embodiments, the methods reduce the level of apoptosis and/or endothelial cell death in an eye sample receiving said methods, compared to a control eye sample, 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%. 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%.

[0135] In some embodiments, the methods provided herein prevent ischemia-reperfusion (IR) injury. Additional methods for detecting IR injury include fluorescein assay, fluorescent zinc 2,2'-dipicolylamine coordination complex PSVue®794, 99mTc glucarate, and electroretinography.

[0136] In some embodiments, the methods provided herein reduce 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. Levels of inflammatory markers can be monitored using an enzyme-linked immunosorbent assay (ELISA), Luminex, cytokine granulometric array, Proteo Plex, FAST Quant, and the like. In some embodiments, the methods provided herein reduce levels of one or more inflammatory markers in an eye sample receiving the methods, compared to a control eye sample, 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%.

[0137] In some embodiments, the methods reduce the level of TNFα in an eye sample receiving the methods, compared to a control eye sample, 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%. In some embodiments, the methods provided herein reduce the level of TNFα by at least 10%. In some embodiments, the methods provided herein reduce the level of TNFα by at least 20%. In some embodiments, the methods provided herein reduce the level of TNFα by at least 30%. In some embodiments, the methods provided herein reduce the level of TNFα by at least 50%. In some embodiments, the methods provided herein reduce the level of TNFα by at least 70%.

[0138] In some embodiments, the methods reduce the level of IL-1β in an eye sample receiving the methods, compared to a control eye sample, 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%. In some embodiments, the methods provided herein reduce the level of IL-1β by at least 10%. In some embodiments, the methods provided herein reduce the level of IL-1β by at least 20%. In some embodiments, the methods provided herein reduce the level of IL-1β by at least 30%. In some embodiments, the methods provided herein reduce IL-1β levels by at least 50%. In some embodiments, the methods provided herein reduce IL-1β levels by at least 70%.

[0139] In some embodiments, the methods reduce the level of IL-6 in an eye sample receiving the methods, compared to a control eye sample, 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%. In some embodiments, the methods provided herein reduce the level of IL-6 by at least 10%. In some embodiments, the methods provided herein reduce the level of IL-6 by at least 20%. In some embodiments, the methods provided herein reduce the level of IL-6 by at least 30%. In some embodiments, the methods provided herein reduce IL-6 levels by at least 50%. In some embodiments, the methods provided herein reduce IL-6 levels by at least 70%.

[0140] In some embodiments, the methods reduce the level of VEGF in an eye sample receiving the methods, compared to a control eye sample, 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%. In some embodiments, the methods provided herein reduce the level of VEGF by at least 10%. In some embodiments, the methods provided herein reduce the level of VEGF by at least 20%. In some embodiments, the methods provided herein reduce the level of VEGF by at least 30%. In some embodiments, the methods provided herein reduce VEGF levels by at least 50%. In some embodiments, the methods provided herein reduce VEGF levels by at least 70%.

[0141] In some embodiments, the methods reduce the level of ICAM-1 in an eye sample receiving the methods, compared to a control eye sample, 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%. In some embodiments, the methods provided herein reduce the level of ICAM-1 by at least 10%. In some embodiments, the methods provided herein reduce the level of ICAM-1 by at least 20%. In some embodiments, the methods provided herein reduce the level of ICAM-1 by at least 30%. In some embodiments, the methods provided herein reduce the level of ICAM-1 by at least 50%. In some embodiments, the methods provided herein reduce the level of ICAM-1 by at least 70%.

[0142] In some embodiments, the methods reduce the level of VCAM-1 in an eye sample receiving the methods, compared to a control eye sample, 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%. In some embodiments, the methods provided herein reduce the level of VCAM-1 by at least 10%. In some embodiments, the methods provided herein reduce the level of VCAM-1 by at least 20%. In some embodiments, the methods provided herein reduce the level of VCAM-1 by at least 30%. In some embodiments, the methods provided herein reduce the level of VCAM-1 by at least 50%. In some embodiments, the methods provided herein reduce the level of VCAM-1 by at least 70%.

[0143] According to some embodiments, the methods reduce the level of MCP1 in an eye sample receiving said methods, compared to a control eye sample, 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%. In some embodiments, the methods provided herein reduce the level of MCP1 by at least 10%. In some embodiments, the methods provided herein reduce the level of MCP1 by at least 20%. In some embodiments, the methods provided herein reduce the level of MCP1 by at least 30%. In some embodiments, the methods provided herein reduce the level of MCP1 by at least 50%. In some embodiments, the methods provided herein reduce the level of MCP1 by at least 70%.

[0144] 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 can prevent typical symptoms of diabetic retinopathy in the retinal vasculature or can prevent further deterioration. Vascular permeability and other indicators of retinal vascular health can be measured, for example, using fluorescein angiography. In some embodiments, the methods provided herein reduce retinal vascular permeability in a subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control subject or compared to a subject before receiving the methods provided herein. 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%.

[0145] According to some embodiments, the methods provided herein improve one or more parameters of vision or prevent a decline in one or more parameters of vision. The parameters of vision include: poor night vision, blurred vision, floaters, black spots or flickering in the field of vision, fluctuating vision, color vision impairment, dark or blank areas in the field of vision, vision loss, sudden severe painless vision loss. According to some embodiments of the present invention, subjects treated according to the methods provided herein may experience one or more of the following effects: improved vision, decreased vision loss, improved night vision, improved low-light vision, improved reading ability, improved peripheral vision, decreased spots in the field of vision, decreased flickering in the field of vision, decreased pain, and improved eye appearance. Many of these parameters can be monitored through a standard eye examination.

[0146] In some embodiments, the methods provided herein improve the Early Treatment Diabetic Retinopathy Score (ETDRS) in subjects with diabetic retinopathy. In some embodiments, the subject is a human. The ETDRS scoring scale uses 3 standard photographs. Standard Photograph 1 shows a moderate degree of microaneurysm and hemorrhage; Standard Photograph 2A shows an intermediate degree of microaneurysm and hemorrhage; and Standard Photograph 2B shows a severe degree of microaneurysm and hemorrhage. In the ETDRS, a microaneurysm is defined as a red spot 125 μm in its longest dimension with sharp edges. This differs from a hemorrhage, which is defined as a red spot with jagged edges and a size greater than 125 μm. Using the area of the retina covered by microaneurysms and/or hemorrhages for comparison with standardized fundus photographs, the grading scale for describing nonproliferative diabetic retinopathy (NPDR) is as follows: grade 0 = no microaneurysm or hemorrhage; grade 1 = equivocal microaneurysm or hemorrhage; grade 2 = definite microaneurysm or hemorrhage, smaller than standard photograph 1; grade 3 = microaneurysm or hemorrhage greater than or equal to standard photograph 1 but smaller than standard photograph 2A; grade 4 = microaneurysm or hemorrhage greater than or equal to standard photograph 2A but smaller than standard photograph 2B. grade 5 = microaneurysms or hemorrhages greater than or equal to standard 2B photograph. In some embodiments, the methods provided herein reduce the average ETDRS score in subjects with DR by at least 0.1, at least 0.2. by at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.2, at least 1.5, at least 1.7, at least 2, at least 2.2, at least 2.5, at least 2.7, at least 3.0, at least 3.2, at least 3.5, at least 3.7, or at least 4 compared to control subjects or compared to subjects before obtaining the methods according to the present disclosure. In some embodiments, the methods provided herein reduce the mean ETDRS score in subjects with DR by at least 0.2. In some embodiments, the methods provided herein reduce the average ETDRS score in subjects with DR by at least 0.5. In some embodiments, the methods provided herein reduce the average ETDRS score in subjects with DR by at least 1. In some embodiments, the methods provided herein reduce the average ETDRS score in subjects with DR by at least 1.5. In some embodiments, the methods provided herein reduce the average ETDRS score in subjects with DR by at least 2. In some embodiments, the methods provided herein reduce the average ETDRS score in subjects with DR by at least 2.5. In some embodiments, the subjects are humans.

[0147] 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 maintaining vision or the absence of typical signs of diabetic retinopathy. For example, subjects who receive anti-ceramide antibodies or antigen-binding fragments thereof prophylactically may not experience or may experience a reduced incidence of one or more of the following: microaneurysms, hemorrhages, intraretinal microvascular abnormalities, segmental retinal vein dilation, cotton-wool spots, new blood vessel formation (neovascularization) elsewhere and on the optic nerve, fibrous proliferation elsewhere and on the optic nerve, preretinal hemorrhage and vitreous hemorrhage, retinal detachment due to scar tissue formation, glaucoma, poor night vision, blurred vision, floaters, black spots or flickering in the visual field, fluctuating vision, color vision disturbances, dark or blank areas in the visual field, vision loss, sudden severe painless vision loss, retinal adhesion detachment, macular edema, vein dilation and intraretinal microvascular disorders.

[0148] In some embodiments, the methods provided herein prevent or reduce macular edema. Macular edema can be observed on slit lamp biomicroscopy as a lifting and blurring of retinal layers.

[0149] In some embodiments, the methods provided herein delay the onset of diabetic retinopathy. Accordingly, the disclosed methods can be used to delay the average onset of diabetic retinopathy by more than 5 years, more than 10 years, more than 11 years, more than 12 years, more than 13 years, more than 14 years, more than 15 years, more than 16 years, more than 17 years, more than 18 years, more than 19 years, or more than 20 years after the initial diagnosis of diabetes.

[0150] In some embodiments, the disclosed methods can be used to reduce, alleviate, lessen the severity of, or eliminate one or more symptoms of diabetic retinopathy. In some embodiments, methods of treating diabetic retinopathy using anti-ceramide antibodies or antigen-binding fragments thereof can reduce, alleviate, lessen the severity of, or eliminate one or more of the following symptoms: microaneurysms, hemorrhages, intraretinal microvascular abnormalities, segmental retinal vein dilation, cotton wool spots, new blood vessel formation (neovascularization) elsewhere and on the optic nerve, fibrous proliferation elsewhere and on the optic nerve, preretinal hemorrhage and vitreous hemorrhage, retinal detachment due to scar tissue formation, vision loss, glaucoma, poor night vision, blurred vision, floaters, black spots or flickering in the visual field, sudden severe painless vision loss, retinal adhesion detachment, macular edema, vein dilation, and intraretinal microvascular abnormalities.

[0151] In some embodiments, the disclosed methods for treating diabetic retinopathy affect one or more parameters of the retinal vasculature, including, but not limited to: permeability, NFkB levels, inflammatory marker levels, apoptosis rate, sphingolipid metabolism, and re-endothelialization. In some embodiments, a method for treating diabetic retinopathy described herein reduces retinal vascular permeability. Retinal vascular permeability can be monitored using fluorescence, indicator dyes, optical testing, and the like. In some embodiments, a method for treating diabetic retinopathy disclosed herein reduces NFkB levels and/or other inflammatory markers in the retinal vasculature. As described above, inflammatory cytokine levels can be measured by conventional means (e.g., ELISA). In some embodiments, the method of treating diabetic retinopathy disclosed herein reduces the incidence of apoptosis in the retinal vasculature. As described above, apoptosis can be measured using conventional means, such as nuclear and non-nuclear staining methods. In some embodiments, the method of treating diabetic retinopathy disclosed herein inhibits or suppresses sphingolipid metabolism. In some embodiments, the method of treating diabetic retinopathy disclosed herein can increase re-endothelialization of healthy tissue.

Examples

Example 1: Anti-ceramide scFv inhibits I/R inflammation and retinal vascular permeability in a mouse model of diabetic retinopathy.

[0152] A mouse model of ischemia-reperfusion (I/R) diabetic retinopathy was used to model the damaging effects of diabetic retinopathy on the retinal vasculature, such as shown in Fig. 1.

[0153] Retinal ischemia-reperfusion (I/R): Each mouse had one eye with I/R and one uninjured eye as a control. Retinal I/R was created by transiently raising the intraocular pressure (IOP) to 90 mmHg as follows. Male C57BL/6J mice weighing 25 to 30 g were anesthetized. The anterior chamber of one eye was cannulated with a 30-gauge needle attached to a saline infusion line. IOP was measured with a portable tonometer (TONO Pen; Medtronic Solan, Jacksonville, FL) in the mouse eyes, and ocular pressure was adjusted to 80 to 90 mmHg with an infusion pump (mfu-surg; Ethox Corp., Buffalo, NY). The other eye of the same animal served as a control. The duration of ischemia was 90 minutes for each mouse. After ischemia, the needle was removed, IOP was normalized, and retinal blood flow was visually documented. Animals were sacrificed at different times after I/R injury. Retinas were harvested 2 or 7 days after retinal I/R.

[0154] Administration of vehicle control or anti-ceramide scFv: Twenty-four hours prior to I/R, control mice received injections of vehicle (phosphate-buffered saline) and test mice received an intravitreal injection of 1 μl of anti-ceramide scFv 6B5 at 1.73 mg/ml.

[0155] Inflammatory Cytokine Levels: At 48 hours post-I/R, expression levels of inflammatory markers for TNFα, IL-1β, IL6, ICAM-1, VCAM-1, and MCP1 were measured using qPCR analysis.

[0156] Additional measurements that can be made: In comparable experiments using this model, permeability, inflammatory markers, and sphingolipid metabolism are measured at 48 hours, and apoptosis, acellular capillaries, and sphingolipid metabolism are measured at 7 days, as shown in Fig. 2A-Fig. 2B.

[0157] Retinal vascular permeability: Retinas were isolated 48 hours after I/R. Briefly, mice were injected with FITC-albumin (0.5 mg in 100 μl PBS) (Sigma-Aldrich, St. Louis, MO). Two hours later, blood was collected from each mouse and centrifuged to obtain plasma; the animal was perfused with 1% formaldehyde and enucleated. Retinas were removed, mounted on a flat four-slot base, and stored on glass slides with Fluoromount fixative medium (Sigma-Aldrich, St. Louis, MO). Images were acquired with an Olympus FluoView 1000 laser scanning confocal microscope, and at least 5 different fields of view were selected for image collection for each sample. Retinas were mechanically disrupted and cleared by centrifugation. FITC-albumin in the supernatant was quantified using a spectrofluorometer and normalized to plasma fluorescence (Kielczewski et al., 2011).

Results

[0158] As shown in Fig. 2A , cytokine expression in I/R eyes treated with anticeramide was lower than in untreated LR eyes for each of the markers tested (TNFα, IL-1β, IL6, ICAM-1, VCAM-1, and MCP1). The reduction observed in TNFα, IL6, ICAM-1, and MCP1 was statistically significant. Fig. 2B demonstrates a clear difference in retinal vascular permeability as demonstrated by fluorescence microscopy imaging and quantitative analysis. Permeability was significantly higher in untreated I/R eyes than in control eyes or in I/R eyes treated with anticeramide.

[0159] This example demonstrates that a single intravitreal injection of anti-ceramide scFv in the setting of diabetes improves the outcome of potential diabetic retinopathy by preventing endothelial cell loss and subsequent retinal damage.

Example 2: Efficacy of anti-ceramide scFv administration in a rat model of diabetic retinopathy.

[0160] The STZ-induced diabetic retinopathy model was used in rats to model the damaging effects of diabetic retinopathy on the retinal vasculature, such as shown in Fig. 3.

[0161] Animals and Induction of STZ-Induced Diabetes: Male Sprague-Dawley rats weighing 237-283 g were induced to become diabetic with a single intraperitoneal injection of 65 mg streptozotocin (STZ)/kg body weight. Rats were maintained on a Harlan-Teklad laboratory diet (#8640) and water ad libitum. Body weight gain and blood glucose levels for the control and STZ-induced diabetic groups were monitored biweekly.

[0162] Administration of vehicle control or anti-ceramide scFv: After confirmation of diabetes (hyperglycemia greater than 250 mg/dL, 7-10 days after STZ injection), diabetic control rats received injections of PBS vehicle and diabetic test rats received an intravitreal injection of 2 μL of anti-ceramide scFv 6 B5 at 1.73 mg/mL. Non-diabetic control rats also received an injection of either vehicle control or anti-ceramide scFv.

[0163] Retinal vascular permeability assay: Retinas were isolated 48 hours after I/R. Briefly, mice were injected with FITC-albumin (0.5 mg in 100 μl PBS) (Sigma-Aldrich, St. Louis, MO). Two hours later, blood was collected from each mouse and centrifuged to obtain plasma; the animal was perfused with 1% formaldehyde and enucleated. Retinas were removed, mounted on a flat four-slot base, and stored on glass slides containing Fluoromount fixative (Sigma-Aldrich, St. Louis, MO). Images were acquired with an Olympus FluoView 1000 laser scanning confocal microscope, and at least 5 different fields of view were selected for image collection for each sample. Retinas were mechanically disrupted and cleared by centrifugation. FITC-albumin in the supernatant was quantified using a spectrofluorometer and normalized to plasma fluorescence (Kielczewski et al., 2011).

[0164] Additional measurements: In comparable experiments, the experiment was performed in a mouse model or the diabetic model is leptin deficiency db/db, as shown in Fig. 3. In addition, 6-8 weeks after induction of diabetes in the STZ-induced model, the retinal vasculature of each animal was assessed for permeability, NFkB and inflammatory markers, apoptosis and sphingolipid metabolism, as shown in Fig. 3.

Results

[0165] The results shown in Fig. 4 demonstrate that diabetic rats given an intravitreal dose of anti-ceramide scFv did not develop vascular leakage indicative of diabetic retinopathy, thus demonstrating that a single intravitreal injection of anti-ceramide scFv is sufficient to reverse hyperglycemia-induced diabetic retinopathy in a rat model of this condition.

Example 3: Intravitreal or systemic administration of anti-ceramide scFv for the treatment of diabetes-induced proinflammatory changes in DR.

[0166] Experiments are performed to compare the effects of intravitreal anti-ceramide therapy versus systemic anti-ceramide therapy on diabetes-induced pro-inflammatory changes in a mouse model of DR. Mice with STZ-induced type 1 diabetes receive a single intravitreal or systemic (intravenous) treatment with anti-ceramide scFv 6B5 at the onset of diabetes. Control, diabetic, and anti-ceramide-treated diabetic animals are sacrificed 6-8 weeks after STZ administration. Retinas are isolated from each animal; inflammatory cytokine, growth factor, and adhesion molecule profiles are determined; and endothelial ASM expression and activity in the retina are measured. These measurements are performed using qPCR, Western blotting, ESI-MS/MS, fluorescence microscopy, and immunogold electron microscopy experiments. An exemplary experimental protocol is shown in Fig. 5.

[0167] A single intravitreal injection of anti-ceramide scFv is expected to have a greater therapeutic effect on the development of DR. Additional experiments are underway to evaluate the ability of a single intravitreal dose of anti-ceramide antibody or its antigen-binding fragment to treat existing DR pathologies.

Example 4: Intravitreal or systemic administration of anti-ceramide scFv for the treatment of vascular dysfunction in DR.

[0168] Experiments are performed to compare the effects of intravitreal and systemic anti-ceramide therapy on retinal vascular permeability in a mouse model of DR. In this example, the effects of anti-ceramide scFv 6B5 administration on diabetes-induced retinal vascular damage are measured. To assess the initial disruption of the blood-retinal barrier, mice are given a single intravitreal or systemic (intravenous) treatment with anti-ceramide scFv at the onset of diabetes. Retinal vascular permeability is assessed 6-8 weeks after diabetes induction using fluorescein as described in Example 2. An exemplary experimental protocol is shown in Fig. 5.

[0169] A single intravitreal injection of scFv is expected to have a greater therapeutic effect on DR vascular dysfunction. Additional experiments are underway to evaluate the ability of a single intravitreal dose of anti-ceramide antibody or its antigen-binding fragment to treat existing DR pathologies.

Example 5: scFv versus ceramide versus DHA or desipramine to reduce proinflammatory changes in DR.

[0170] Experiments are performed to compare the effects of a single intravitreal or systemic treatment with anti-ceramide scFv with the effects of a single treatment with DHA-rich fish oil or desipramine on diabetes-induced proinflammatory changes in a mouse model of DR. Mice with STZ-induced type 1 diabetes receive a single dose of DHA-rich fish oil by gavage, a single intravenous injection of desipramine, a single intravitreal injection of anti-ceramide scFv, or a systemic injection of anti-ceramide scFv at the onset of diabetes. Animals are sacrificed 6-8 weeks after STZ-induced diabetes and their retinas are isolated. Profiling of inflammatory cytokines, growth factors, and adhesion molecules is performed. Endothelial ASM expression and activity is measured. These measurements are performed in a series of qPCR, Western blotting, ES-MS/MS, fluorescence microscopy, and immunogold electron microscopy experiments as described in Example 3. An illustrative experimental protocol is shown in Fig. 6.

[0171] A single intravitreal injection of anti-ceramide scFv is expected to have a greater therapeutic effect on the development of DR. Additional experiments are underway to evaluate the ability of a single intravitreal dose of anti-ceramide antibody or its antigen-binding fragment to treat existing DR pathologies.

Example 6: Administration of scFv against ceramide versus DHA or desipramine for the treatment of vascular dysfunction DR.

[0172] Experiments are performed to compare the effects of a single intravitreal or systemic treatment with anti-ceramide scFv compared to a single treatment with DHA-rich fish oil or desipramine on retinal vascular permeability in a mouse model of DR. Mice with STZ-induced type 1 diabetes are given a single dose of DHA-rich fish oil by gavage, a single intravenous injection of desipramine, a single intravitreal injection of anti-ceramide scFv, or a systemic injection of anti-ceramide scFv at the onset of diabetes. Retinal vascular permeability is assessed 6-8 weeks after diabetes induction using fluorescein as described in Example 4. An exemplary experimental protocol is shown in Fig. 6.

[0173] A single intravitreal injection of scFv is expected to have a greater therapeutic effect on DR vascular dysfunction. Additional experiments are underway to evaluate the ability of a single intravitreal dose of anti-ceramide antibody or its antigen-binding fragment to treat existing DR pathologies.

Example 7: Administration of anti-ceramide scFv inhibits stress-induced apoptosis.

[0174] Experiments are conducted to demonstrate that anti-ceramide scFv is effective in human retinal endothelial cells (HREC) in culture to inhibit stress-induced apoptosis under conditions where anti-angiogenic drugs are ineffective. The mechanism of DR therapy with anti-ceramide scFv and anti-angiogenic drugs is distinct from previously characterized mechanisms. Without being limited by theory, it is believed that diabetes involves ongoing cell death and remodeling that is not reversed by anti-VEGF treatment but is unresponsive to anti-ceramide. Therefore, while anti-VEGF therapy can only prevent neoangiogenesis, administration of anti-ceramide antibodies can prevent ongoing endothelial cell death by enhancing vascular repair and preventing hypoxia and subsequent neoangiogenesis. To test whether the anti-ceramide scFv would work in vitro under conditions where anti-angiogenic drugs are ineffective, HREC were exposed to various stress conditions that induce ASM/ceramide-mediated apoptotic death (e.g., exposure to IL-1β and TNF cytokines or _H2O2 ). HREC were treated with anti-VEGF inhibitors (anti-VEGFR2 DC101 Ab or the VEGFR inhibitor TK Sorafenib) or _anti -ceramide antibodies.

[0175] Administration of anti-ceramide scFv is expected to have a greater therapeutic effect on inhibiting endothelial cell death.

Example 8: Desipramine treatment of diabetic mice

[0176] Diabetes was induced in male C57BL/6J mice (20-25 g) by intraperitoneal injections of SZT (65 mg/kg) for five consecutive days. Control animals received injections of citric acid vehicle (pH 4.5). Two weeks after the last injection, blood glucose levels were measured from a spot of blood collected from the dorsal foot vein. Diabetes was confirmed by blood glucose levels ≥ 300 mg/dL. Weight loss, polyuria, water and food intake were monitored daily. On the first day when diabetes was confirmed, desipramine was added to the treatment initiation water, ~2 weeks after the first SZT injection. Desipramine treatment had no effect on control animals. However, diabetic animals showed dehydration and lost more than 25% of their body weight within 2-4 days, after which the experiment had to be terminated in accordance with the IACUC Humane Animal Use Guidelines.

[0177] Because the diabetic animals had polydipsia and polyuria, desipramine in the water could interfere with drinking patterns, causing dehydration. Therefore, the experiment was repeated using intraperitoneal injections of 2 mg/mL desipramine at a dose of 20 mg/kg. As with the desipramine in the water treatment, the control animals tolerated the drug well. However, the diabetic animals exhibited abnormal tremors, became weak, stopped grooming, eating, and drinking, and lost more than 25% of their body weight within 5-7 days of the daily injections, at which point the experiment had to be terminated in accordance with the IACUC Humane Animal Use Guidelines.

Example 9: DHA treatment of wild-type mice

[0178] Male Sprague-Dawley rats (237-283) were fed either a control purified rodent diet AIN-93M with 10% of the calories supplied as soybean oil containing 50.8% linoleic acid from Dyets Inc. (Bethlehem, PA), a DHA-rich fish oil diet in which half of the calories were supplied as soybean oil or 5% of the calories were supplied as American herring oil containing 10.26% DHA and 14.16% EPA. After 9 months of diet treatment, the retinal vasculature of the rats was isolated by trypsin digestion and acellular capillaries were systematically counted in the middle of the retina by two independent observers. As shown in Fig. 7, DHA-rich fish oil treatment was detrimental to the control animals by inducing the development of acellular capillaries.

[0179] Although the health benefits of fish oil as a triglyceride-lowering agent are well recognized, the dose required for the retinal effects was -5% of caloric intake, equivalent to -12 g fish oil per day, 3 times the recommended human intake. High dose DHA in humans, especially in diabetes, is associated with a number of well-documented side effects, including higher blood glucose levels, excessive bleeding, decreased wound healing, frequent infections, gastrointestinal problems, and weight gain. Because of these concerns, DHA is not recommended for diabetic complications.

Additional numbered embodiments of the invention

[0180] Additional embodiments of the present invention are presented in the numbered embodiments below:

[0181] Embodiment 1. A method of treating or preventing diabetic retinopathy in a subject in need thereof, comprising ocularly administering to the subject an anti-ceramide antibody or antigen-binding fragment thereof.

[0182] Embodiment 2. The method according to embodiment 1, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

[0183] Embodiment 3. The method according to embodiment 1 or 2, characterized in that the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber through the temporal margin, intrastromal administration, intracorneal administration, subretinal administration, injection into the intraocular fluid, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0184] Embodiment 4. The method according to any of embodiments 1-3, characterized in that said administration is intravitreal administration.

[0185] Embodiment 5. The method according to any one of embodiments 1-4, characterized in that said subject has received previous treatment for diabetic retinopathy.

[0186] Embodiment 6. The method of embodiment 5, wherein said subject has not responded to prior treatment for diabetic retinopathy.

[0187] Embodiment 7. The method according to embodiment 5 or 6, characterized in that the prior treatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from a steroid and anti-vascular endothelial growth factor (VEGF) therapy.

[0188] Embodiment 7. The method of embodiment 7, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

[0189] Embodiment 9. The method according to any one of embodiments 1-8, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered as a single dose.

[0190] Embodiment 10. The method according to any one of embodiments 1-8, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least two weeks, at least three weeks, or at least four weeks.

[0191] Embodiment 11. The method according to any one of embodiments 1-8, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about two weeks to about four weeks.

[0192] Embodiment 12. The method of any one of embodiments 1-8, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least 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.

[0193] Embodiment 13. The method according to any one of embodiments 1-8, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about one month to about six months.

[0194] Embodiment 14. The method according to any one of embodiments 1-8, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least one year.

[0195] Embodiment 15. The method according to any one of embodiments 1-14, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of diabetic retinopathy.

[0196] Embodiment 16. The method according to any one of embodiments 1-14, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered after the occurrence of one or more symptoms of diabetic retinopathy.

[0197] Embodiment 17. The method according to any one of embodiments 1-14, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative stage of diabetic retinopathy.

[0198] Embodiment 18. The method according to embodiment 17, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative stage of diabetic retinopathy.

[0199] Embodiment 19. A method of treating or preventing diabetic retinopathy in a subject in need thereof, comprising administering an anti-ceramide antibody or antigen-binding fragment thereof to the subject, wherein said subject has received prior treatment for diabetic retinopathy.

[0200] Embodiment 20. The method of embodiment 19, wherein said subject has not responded to prior treatment for diabetic retinopathy.

[0201] Embodiment 21. The method of embodiment 19 or 20, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

[0202] Embodiment 22. The method according to any one of embodiments 19-21, characterized in that said administration is ocular administration.

[0203] Embodiment 23. The method of embodiment 22, wherein the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, anterior chamber injection through the temporal margin, intrastromal administration, intracorneal administration, subretinal administration, injection into aqueous humor, sub-Tenon administration, suprachoroidal space (SCS) administration, supraciliary space administration, and intravitreal administration.

[0204] Embodiment 24. The method according to embodiment 22 or 23, characterized in that the ocular administration is intravitreal administration.

[0205] Embodiment 25. The method according to any one of embodiments 19-24, characterized in that the prior treatment was vitrectomy, laser surgery, steroid, and/or anti-vascular endothelial growth factor (VEGF) therapy.

[0206] Embodiment 26. The method of embodiment 25, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

[0207] Embodiment 27. The method according to any one of embodiments 19-26, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered as a single dose.

[0208] Embodiment 28. The method of any one of embodiments 19-26, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least two weeks, at least three weeks, or at least four weeks.

[0209] Embodiment 29. The method of any one of embodiments 19-26, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about two weeks to about four weeks.

[0210] Embodiment 30. The method of any one of embodiments 19-26, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least 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.

[0211] Embodiment 31. The method of any one of embodiments 19-26, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about one month to about six months.

[0212] Embodiment 32. The method according to any one of embodiments 19-26, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least one year.

[0213] Embodiment 33. The method according to any one of embodiments 19-32, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered before the onset of one or more symptoms of diabetic retinopathy.

[0214] Embodiment 34. The method according to any one of embodiments 19-32, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered after the occurrence of one or more symptoms of diabetic retinopathy.

[0215] Embodiment 35. The method according to any one of embodiments 19-32, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative stage of diabetic retinopathy.

[0216] Embodiment 36. The method according to any one of embodiments 19-32, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative stage of diabetic retinopathy.

[0217] Embodiment 37. A method of treating or preventing diabetic retinopathy in a subject, comprising administering to the subject a single dose of an anti-ceramide antibody or antigen-binding fragment thereof.

[0218] Embodiment 38. A method of treating or preventing diabetic retinopathy in a subject, comprising administering to the subject two or more doses of an anti-ceramide antibody or antigen-binding fragment thereof, wherein the two or more doses are separated by at least two weeks.

[0219] Embodiment 39. The method of embodiment 38, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least two weeks, at least three weeks, or at least four weeks.

[0220] Embodiment 40. The method of embodiment 38, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about two weeks to about four weeks.

[0221] Embodiment 41. The method of embodiment 38, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least 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.

[0222] Embodiment 42. The method of embodiment 38, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about one month to about six months.

[0223] Embodiment 43. The method of embodiment 38, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least one year.

[0224] Embodiment 44. The method of any one of embodiments 37-43, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

[0225] Embodiment 45. The method according to any one of embodiments 37-44, characterized in that said administration is ocular administration.

[0226] Embodiment 46. The method of embodiment 45, wherein the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, anterior chamber injection through the temporal margin, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, suprachoroidal space (SCS) administration, supraciliary space administration, or intravitreal administration.

[0227] Embodiment 47. The method according to embodiment 45 or 46, characterized in that the ocular administration is intravitreal administration.

[0228] Embodiment 48. The method according to any one of embodiments 37-47, characterized in that said subject has received prior treatment for diabetic retinopathy.

[0229] Embodiment 49. The method of embodiment 48, wherein said subject has not responded to prior treatment for diabetic retinopathy.

[0230] Embodiment 50. The method of embodiment 48 or 49, wherein the prior treatment was vitrectomy, laser surgery, steroid, and/or anti-vascular endothelial growth factor (VEGF) therapy.

[0231] Embodiment 51. The method of embodiment 50, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

[0232] Embodiment 52. The method of any one of embodiments 37-51, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered prior to the onset of one or more symptoms of diabetic retinopathy.

[0233] Embodiment 53. The method of any one of embodiments 37-51, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered after the onset of one or more symptoms of diabetic retinopathy.

[0234] Embodiment 54. The method according to any one of embodiments 37-51, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered during the non-proliferative stage of diabetic retinopathy.

[0235] Embodiment 55. The method according to any one of embodiments 37-51, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is administered during the proliferative stage of diabetic retinopathy.

[0236] Embodiment 56. A method of treating an inflammatory disease of the eye comprising ocular administration of an anti-ceramide antibody or antigen-binding fragment thereof.

[0237] Embodiment 57. The method of embodiment 56, wherein the anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

[0238] Embodiment 58. The method of embodiment 56 or 57, wherein the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber through the temporal margin, intrastromal administration, intracorneal administration, subretinal administration, injection into the aqueous humor, sub-Tenon's administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, or intravitreal administration.

[0239] Embodiment 59. The method of any one of embodiments 56-58, wherein said 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, cystoid macular edema, optic disc edema, recurrent ischemia, ocular hemorrhage, and proliferative vitreoretinopathy.

[0240] Embodiment 60. The method according to any one of embodiments 56-59, characterized in that said ocular administration is intravitreal administration.

[0241] Embodiment 61. The method according to any one of embodiments 56-60, characterized in that said subject has received prior treatment for diabetic retinopathy.

[0242] Embodiment 62. The method of embodiment 61, wherein said subject has not responded to prior treatment for diabetic retinopathy.

[0243] Embodiment 63. The method of embodiment 61 or 62, wherein the prior treatment was a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroid therapy and anti-vascular endothelial growth factor (VEGF) therapy.

[0244] Embodiment 64. The method of embodiment 63, wherein the anti-VEGF therapy is an anti-VEGF antibody selected from the group consisting of bevacizumab, ranibizumab, and aflibercept.

[0245] Embodiment 65. The method according to any one of embodiments 56-64, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered as a single dose.

[0246] Embodiment 66. The method of any one of embodiments 56-64, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least two weeks, at least three weeks, or at least four weeks.

[0247] Embodiment 67. The method of any one of embodiments 56-64, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about two weeks to about four weeks.

[0248] Embodiment 68. The method of any one of embodiments 56-64, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least 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.

[0249] Embodiment 69. The method of any one of embodiments 56-64, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about one month to about six months.

[0250] Embodiment 70. The method of any one of embodiments 56-64, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least one year.

[0251] Embodiment 71. The method of any one of embodiments 56-70, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered prior to the onset of one or more symptoms of an inflammatory disease of the eye.

[0252] Embodiment 72. The method of any one of embodiments 56-70, wherein the anti-ceramide antibody or antigen-binding fragment thereof is administered after the occurrence of one or more symptoms of an inflammatory disease of the eye.

[0253] Embodiment 73. The method of any one of embodiments 1-72, wherein the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain ( _VH ) and a variable light chain ( _VL ), wherein _VH comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1), HCDR2 comprising the amino acid sequence YNYPRDGSTKYNEKFKG (SEQ ID NO: 2), and HCDR3 comprising the amino acid sequence GFITTVVPSAY (SEQ ID NO: 3), and wherein _VL comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence RASKSISKYLA (SEQ ID NO: 4), LCDR2 comprising the amino acid sequence SGSTLQS (SEQ ID NO: 5), and LCDR3, containing the amino acid sequence QQHNEYPWT (SEQ ID NO: 6).

[0254] Embodiment 74. The method according to any one of embodiments 1-72, characterized in that V _H comprises the amino acid sequence of SEQ ID NO: 7, and wherein V _L comprises the amino acid sequence of SEQ ID NO: 8.

[0255] Embodiment 75. The method of any one of embodiments 1-74, wherein the anti-ceramide antibody or antigen-binding fragment thereof is the 6B5 antibody.

[0256] Embodiment 76. The method of any one of embodiments 1-74, wherein the anti-ceramide antibody or antigen-binding fragment thereof is scFv 6B5.

[0257] Embodiment 77. The method of any one of embodiments 1-72, wherein the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V _H ) and a variable light chain (V _L ), wherein V _H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence GYTFTNYWMH (SEQ ID NO: 33), HCDR2 comprising the amino acid sequence AIYPGDSDTSYNQKFKG (SEQ ID NO: 34), and HCDR3 comprising the amino acid sequence GLYYGYD (SEQ ID NO: 35), and wherein V _L comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence KSSQSLIDSDGKTFLN (SEQ ID NO: 36), LCDR2 comprising the amino acid sequence LVSKLDS (SEQ ID NO: 37), and LCDR3, containing the amino acid sequence WQGTHFPYT (SEQ ID NO: 38).

[0258] Embodiment 78. The method according to any one of embodiments 1-72, characterized in that V _H comprises the amino acid sequence of SEQ ID NO: 39, and wherein V _L comprises the amino acid sequence of SEQ ID NO: 40.

[0259] Embodiment 79. The method according to any one of embodiments 1-72 and 77-78, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is antibody 2A2.

[0260] Embodiment 80. The method according to any one of embodiments 1-72 and 77-78, characterized in that the anti-ceramide antibody or antigen-binding fragment thereof is scFv 2A2.

[0261] Embodiment 81. The method of any one of embodiments 1-72, wherein the anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) and a variable light chain (VL),

a) wherein VH comprises a heavy chain complementarity determining region 1 (HCDR1) comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1 and 43, HCDR2 comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 44-47, and HCDR3 comprising or consisting of the amino acid sequence GFITTVVPSAY (SEQ ID NO: 3), and

b) wherein VL comprises a light chain complementarity determining region 1 (LCDR1) comprising or consisting of the amino acid sequence RASKSISKYLA (SEQ ID NO: 4), LCDR2 comprising or consisting of the amino acid sequence SGSTLQS (SEQ ID NO: 5), and LCDR3 comprising or consisting of the amino acid sequence QQHNEYPWT (SEQ ID NO: 6).

[0262] Embodiment 82. The method of embodiment 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 YNYPRDGSTKYNEKFQG (SEQ ID NO: 44).

[0263] Embodiment 83. The method of embodiment 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 YNYPREGSTKYNEKFQG (SEQ ID NO: 45).

[0264] Embodiment 84. The method of embodiment 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).

[0265] Embodiment 85. The method of embodiment 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 YNYPRDGSTKYAEKFQG (SEQ ID NO: 47).

[0266] Embodiment 86. The method of embodiment 81, wherein 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).

[0267] Embodiment 87. The method of embodiment 81, wherein HCDR1 comprises or consists of the amino acid sequence GYTFTDHTMH (SEQ ID NO: 43), and HCDR2 comprises or consists of the amino acid sequence YNYPREGSTK YNEKFQG (SEQ ID NO: 45).

[0268] Embodiment 88. The method of embodiment 81, wherein HCDR1 comprises or consists of the amino acid sequence GYTFTDHTMH (SEQ ID NO: 43), and HCDR2 comprises or consists of the amino acid sequence YNYPRDVSTK YNEKFQG (SEQ ID NO: 46).

[0269] Embodiment 89. The method of embodiment 81, wherein 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).

[0270] Embodiment 90. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 48, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 53.

[0271] Embodiment 91. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 48, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 54.

[0272] Embodiment 92. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 48, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 55.

[0273] Embodiment 93. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 49, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 53.

[0274] Embodiment 94. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 49, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 54.

[0275] Embodiment 95. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 49, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 55.

[0276] Embodiment 96. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 50, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 53.

[0277] Embodiment 97. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 50, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 54.

[0278] Embodiment 98. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 50, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 55.

[0279] Embodiment 99. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 51, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 53.

[0280] Embodiment 100. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 51, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 54.

[0281] Embodiment 101. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 51, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 55.

[0282] Embodiment 102. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 52, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 53.

[0283] Embodiment 103. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 52, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 54.

[0284] Embodiment 104. The method of embodiment 81, wherein VH comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 52, and VL comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 55.

[0285] Embodiment 105. The method of any one of embodiments 81-104, wherein said anti-ceramide antibody or antigen-binding fragment thereof is humanized 6B5 (h6B5).

[0286] Embodiment 106. The method of any one of embodiments 81-104, wherein the anti-ceramide antibody or antigen-binding fragment thereof is scFv h6B5.

[0287] Embodiment 107. The method of any one of embodiments 1-72, wherein the anti-ceramide antibody or 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 wherein VL comprises the amino acid sequence of SEQ ID NO: 53.

[0288] Embodiment 108. The method of any one of embodiments 1-72, wherein the anti-ceramide antibody or 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 wherein VL comprises the amino acid sequence of SEQ ID NO: 55.

[0289] Embodiment 109. The method of any one of embodiments 1-72, wherein the anti-ceramide antibody or 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 wherein VL comprises the amino acid sequence of SEQ ID NO: 53.

[0290] Embodiment 110. The method of any one of embodiments 1-72, wherein the anti-ceramide antibody or 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 wherein VL comprises the amino acid sequence of SEQ ID NO: 54.

[0291] Embodiment 111. The method of any one of embodiments 1-72, wherein the anti-ceramide antibody or 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 wherein VL comprises the amino acid sequence of SEQ ID NO: 53.

[0292] Embodiment 112. The method of any one of embodiments 1-72, wherein the anti-ceramide antibody or 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 wherein VL comprises the amino acid sequence of SEQ ID NO: 54.

[0293] Embodiment 113. The method of any one of embodiments 1-72, wherein the anti-ceramide antibody or 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 wherein VL comprises the amino acid sequence of SEQ ID NO: 53.

[0294] Embodiment 114. The method of any one of embodiments 1-72, wherein the anti-ceramide antibody or 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 wherein VL comprises the amino acid sequence of SEQ ID NO: 53.

[0295] Embodiment 115. The method of any one of embodiments 107-114, wherein said anti-ceramide antibody or antigen-binding fragment thereof is a humanized antibody.

[0296] Embodiment 116. The method of any one of embodiments 107-114, wherein said anti-ceramide antibody or antigen-binding fragment thereof is a humanized scFv.

[0297] Embodiment 117. The method of any one of the preceding embodiments 1-116, wherein preventing diabetic retinopathy or an inflammatory eye disease comprises delaying the onset of diabetic retinopathy or an inflammatory eye disease.

[0298] Embodiment 118. The method of any one of the preceding embodiments 1-117, wherein one or more symptoms of diabetic retinopathy or inflammatory eye disease in the subject are reduced compared to the subject in a control or compared to the subject before treatment with the anti-ceramide antibody or antigen-binding fragment thereof.

[0299] Embodiment 119. The method of embodiment 118, wherein the one or more symptoms of diabetic retinopathy or inflammatory eye disease are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemia-reperfusion injury, retinal leakage area, and occludin disorder.

[0300] Embodiment 120. The method of embodiment 118 or 119, wherein one or more symptoms of diabetic retinopathy or inflammatory eye disease are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject before treatment with an anti-ceramide antibody or antigen-binding fragment thereof.

[0301] Embodiment 121. The method of any one of the preceding embodiments 1-120, wherein the expression level of one or more inflammatory markers in the eye is reduced compared to the expression level in the eye of the subject in a control or compared to the expression level in the eye of the subject prior to treatment with the anti-ceramide antibody or antigen-binding fragment thereof.

[0302] Embodiment 122. The method of embodiment 121, wherein the one or more inflammatory markers are selected from a cytokine, a growth factor, and an adhesion molecule.

[0303] Embodiment 123. The method of embodiment 122, wherein the cytokine is selected from TNFα, IL-1β, IL-6, or MCP1.

[0304] Embodiment 124. The method of embodiment 122, wherein the growth factor is VEGF.

[0305] Embodiment 125. The method of embodiment 122, wherein the adhesion molecule is ICAM-1 or VCAM-1.

[0306] Embodiment 126. The method of any one of embodiments 121-125, 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 subject in a control or compared to the expression level in the eye of the subject before treatment with the anti-ceramide antibody or antigen-binding fragment thereof.

[0307] Embodiment 127. The method of any one of the preceding embodiments 1-126, wherein the subject has an increase in one or more visual parameters compared to the subject's visual parameters in a control or compared to the subject's visual parameters before treatment with an anti-ceramide antibody or antigen-binding fragment thereof.

[0308] Embodiment 128. The method of embodiment 127, wherein one or more vision parameters are selected from peripheral vision; night vision; color vision; far vision; near vision; and visual clarity.

[0309] Embodiment 129. The method of any one of the preceding embodiments 1-128, wherein the retinal vascular permeability in the subject's eye is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the retinal vascular permeability in the subject's eye in a control or compared to the expression level in the subject's eye before treatment with the anti-ceramide antibody or antigen-binding fragment thereof.

[0310] Embodiment 130. The method of any one of preceding embodiments 1-129, wherein the mean early diabetic retinopathy score (ETDRS) in the group of subjects 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 score in the group of subjects in a control or compared to the mean ETDRS score in the group of subjects before treatment with the anti-ceramide antibody or antigen-binding fragment thereof.

INCLUSION BY LINK

[0311] All references, articles, publications, patents, patent publications and patent applications cited in this application are hereby incorporated by reference in their entirety for all purposes. However, citation of any reference, article, publication, patent, patent publication and patent application cited in this application does not constitute and should not be construed as an endorsement or any form of suggestion that they constitute prior art or are part of the common knowledge in any country in the world.

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SEQUENCE LIST

<110> MEMORIAL SLOAN KETTERING CANCER CENTER

BOARD OF TRUSTEES OF MICHIGAN STATE UNIVERSITY

<120> METHODS AND COMPOSITIONS FOR THE TREATMENT OF DIABETIC RETINOPATHY

<130> 115872-1233

<150> US 62/907,287

<151> 2019-09-27

<160> 55

<170> PatentIn version 3.5

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<220>

<223> Antibody 7B10 HCDR1

<400> 17

Gly Tyr Thr Phe Thr Ser Tyr Trp Met His

1 5 10

<210> 18

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 7B10 HCDR2

<400> 18

Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Phe Lys Asp

1 5 10 15

<210> 19

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 7B10 HCDR3

<400> 19

Gly Gly Tyr Tyr Gly Phe Ala Tyr

1 5

<210> 20

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 7B10 LCDR1

<400> 20

Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr

1 5 10

<210> 21

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 7B10 LCDR2

<400> 21

Leu Thr Ser Asn Leu Ala Ser

1 5

<210> 22

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 7B10 LCDR3

<400> 22

Gln Gln Trp Ser Ser Asn Pro Leu Thr

1 5

<210> 23

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 7B10 VH

<400> 23

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Tyr Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Tyr Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115

<210> 24

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 7B10 VL

<400> 24

Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 25

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 9H10 HCDR1

<400> 25

Gly Phe Ser Leu Thr Gly Tyr Gly Val His

1 5 10

<210> 26

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 9H10 HCDR2

<400> 26

Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile Ser

1 5 10 15

<210> 27

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 9H10 HCDR3

<400> 27

Asn Tyr Gly Tyr Asp Tyr Ala Met Asp Tyr

1 5 10

<210> 28

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 9H10 LCDR1

<400> 28

Arg Ala Ser Gln Ser Ile Gly Thr Ser Ile His

1 5 10

<210> 29

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 9H10 LCDR2

<400> 29

Tyr Ala Ser Glu Ser Ile Ser

1 5

<210> 30

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 9H10 LCDR3

<400> 30

Gln Gln Ser Asn Ser Trp Pro Phe Thr

1 5

<210> 31

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 9H10 VH

<400> 31

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Val Gln Pro Ser Ser Leu

1 5 10 15

Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val

20 25 30

His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val

35 40 45

Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile Ser Arg

50 55 60

Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met

65 70 75 80

Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Asn

85 90 95

Tyr Gly Tyr Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val

100 105 110

Thr Val Ser Ser

115

<210> 32

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 9H10 VL

<400> 32

Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Ser

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 33

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 2A2 HCDR1

<400> 33

Gly Tyr Thr Phe Thr Asn Tyr Trp Met His

1 5 10

<210> 34

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 2A2 HCDR2

<400> 34

Ala Ile Tyr Pro Gly Asp Ser Asp Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 35

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 2A2 HCDR3

<400> 35

Gly Leu Tyr Tyr Gly Tyr Asp

1 5

<210> 36

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 2A2 LCDR1

<400> 36

Lys Ser Ser Gln Ser Leu Ile Asp Ser Asp Gly Lys Thr Phe Leu Asn

1 5 10 15

<210> 37

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 2A2 LCDR2

<400> 37

Leu Val Ser Lys Leu Asp Ser

1 5

<210> 38

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody 2A2 LCDR3

<400> 38

Trp Gln Gly Thr His Phe Pro Tyr Thr

1 5

<210> 39

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> Mouse antibody 2A2 VH

<400> 39

Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Val Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asp Ser Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Lys Leu Thr Ala Val Thr Ser Thr Ser Thr Ala Phe

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Gly Leu Tyr Tyr Gly Tyr Asp Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser

115

<210> 40

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> Mouse antibody 2A2 VL

<400> 40

Asp Val Leu Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Ile Asp Ser

20 25 30

Asp Gly Lys Thr Phe Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 41

<211> 463

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized antibody heavy chain 2A2 (h2A2)

<400> 41

Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly

1 5 10 15

Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Ala Ile Tyr Pro Gly Asp Ser Asp Thr Ser Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

85 90 95

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Leu Tyr Tyr Gly Tyr Asp Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

<210> 42

<211> 239

<212> Protein

<213> Artificial sequence

<220>

<223>Humanized antibody light chain 2A2 (h2A2)

<400> 42

Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Pro

1 5 10 15

Gly Ser Ser Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro

20 25 30

Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser

35 40 45

Leu Ile Asp Ser Asp Gly Lys Thr Phe Leu Asn Trp Phe Gln Gln Arg

50 55 60

Pro Gly Gln Ser Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr

100 105 110

Cys Trp Gln Gly Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 43

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 HCDR1 (IH-MH)

<400> 43

Gly Tyr Thr Phe Thr Asp His Thr Met His

1 5 10

<210> 44

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 HCDR2 (DG)

<400> 44

Tyr Asn Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe Gln

1 5 10 15

Gly

<210> 45

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 HCDR2 (DG-EG)

<400> 45

Tyr Asn Tyr Pro Arg Glu Gly Ser Thr Lys Tyr Asn Glu Lys Phe Gln

1 5 10 15

Gly

<210> 46

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 HCDR2 (DG-DV)

<400> 46

Tyr Asn Tyr Pro Arg Asp Val Ser Thr Lys Tyr Asn Glu Lys Phe Gln

1 5 10 15

Gly

<210> 47

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 HCDR2 (NE-AE)

<400> 47

Tyr Asn Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Ala Glu Lys Phe Gln

1 5 10 15

Gly

<210> 48

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 VH-1

<400> 48

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Asn Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Ala Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Phe Ile Thr Thr Val Val Pro Ser Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 49

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 VH-2

<400> 49

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His

20 25 30

Thr Ile His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Asn Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Phe Ile Thr Thr Val Val Pro Ser Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 50

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 VH-3

<400> 50

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His

20 25 30

Thr Ile His Trp Met Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Tyr Asn Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Phe Ile Thr Thr Val Val Pro Ser Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 51

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 VH-4

<400> 51

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His

20 25 30

Thr Ile His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Asn Tyr Pro Arg Glu Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Phe Ile Thr Thr Val Val Pro Ser Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 52

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 VH-5

<400> 52

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp His

20 25 30

Thr Ile His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Asn Tyr Pro Arg Asp Val Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Phe Ile Thr Thr Val Val Pro Ser Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 53

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 VL-1

<400> 53

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 54

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 VL-2

<400> 54

Asp Val Gln Ile Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Asn Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 55

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody h6B5 VL-3

<400> 55

Asp Val Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Trp

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<---

Claims (85)

1. A method for treating or preventing diabetic retinopathy in a subject in need thereof, comprising ocularly administering to said subject a therapeutically effective amount of an anti-ceramide antibody or antigen-binding fragment thereof, wherein said anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V _H ) and a variable light chain (V _L ), a) wherein V _H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1), HCDR2 comprising the amino acid sequence YNYPREGSTKYNEKFQG (SEQ ID NO: 45), and HCDR3 comprising the amino acid sequence GFITTVVPSAY (SEQ ID NO: 3), and b) wherein V _L comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence RASKSISKYLA (SEQ ID NO: 4), LCDR2 comprising the amino acid sequence SGSTLQS (SEQ ID NO: 5), and LCDR3 comprising the amino acid sequence QQHNEYPWT (SEQ ID NO: 6). 2. The method according to claim 1, characterized in that said antibody against ceramide or its antigen-binding fragment is a single-chain variable fragment (scFv). 3. The method according to claim 1 or 2, characterized in that said ocular administration is selected from the group consisting of local administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber through the temporal margin, intrastromal administration, intracorneal administration, subretinal administration, injection into the intraocular fluid, sub-Tenon administration, administration into the suprachoroid space (SCS), administration into the supraciliary space, and intravitreal administration. 4. The method according to any one of paragraphs 1-3, characterized in that said administration is intravitreal administration. 5. The method according to any one of claims 1-4, characterized in that said subject has received previous treatment for diabetic retinopathy. 6. The method of claim 5, wherein said subject has not responded to said previous treatment for diabetic retinopathy. 7. The method according to claim 5 or 6, characterized in that said previous treatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from a steroid and anti-vascular endothelial growth factor (VEGF) therapy. 8. The method according to claim 7, characterized in that said 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 1-8, characterized in that said antibody against ceramide or antigen-binding fragment thereof is administered as a single dose. 10. The method according to any one of claims 1-8, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least two weeks, at least three weeks, or at least four weeks. 11. The method according to any one of claims 1-8, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about two weeks to about four weeks. 12. The method according to any one of claims 1-8, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by 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 1-8, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about one month to about six months. 14. The method according to any one of claims 1-8, characterized in that said antibody against ceramide or antigen-binding fragment thereof is administered in two or more doses separated by at least one year. 15. The method according to any one of claims 1-14, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered before the occurrence of one or more symptoms of diabetic retinopathy. 16. The method according to any one of claims 1-14, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered after the occurrence of one or more symptoms of diabetic retinopathy. 17. The method according to any one of claims 1-14, characterized in that said antibody against ceramide or its antigen-binding fragment is administered during the non-proliferative stage of diabetic retinopathy. 18. The method according to claim 17, characterized in that said antibody against ceramide or its antigen-binding fragment is administered during the proliferative stage of diabetic retinopathy. 19. A method for treating or preventing diabetic retinopathy in a subject in need thereof, comprising administering a therapeutically effective amount of an anti-ceramide antibody or antigen-binding fragment thereof to said subject, wherein said subject has received prior treatment for diabetic retinopathy, wherein said anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V _H ) and a variable light chain (V _L ), a) wherein V _H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1), HCDR2 comprising the amino acid sequence YNYPREGSTKYNEKFQG (SEQ ID NO: 45), and HCDR3 comprising the amino acid sequence GFITTVVPSAY (SEQ ID NO: 3), and b) wherein V _L comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence RASKSISKYLA (SEQ ID NO: 4), LCDR2 comprising the amino acid sequence SGSTLQS (SEQ ID NO: 5), and LCDR3 comprising the amino acid sequence QQHNEYPWT (SEQ ID NO: 6). 20. The method of claim 19, wherein said subject has not responded to said previous treatment for diabetic retinopathy. 21. The method according to claim 19 or 20, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). 22. The method according to any one of paragraphs 19-21, characterized in that said administration is ocular administration. 23. The method of claim 22, wherein said ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber through the temporal margin, intrastromal administration, intracorneal administration, subretinal administration, injection into the intraocular fluid, sub-Tenon administration, administration into the suprachoroid space (SCS), administration into the supraciliary space, and intravitreal administration. 24. The method according to claim 22 or 23, characterized in that said ocular administration is intravitreal administration. 25. The method according to any one of claims 19-24, characterized in that said previous treatment was vitrectomy, laser surgery, steroid and/or anti-vascular endothelial growth factor (VEGF) therapy. 26. The method according to claim 25, characterized in that said 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 paragraphs 19-26, characterized in that said antibody against ceramide or antigen-binding fragment thereof is administered as a single dose. 28. The method according to any one of claims 19-26, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least two weeks, at least three weeks, or at least four weeks. 29. The method according to any one of claims 19-26, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about two weeks to about four weeks. 30. The method according to any one of claims 19-26, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by 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 19-26, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about one month to about six months. 32. The method according to any one of claims 19-26, characterized in that said antibody against ceramide or antigen-binding fragment thereof is administered in two or more doses separated by at least one year. 33. The method according to any one of claims 19-32, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered before the occurrence of one or more symptoms of diabetic retinopathy. 34. The method according to any one of claims 19-32, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered after the occurrence of one or more symptoms of diabetic retinopathy. 35. The method according to any one of claims 19-32, characterized in that said antibody against ceramide or its antigen-binding fragment is administered during the non-proliferative stage of diabetic retinopathy. 36. The method according to any one of paragraphs 19-32, characterized in that said antibody against ceramide or its antigen-binding fragment is administered during the proliferative stage of diabetic retinopathy. 37. A method of treating or preventing diabetic retinopathy in a subject, comprising administering to said subject a single therapeutically effective dose of an anti-ceramide antibody or antigen-binding fragment thereof, wherein said anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V _H ) and a variable light chain (V _L ), a) wherein V _H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1), HCDR2 comprising the amino acid sequence YNYPREGSTKYNEKFQG (SEQ ID NO: 45), and HCDR3 comprising the amino acid sequence GFITTVVPSAY (SEQ ID NO: 3), and b) wherein V _L comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence RASKSISKYLA (SEQ ID NO: 4), LCDR2 comprising the amino acid sequence SGSTLQS (SEQ ID NO: 5), and LCDR3 comprising the amino acid sequence QQHNEYPWT (SEQ ID NO: 6). 38. A method for treating or preventing diabetic retinopathy in a subject, comprising administering to said subject two or more therapeutically effective doses of an anti-ceramide antibody or antigen-binding fragment thereof, wherein said two or more doses are separated by at least two weeks, wherein said anti-ceramide antibody or antigen-binding fragment thereof comprises a variable heavy chain (V _H ) and a variable light chain (V _L ), a) wherein V _H comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence GYTFTDHTIH (SEQ ID NO: 1), HCDR2 comprising the amino acid sequence YNYPREGSTKYNEKFQG (SEQ ID NO: 45), and HCDR3 comprising the amino acid sequence GFITTVVPSAY (SEQ ID NO: 3), and b) wherein V _L comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence RASKSISKYLA (SEQ ID NO: 4), LCDR2 comprising the amino acid sequence SGSTLQS (SEQ ID NO: 5), and LCDR3 comprising the amino acid sequence QQHNEYPWT (SEQ ID NO: 6). 39. The method according to claim 38, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by at least two weeks, at least three weeks, or at least four weeks. 40. The method of claim 38, wherein said anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about two weeks to about four weeks. 41. The method according to claim 38, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by 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 of claim 38, wherein said anti-ceramide antibody or antigen-binding fragment thereof is administered in two or more doses separated by a period of from about one month to about six months. 43. The method according to claim 38, characterized in that said antibody against ceramide or its antigen-binding fragment is administered in two or more doses separated by at least one year. 44. The method according to any one of claims 37-43, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv). 45. The method according to any one of paragraphs 37-44, characterized in that said administration is ocular administration. 46. The method of claim 45, wherein said ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber through the temporal margin, intrastromal administration, intracorneal administration, subretinal administration, injection into the aqueous humor, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, or intravitreal administration. 47. The method according to claim 45 or 46, characterized in that said ocular administration is intravitreal administration. 48. The method according to any one of paragraphs 37-47, characterized in that said subject has received previous treatment for diabetic retinopathy. 49. The method of claim 48, wherein said subject has not responded to said previous treatment for diabetic retinopathy. 50. The method of claim 48 or 49, wherein said prior treatment was vitrectomy, laser surgery, steroid and/or anti-VEGF therapy. 51. The method according to claim 50, characterized in that said 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 37-51, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered before the occurrence of one or more symptoms of diabetic retinopathy. 53. The method according to any one of claims 37-51, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is administered after the occurrence of one or more symptoms of diabetic retinopathy. 54. The method according to any one of paragraphs 37-51, characterized in that said antibody against ceramide or its antigen-binding fragment is administered during the non-proliferative stage of diabetic retinopathy. 55. The method according to any one of paragraphs 37-51, characterized in that said antibody against ceramide or its antigen-binding fragment is administered during the proliferative stage of diabetic retinopathy. 56. The method according to any one of claims 1 to 55, characterized in that V _H comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 51, and V _L comprises or consists of an amino acid sequence that is at least 90% identical, at least 95% identical, at least 97% identical, or 100% identical to SEQ ID NO: 53. 57. The method according to any one of claims 1-55, characterized in that V _H comprises the amino acid sequence of SEQ ID NO: 51 and V _L comprises the amino acid sequence of SEQ ID NO: 53. 58. The method according to any one of claims 1-57, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is a humanized antibody. 59. The method according to any one of claims 1-57, characterized in that said anti-ceramide antibody or antigen-binding fragment thereof is a humanized scFv. 60. The method according to any one of the preceding claims, characterized in that preventing diabetic retinopathy or inflammatory eye disease includes delaying the onset of diabetic retinopathy or inflammatory eye disease. 61. The method according to any one of the preceding claims, characterized in that one or more symptoms of diabetic retinopathy or inflammatory eye disease in said subject is reduced compared to a control subject or compared to said subject before treatment with said anti-ceramide antibody or antigen-binding fragment thereof. 62. The method of claim 61, wherein said one or more symptoms of diabetic retinopathy or inflammatory eye disease are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemia-reperfusion injury, retinal leakage area, and occludin disorder. 63. The method according to claim 61 or 62, characterized in that said one or more symptoms of diabetic retinopathy or inflammatory eye disease are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to said subject before treatment with said anti-ceramide antibody or antigen-binding fragment thereof. 64. The method according to any one of the preceding claims, characterized in that the level of expression of one or more inflammatory markers in the eye is reduced compared to the level of expression in the eye of the subject in the control or compared to the level of expression in the eye of said subject before treatment with said anti-ceramide antibody or antigen-binding fragment thereof. 65. The method according to claim 64, characterized in that one or more inflammatory markers are selected from a cytokine, a growth factor, and an adhesion molecule. 66. The method according to claim 65, characterized in that said cytokine is selected from TNFα, IL-1β, IL-6 or MCP1. 67. The method according to claim 65, characterized in that said growth factor is VEGF. 68. The method according to claim 65, characterized in that said adhesion molecule is ICAM-1 or VCAM-1. 69. The method according to any one of claims 64-68, characterized in that the level of expression of one or more inflammatory markers in the eye of said subject is reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the level of expression in the eye of the subject in the control or compared to the level of expression in the eye of said subject before treatment with said anti-ceramide antibody or antigen-binding fragment thereof. 70. The method according to any one of the preceding claims, characterized in that said subject has an increase in one or more visual parameters compared to the visual parameters of the subject in the control or compared to the visual parameters of said subject before treatment with said anti-ceramide antibody or antigen-binding fragment thereof. 71. The method according to claim 70, characterized in that said one or more parameters of vision are selected from peripheral vision; night vision; color vision; far vision; near vision; and visual clarity. 72. The method according to any one of the preceding claims, characterized in that the retinal vascular permeability in the eye of said subject 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 eye of the subject in a control or compared to the expression level in the eye of said subject before treatment with said anti-ceramide antibody or antigen-binding fragment thereof. 73. The method according to any one of the preceding claims, characterized in that the mean early diabetic retinopathy score (ETDRS) in the group of said subjects 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 score in the group of subjects in the control or compared to the mean ETDRS score in the group of said subjects before treatment with said anti-ceramide antibody or antigen-binding fragment thereof.