Anti ROBO1 and ROBO2 antibodies and uses thereof

Abstract

The present disclosure provides monoclonal antibodies that bind Robo1 and / or Robo2. In some aspects, the invention comprises a method of treating an ocular neovascular disease, a blinding eye disease, and / or cancer in a subject, comprising administering any of the antibodies of the invention to the subject. In yet other aspects, the invention comprises methods of detecting Robo1 and / or Robo2 using any of the antibodies of the invention.

Description

[0001] Attorney Docket No. 047162-7536W01(02740)

[0002] TITLE OF THE INVENTION

[0003] Anti Robol and Robo2 Antibodies and Uses Thereof

[0004] CROSS REFERENCE TO RELATED APPLICATIONS

[0005] The present application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 708,560 fded October 17, 2024, which is hereby incorporated by reference herein in its entirety.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with government support under number HL 125811 awarded by National Institutes of Health. The government has certain rights in the invention.

[0008] SEQUENCE LISTING

[0009] This application is being filed electronically via Patent Center and includes an electronically submitted sequence listing in .xml format. The .xml file contains a sequence listing entitled ■‘047162-7536WOl.xml’’ created on September 24, 2025 and having a size of 19,144 bytes. The sequence listing contained in this .xml file is part of the specification and is herein incorporated by reference in its entirety.

[0010] BACKGROUND OF THE INVENTION

[0011] Roundabout (Robo) 1 and 2 are transmembrane receptors that bind secreted Slit ligands through their extracellular domains (ECD), and signal through their cytoplasmic domains to modulate the cytoskeleton and regulate cell migration, adhesion, and proliferation. Slit-Robo signaling was discovered as a guidance cue for axons in the developing nervous system, but Robo receptors are expressed by many additional cell types and more broadly regulate organ morphogenesis, as well as cancer and pathological ocular neovascularization.

[0012] Slit-Robo signaling is an important driver of angiogenesis in the retina, kidney, and bone and plays a role in tumor angiogenesis and cancer progression. During retinal angiogenesis, activation of Robol / 2 signaling by Slit2 drives tip cell front-rear polarization and migration of endothelial cells (ECs) towards hypoxic avascular areas. Global inducible knockout of Sht2 or of Robol / 2 disrupts developmental retinal angiogenesis. Slit2 expression increases in oxygen-induced retinopathy (OIR), and global inducible knockout of Slit2 or of Attorney Docket No. 047162-7536W01(02740)

[0013] Robo L2 inhibits OIR neovascularization, demonstrating that this pathway also drives pathological ocular neovascularization.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, the drawings show specific embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0016] FIGs. 1A-1I show anti-Robol / 2 antibodies prevent Slit2 signaling in vitro. FIG. 1A shows BLI binding kinetics for Anti-Robol. Anti-Robo2 and Anti-Robol / 2 antibodies binding to murine and human Robol and Robo2. FIG. IB shows trans well migration assay of RAW267.4 macrophages response to Slit2 or carrier (CTRL) loaded in the bottom chamber after pretreatment with different Anti-Robo mAbs (n = 4 or 8, Two-way ANOVA) or Ctrl IgG. FIGs. 1C-1D show trans well migration assay of BMDMs (FIG. 1C) and of murine primary microglia (FIG. ID) in response to Slit2 after pretreatment as indicated (FIG. 1C. n = 8; FIG. ID, n=6, Two-way ANOVA). FIGs. IE- IF show Western blot quantification of Slit2 downstream signaling targets in cultured BMDMs pretreated with Ctrl IgG or Anti- Robol / 2 ( = 4, Two-way ANOVA). FIG. 1G shows qPCR analysis of BMDM cultures following Slit2 treatment with control IgG or with Anti-Robol / 2 mAb (n =5. Two-Way ANOVA). FIG. 1H shows transwell migration assay of BMDMs in response to indicated chemoattractants after pretreatment with IgG or Anti-Robol / 2 mAb (n = 3, Two-way ANOVA). FIG. II shows transwell migration assay of HUVECs in response to Slit2 or carrier (CTRL) after pretreatment as indicated (w = 4 or 8, Two-way ANOVA). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

[0017] FIGs 2A-2J show anti-Robol / 2 prevents retinal neovascularization in OIR. FIG. 2A shows IB-4 staining of Pl 7 retinal flat mounts isolated from mice subjected to OIR. Yellow7lines delineate avascular area and white arrows indicate NVTs. FIGs. 2B-2C show quantification of NVT area (FIG. 2B) and avascular area (FIG. 2C) from retinas illustrated in (FIG. 2A) (n = 22 / 18, Mann- Whitney U Test). FIG. 2D shows IB-4, Ibal and smooth muscle actin (SMA) staining of NVTs from P17 OIR retinal flat mounts. FIGs. 2E-2F show7quantification of Ibal fluorescence intensity normalized to retinal area (FIG. 2E) and MRC1+ macrophages (FIG. 2F) in P17 OIR retinal flat mounts (n = 22 / 18, Mann-Whitney U Test). FIGs. 2G-2J show FACS analysis of P17 OIR retinas quantifying myeloid cells Attorney Docket No. 047162-7536W01(02740)

[0018] (CD45+CD1 lb+CD3-, FIG. 2G), macrophages (CD45+CDllb+CD3-Ly6G-, FIG. 2H), B lymphocytes (CD45+CD1 lb-CD3-CD19+, FIG. 21) and T lymphocytes (CD45+CD11b- CD3+CD19-, FIG. 2J) (n = 4, Mann- Whitney U Test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

[0019] FIGs. 3A-3D show anti-Robol / 2 modifies OIR endothelial cell and pericyte transcriptomes. FIG. 3A shows UMAP plot of retinal cell clusters from Pl 7 OIR retinas divided by treatment. FIG. 3B shows gene set signatures represented by Normalized Enrichment Score (NES) and -loglO p value of NES comparing transcripts from Isotype control and Anti-Robol / 2-treated ECs. FIG. 3C shows gene set signatures involved in BRB, Wnt and Tgfp signaling represented by Normalized Enrichment Score (NES) and -loglO p value of NES comparing transcripts from Isotype control and Anti-Robol / 2-treated ECs. FIG. 3D shows gene set signatures represented by Normalized Enrichment Score (NES) and -loglO p value of NES comparing transcripts from Isotype control and Anti-Robol / 2-treated pericytes.

[0020] FIGs. 4A-4G show anti-Robol / 2 reduces ’M2-like' macrophage activation in OIR retinas. FIG. 4A shows UMAP plot of subclustered immune cells from P17 Normoxia and OIR retinas. FIG. 4B shows bar plot showing the percentage composition of each immune cell cluster from (FIG. 4A). FIG. 4C shows gene set signatures represented by NES and - loglO p value of NES comparing transcripts from immune cell clusters of Isotype control treated OIR to normoxia. FIG. 4D shows gene set signatures comparing transcripts from immune cell clusters of Anti -Robo 1 / 2 -treated OIR to Isotype control -treated OIR transcripts. FIG. 4E shows gene set signatures comparing transcripts from cluster Microglia / Infiltrating Macrophages 1 to those of all other clusters. FIG. 4F shows gene set signatures represented by NES and -loglO p value of NES comparing transcripts from cluster Microglia / Infiltrating Macrophages 6 to those of all other clusters. FIG. 4G shows circle plot demonstrating the differential number of interactions between different immune cell subclusters and endothelial cells comparing Isot pc control and Anti-Robol / 2-treated OIR retinas.

[0021] FIGs. 5A-5D show anti-Robol / 2 enhances efficacy of Anti-Vegfa in OIR. FIG. 5A shows IB-4 and MRC1 staining of P17 retinal flat mounts of mice subjected to oxygen- induced retinopathy after treatment with Isotype control, Anti-Vegfa and / or Anti-Robol / 2 mAbs. FIGs. 5B-5D show quantification of NVT area (FIG. 5B), avascular area (FIG. 5C) and Ibal fluorescence intensity normalized by retinal area (FIG. 5D) from (FIG. 5A) (n = 9- 18, Mann-Whitney U Test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 Attorney Docket No. 047162-7536W01(02740)

[0022] FIGs. 6A-6E show macrophage-specific Robo 1 / 2 knockout prevents retinal neovascularization. FIG. 6A shows IB-4, Ibal and MRC1 staining of P17 retinal flat mounts of mice subjected to OIR, yellow lines delineate avascular area and white arrows indicate neovascular tufts. FIGs. 6B-6E shows quantification ofNVT area (FIG. 6B), avascular area (FIG. 6C), Ibal fluorescence intensity normalized by retinal area (FIG. 6D), and MRC1+ macrophages (FIG. 6E) from (FIG. 6A) (n = 4 / 5, Mann- Whitney U Test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

[0023] FIGs. 7A-7C show Slit2 treatment induced BMDM activation in vitro. FIG. 7A shows volcano plot demonstrating differentially expressed genes on BMDMs upon Slit2 treatment. FIG. 7B shows a heatmap of the 15 top upregulated and 15 top downregulated genes on BMDMs upon Slit2 treatment as log2 fold change gene expression. FIG. 7C shows gene set signatures induced by Slit2 treatment in BMDMs represented by NES and -loglO p value of NES.

[0024] FIGs. 8A-8H show anti -Robo 1 / 2 prevents laser-induced CNV progression. FIG. 8A shows Optical Coherence Tomography (OCT) images 3- and 7-days post laser injury demonstrating laser-induced CNV lesions (y ellow boxes). FIGs. 8B-8C show quantification of (FIG. 8A) (n =9 / 6, Mann-Whitney U Test). FIG. 8D shows Fundus fluorescein angiography 1160 images 7dpi demonstrating laser-induced CNV lesions and associated vascular leakage. FIGs. 8E-8G show quantification from (FIG. 8D) of lesion area (FIG. 8E) and vascular leakage (FIG. 8F) from retinas illustrated in (FIG. 8D) (n = 7 / 6, Mann- Whitney U Test). FIG. 8G shows CD31 and Ibal staining of choroidal flat mounts 7 days post laser injury. FIG. 8H shows quantification of Ibal fluorescence intensity normalized by retinal area from (FIG. 8G) (n = 4, Mann- Whitney U Test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

[0025] FIGs. 9A-9J show macrophage-specific Robo 1 / 2 or PI3Ky knockout prevent CNV progression. FIG. 9A shows Fundus fluorescein angiography images 7-days post laser injury demonstrating laser induced CNV lesions and associated leakage. FIGs. 9B-9C show quantification of CNV lesion area from iRoboMacKO (FIG. 9B) and PIK3CG KO (FIG. 9C) mice (n = 3 mice per group, Mann- Whitney U Test). FIGs. 9D-9E show quantification of leakage in CNV lesions from iRoboMacKO (FIG. 9D) and PIK3CG KO (FIG. 9E) mice (n = 3 mice per group, Mann- Whitney U Test). FIG. 9F shows CD31 and Ibal staining of choroidal flat mounts 7 days post laser injury. FIGs. 9G-9H show quantification of lesion area from iRoboMacKO (FIG. 9G) and PIK3CG KO (FIG. 9H) mice (n = 3 mice per group, Mann- Whitney U Test). FIGs. 9I-9J show quantification of Ibal fluorescence intensity Attorney Docket No. 047162-7536W01(02740) normalized by retinal area from iRoboMacKO (FIG. 91) and PIK3CG KO (FIG. 9J) mice (n = 3 mice per group, Mann- Whitney U Test). Data are presented as mean ± s.e.m. * P < 0.05. ** P < 0.01, *** P < 0.001

[0026] FIGs. 10A-10F show affinity to human and rodent Robol and Robo2 of candidate antibodies. FIG. 10A shows BLI curves of affinity7to murine and human Robol and Robo2 of Anti-Robol. Anti-Robo2 and Anti-Robol / 2 antibodies. FIGs. 10B-10C shows transwell migration assay of murine (FIG. 10B) and human (FIG. 10C) microglia cell lines in response to Slit2 after pretreatment with Anti-Robol / 2 mAbs (n = 4, Two-way ANOVA). FIG. 10D shows Western blot analysis of Slit2 downstream signaling targets in cultured BMDMs pretreated with Anti-Robol / 2 antibodies (quantified on FIGs. 1E-1F). FIG. 10E shows high magnification image of IB-4 staining in retinal vasculature of Isotype control or Anti- Robol / 2 treated Pl 7 OIR retinas from FIG. 2A. FIG. 10F shows MRC1 staining of Pl 7 retinal flat mounts isolated from mice subjected to oxygen-induced retinopathy and treated with control IgG or Anti-Robol / 2 mAbs (quantified on FIG. 2F).

[0027] FIGs. 11A-11F show single anti-Robol / 2 intravitreal injection prevents neovascularization. FIG. 11A shows IB-4, MRC1, IgG and TERI 19 staining of P17 OIR retina flat mounts after intravitreal treatment with Anti-Robol / 2 or isotype control IgG (white arrow s indicate neovascular tufts). FIGs. 11B-1F show quantification from (FIG. 11A) of neovascular tuft area (FIG. 11B), avascular area (FIG. 11C), MRC1+ cells (FIG. 11D), Ibal fluorescence intensity normalized by retinal area (FIG. HE) and TERI 19+ red blood cells (RBCs, FIG. 1 IF) in the vascular plexus (n = 1 / 6, Mann-Whitney U Test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

[0028] FIGs. 12A-12C show7FACS analysis of Anti-Robol / 2-treated OIR P17 retinas. FIG. 12A shows flow cytometry-gating strategy7for FIGs. 2G-2J and FIGs. 12A-12C. FIG. 12B shows FACS quantification of neutrophils (CD45+CD1 lb+Ly6G+). FIG. 12C show's the ratio of CD4+ / CD8+ T lymphocytes between control- and Anti-Robol / 2 treated P17 OIR retinas (n = 4, Mann-Whitney U Test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** < 0.001

[0029] FIGs. 13A-13J show the effects of anti-Robol / 2 antibody in retinal cells. FIG. 13A show's dot plot of expression amount and frequency among cell clusters of selected genes in P17 OIR retinal cells. FIG. 13B show s cell cycle analysis of retinal cells from P17 OIR retinas. FIG. 13C shows gene set signatures represented by Normalized Enrichment Score (NES) and -log 10 p value of NES comparing transcripts from Isotype control and AntiRobo 1 / 2 -treated photoreceptors. FIG. 13D shows a volcano plot of differentially expressed Attorney Docket No. 047162-7536W01(02740) gene transcripts from Anti-Robol / 2-treated ECs compared to Isotype control-treated ECs. FIG. 13E shows a volcano plot of differentially expressed gene transcripts from AntiRobo 1 / 2 -treated pericytes when compared to Isotype control-treated. FIG. 13F shows a transwell migration assay of HBPCs in response to Slit2 or carrier (CTRL) after pretreatment with different anti-Robo mAbs (n = 4 or 8, Two-way ANOVA). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

[0030] FIGs. 14A-14L show the effect of Anti-Robol / 2 treatment on macrophage heterogeneity in OIR retinas. FIG. 14A shows a dot plot of transcript expression level and frequency across immune cell clusters of selected genes in Pl 7 OIR retinas. FIG. 14B shows a UMAP plot of immune cells from P17 retinas separated by experimental group / treatment. FIG. 14C shows cell cycle analysis of subclustered immune cells from P17 OIR retinas. FIG. 14D shows volcano plots comparing transcripts from cluster Microglia / Infiltrating Macrophages 6 to those of all other clusters. FIG. 14E shows a volcano plot comparing transcripts from cluster Microglia / Infiltrating Macrophages 1 to those of all other clusters. FIG. 14F shows a plot demonstrating changes on incoming and outgoing signaling pathways in cluster Microglia / Infiltrating Macrophages 1 from Anti-Robol / 2 treated OIR retinas when compared to Isotype control. FIG. 14G shows gene set signatures represented by Normalized Enrichment Score (NES) and -loglO p value of NES comparing transcripts from immune cell clusters of Anti-Robo 1 / 2-treated OIR to Isotype control-treated OIR retinas. FIG. 14H shows a heatmap demonstrating the differential number of interactions between different immune cell subclusters and endothelial cells comparing Isotype control and Anti-Robo I / 2-treated OIR retinas (related to circle plot on FIG. 4G). FIG. 141 shows a dot plot demonstrating pathways with increased signaling between Microglia / Infiltrating Macrophages subclusters and ECs on Anti-Robo 1 / 2-treated retinas when compared to Isotype control-treated. FIG. 14J shows a dot plot demonstrating pathways with decreased signaling between Microglia / Infiltrating Macrophages subclusters and ECs on Anti-Robo 1 / 2-treated retinas when compared to Isoty pe control -treated. FIGs. 14K-14L show gene set signatures (FIG. 14K) and Volcano plot (FIG. 14L) comparing transcripts from Isotype control and Anti- Robo 1 / 2-treated Neutrophils.

[0031] FIGs. 15A-15C shows the evaluation of the Anti-VEGFa mAb. FIG. 15A shows quantification of Biolayer interferometry' (BLI) demonstrating that the anti-Vegfa mAb (200 nM) binds to human VEGF121 and to human and mouse VEGF165, but not human VEGFB, C, or D. FIG. 15B shows the binding of the anti-Vegfa mAb to human VEGF121 is blocked completely by VEGFRl-Fc. Quantification BLI demonstrating binding to VEGF 121 -coated Attorney Docket No. 047162-7536W01(02740)

[0032] BLI sensors by 200 nM Anti-Vegfa mAb is completely prevented when the sensors are prebound by saturating hVEGRl-Fc (200 nM), but not when prebound by a non-binding control protein (200 nM). The anti-Vegfa mAb does not bind to tips that are not coated with VEGF (no VEGF). FIG. 15C shows high magnification image of IB-4 staining in retinal vasculature of P17 OIR retinas from FIG. 5 A.

[0033] FIGs. 16A-16O show PI3Kgamma knockout prevents retinal neovascularization.

[0034] FIG. 16A shows a transwell migration assay of BMDMs from PIK.3CG 1253 wild-type (WT) and knockout (KO) mice in response to various chemoattractants (n = 3, Two-way ANOVA). FIG. 16B shows a UMAP plot of P10 retinal cells from 53. FIG. 16C shows a dot plot of expression level and frequency of different PI3K isoforms among cell clusters from (FIG. 16B). FIG. 16D shows IB-4 and MRC1 staining of P17 retinal flat mounts of mice subjected to oxygen-induced retinopathy (OIR, yellow lines delineate avascular area and white arrows indicate neovascular tufts). FIGs. 16E-16H show the quantification of neovascular tuft area (FIG. 16E), avascular area (FIG. 16F), Ibal fluorescence intensity normalized by retinal area (FIG. 16G) and MRC1+ macrophages (FIG. 16H) from retinas illustrated in (FIG. 16D) (n = 4, Mann-Whitney U Test). FIGs. 161-160 show flow cytometry -gating strategy and analysis of P17 OIR retinas quantifying myeloid cells (CD45+CD1 lb+CD3-, FIG. 16J), macrophages (CD45+CDllb+CD3-Ly6G-, FIG. 16K), type 1 dendritic cells (CD45+CDl lb+CDl lc+, FIG. 16L), type 2 dendritic cells (CD45+CDl lb-CDl lc+, FIG. 16M) B lymphocytes (CD45+CDl lb-CD3-CD19+. FIG. 16N) and T lymphocytes (CD45+CD1 lb-CD3+CD19-, FIG. 160) (w = 7 / 6, Mann-Whitney U Test). Data are presented as mean ± s.e.m. * P < 1266 0.05, ** P < 0.01, *** P < 0.001

[0035] FIGs. 17A-17B shows Slit2 induces CD38 and IL-ls expression, but does not change activation of other Ml polarization pathways. FIG. 17A shows a volcano plot demonstrating differential expression of Ml polarization-related genes on BMDMs upon Slit2 treatment. FIG. 17B shows gene set signatures of Ml -related pathways in BMDMs represented by Normalized Enrichment Score (NES) and -loglO p value of NES comparing control and Slit2 -treated BMDMs.

[0036] FIGs. 18A-18J show anti-Robol / 2 mAbs inhibit retinal angiogenesis. FIG. 18A shows an experimental design: newborn Cx3crl-YFP mice were 1276 treated with 10 mg / kg Anti-Robol / 2 mAb i.p. at postnatal days 0, 2 and 4 and analyzed at postnatal day 5. FIG. 18B shows CD31 staining of P5 retinal flat mounts. FIG. 18C shows quantification of vascular outgrowth from retinas illustrated in (FIG. 18B) (n = 10 / 11. Mann- Whitney U Test). FIG. 18D shows CD31 and YFP staining of vascular fronts and plexus from P5 retinal flat Attorney Docket No. 047162-7536W01(02740) mounts. FIGs. 18E-18G shows quantification from (FIG. 18D) of sprouts (FIG. 18E), branchpoints (FIG. 18F), CX3CR1+ cells in the vascular front and in the plexus (FIG. 18G) (n = 10 / 1 1, Mann- Whitney U Test). FIGs. 18H-18J show quantifications of the interactions between CX3CR1+ macrophages and sprouts in the vascular fronts (yellow arrows in (FIG. 18D), n = 10 / 11, Mann-Whitney U Test). Data are presented as mean ± s.e.m. * P < 0.05, ** < 0.01, *** < 0.001

[0037] FIGs. 19A-19N show macrophage-specific Robol / 2 knockout and PI3gamma knockout impairs retinal angiogenesis. FIG. 19A shows IB-4 staining of P6 retinal flat mounts isolated from iRoboMacKO and P5 retinal flat mounts isolated from PIK3CG KO mice. FIGs. 19B-19C shows quantification of vascular outgrowth from (FIG. 19A) ( = 6-28, Mann- Whitney U Test). FIG. 19D show IB-4 and Ibal staining of vascular fronts and plexus from retinal flat mounts of iRoboMacKO and PIK3CG KO mice. FIGs. 19E-19H show quantifications from (FIG. 19D) of sprouts (FIGs. 19E-19F) and Ibal+cells in the vascular front and plexus (FIG. 19G-19H) (n = 6-23, Mann- Whitney U Test). FIGs. 19I-19N show quantifications of the interactions between Ibal macrophages and sprouts in the vascular fronts from (FIG. 19D) n = 4-6, Mann-Whitney U Test). Data are presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] Definitions

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0041] As used herein, each of the following terms has the meaning associated with it in this section.

[0042] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in Attorney Docket No. 047162-7536W01(02740) cell culture, molecular genetics, analytical chemistry, immunology, and nucleic acid chemistry and hybridization are those well-known and commonly employed in the art. Standard techniques or modifications thereof are used for chemical syntheses and chemical analyses.

[0043] The articles “a” and “an’' are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element" means one element or more than one element.

[0044] As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a concentration, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0045] As used herein, the term “affinity ” for a molecule towards another refers to the degree (or tightness) of binding between the two molecules. A higher affinity means tighter binding between the two molecules. Affinity can be quantified in terms of dissociation constant (or Ka), where a Kd value that is lower in magnitude (closer to zero) indicates a higher affinity.

[0046] An “amino acid” as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. “Standard amino acid” means any of the twenty L- amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid residues” means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, “synthetic amino acid” also encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change a peptide’s circulating half life without adversely affecting activity of the peptide. Additionally, a disulfide linkage may be present or absent in the peptides.

[0047] The term “antibody,” as used herein, refers to an immunoglobulin molecule able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular Attorney Docket No. 047162-7536W01(02740) antibodies (“intrabodies”), Fv, Fab and F(ab)2, as well as single chain antibodies (scFv), camelid antibodies and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). As used herein, a “neutralizing antibody” is an immunoglobulin molecule that binds to and blocks the biological activity of the antigen.

[0048] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated or synthesized, or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0049] As used herein, the term “blinding eye disease” refers to diseases that can cause blindness in the eye. These disease can overlap, but do not necessarily have to be classified as an ocular neovascular disease. Symptoms include, but are not limited to blurry vision, eye pain, floaters and flashers in the line of sight, sensitivity to light, sudden loss of vision, or the sudden appearance of black spots in your vision.

[0050] A “coding region” of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene that are homologous with or complementary to, respectively, the coding region of an mRNA molecule produced by transcription of the gene.

[0051] A “coding region” of an mRNA molecule also consists of the nucleotide residues of the mRNA molecule that are matched with an anti-codon region of a transfer RNA molecule Attorney Docket No. 047162-7536W01(02740) during translation of the mRNA molecule or that encode a stop codon. The coding region may thus include nucleotide residues corresponding to amino acid residues not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0052] “Complementary” as used herein to refer to a nucleic acid, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[0053] The term “delivery vehicle” is used herein as a generic reference to any delivery vehicle capable of delivering a compound to a subject, including, but not limited to, dermal delivery vehicles and transdermal delivery vehicles.

[0054] The term “DNA” as used herein is defined as deoxyribonucleic acid.

[0055] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein, effective to achieve a particular biological result. Such results may include, but are not limited to, treatment of a disease or condition as determined by any means suitable in the art.

[0056] “Encoding” refers to the inherent property7of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to sen e as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the Attorney Docket No. 047162-7536W01(02740) biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0057] As used herein, the term "fragment.” as applied to a protein or peptide, refers to a subsequence of a larger protein or peptide. A “fragment” of a protein or peptide can be at least about 20 amino acids in length; for example at least about 50 amino acids in length; at least about 100 amino acids in length, at least about 200 amino acids in length, at least about 300 amino acids in length, and at least about 400 amino acids in length (and any integer value in between). As used herein, an antibody fragment refers to active fragments thereof, i.e., fragments having the same characteristics that are used for the definition of an antibody according to the invention, in certain embodiments high affinity for a-Syn fibrils (composed of misfolded a-Syn) and low or high binding affinity to a-Syn monomers. For convenience when the term antibody is used, fragments thereof exhibiting the same characteristic are also being considered.

[0058] As used herein, the term “fragment,” as applied to a nucleic acid, refers to a subsequence of a larger nucleic acid. A “fragment” of a nucleic acid can be at least about 15 nucleotides in length; for example, at least about 50 nucleotides to about 100 nucleotides; at least about 100 to about 500 nucleotides, at least about 500 to about 1000 nucleotides, at least about 1000 nucleotides to about 1500 nucleotides; or about 1500 nucleotides to about 2500 nucleotides; or about 2500 nucleotides (and any integer value in between).

[0059] Conventional notation is used herein to describe polynucleotide sequences: the lefthand end of a single-stranded polynucleotide sequence is the 5 ’-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5’-direction.

[0060] The direction of 5’ to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand”; sequences on the DNA strand which are located 5’ to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3‘ to a reference point on the DNA are referred to as "downstream sequences.”

[0061] An “individual”, “patient” or “subject”, as that term is used herein, includes a member of any animal species including, but are not limited to, birds, humans and other primates, and Attorney Docket No. 047162-7536W01(02740) other mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs. Preferably, the subject is a human.

[0062] ■‘Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition and / or compound of the invention in a kit. The instructional material of the kit may. for example, be affixed to a container that contains the compound and / or composition of the invention or be shipped together with a container which contains the compound and / or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.

[0063] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0064] An “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in anaturally occurring state, z.e., a DNA fragment which has been removed from the sequences which are normally adj acent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzy me digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

[0065] In the context of the present invention, the following abbreviations for the commonly- occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine. Attorney Docket No. 047162-7536W01(02740)

[0066] By “nucleic acid'’ is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

[0067] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0068] As used herein, the term “ocular neovascular disease” refers to a group of diseases or conditions that occurs when new blood vessels grow in the eye in the wrong place or in excess. Symptoms include, but are not limited objects appear different sizes in the eye, colors appearing less bright or different in each eye, light flashes or flickering in central vision, light sensitivity, blurry vision, pain, and eye redness.

[0069] The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 60 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”

[0070] As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound of the invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

[0071] “Pharmaceutically acceptable” refers to those properties and / or substances that are acceptable to the patient from a pharmacological / toxicological point of view and to the manufacturing pharmaceutical chemist from a physical / chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability. “Pharmaceutically acceptable carrier” refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is Attorney Docket No. 047162-7536W01(02740) administered.

[0072] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i. e. , the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0073] As used herein, the terms “protein”, “peptide” and “polypeptide” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. The term "peptide bond" means a covalent amide linkage formed by loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that may comprise the sequence of a protein or peptide. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many ty pes. “Proteins” include, for example, biologically active fragments, substantially homologous proteins, oligopeptides, homodimers, heterodimers, variants of proteins, modified proteins, derivatives, analogs, and fusion proteins, among others. The proteins include natural proteins, recombinant proteins, synthetic proteins, or a combination thereof. A protein may be a receptor or a non-receptor.

[0074] The term “recombinant DNA” as used herein is defined as DNA produced by joining pieces of DNA from different sources.

[0075] The term “recombinant polypeptide” as used herein is defined as a polypeptide produced by using recombinant DNA methods.

[0076] The term “RNA” as used herein is defined as ribonucleic acid.

[0077] As used herein, the terms “Robot,” “Robo2,” “Robol / 2,” “Roundabout 1,” and “Roundabout 2” refer to proteins that serve as axon guidance receptors and cell adhesion receptors, known to be involved in the decision by axons to cross the central nervous system Attorney Docket No. 047162-7536W01(02740) midline. They bind to Slit proteins Slitl and Slit2 and activate Robo / Slit signaling pathways to promote angiogenesis and immune cell migration and modulate the cytoskeleton, in turn regulating cell migration, adhesion, and proliferation.

[0078] As used herein, the terms “Slit 1 ” and “Slit2” refer to proteins that bind via the second leucine-rich repeat region (D2) to the Igl domain of ROBO1 and ROBO2. Slitl and / or Slit2 binding triggers recruitment of adaptor proteins to the ROBO cytoplasmic domain that modulate the cytoskeleton, in turn regulating cell migration, adhesion, and proliferation.

[0079] The term “therapeutic” as used herein means a treatment and / or prophylaxis.

[0080] The term to “treat,” as used herein, means reducing the frequency with which symptoms are experienced by a subject or administering an agent or compound to reduce the frequency and / or severity with which symptoms are experienced. As used herein, “alleviate” is used interchangeably with the term “treat.”

[0081] As used herein, “treating a disease, disorder or condition” means reducing the frequency or severity with which a symptom of the disease, disorder or condition is experienced by a subject. Treating a disease, disorder or condition may or may not include complete eradication or elimination of the symptom.

[0082] The following abbreviations are used herein: CDR, complementary -determining region; VH, heavy chain variable region; VL, light chain variable region.

[0083] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0084] Description

[0085] Slit ligands are evolutionary conserved secreted polypeptides that bind to transmembrane Roundabout (Robo) receptors and signal through the recruitment of adaptor proteins to the Robo cytoplasmic domain that modulate the cytoskeleton, in turn regulating cell migration, adhesion and proliferation. There are three mammalian Slits and four Robo receptors, but only Robol and 2 bind Slits. Slit-Robo signaling was discovered as a guidance Attorney Docket No. 047162-7536W01(02740) cue for axonal grow th cones, but this pathway affects various cellular processes in multiple tissue types, including the brain, kidney, heart, lung, pancreas and bone. In addition, Slit- Robo signaling has also been implicated in multiple human pathologies including cancer and inflammation.

[0086] Slit-Robo signaling is an important driver of angiogenesis in the retina, kidney and bone and plays a role in tumor angiogenesis and cancer progression. During retinal angiogenesis, activation of Robo 1 / 2 signaling by Slit2 drives tip cell front-rear polarization and migration of endothelial cells (ECs) tow ards hypoxic avascular areas, via Endophilin-A2 dependent internalization and activation of the adaptor proteins Nek and srGAPl, resulting in PAK dependent activation of small GTPases that drive cytoskeletal rearrangements required for tip cell polarization and migration. Global inducible knockout of Slit2 or of Robo 1 / 2 disrupts developmental retinal angiogenesis. Slit2 expression increases in oxygen-induced retinopathy (OIR), and global inducible knockout of Slit2 or of Robo 1 / 2 inhibits OIR neovascularization, demonstrating that this pathway also drives pathological ocular neovascularization. However, EC-specific Robol / 2 knockout has no effect on ocular neovascularization, suggesting the implication of other Sht2-responsive cells.

[0087] Slit-Robo signaling has also been implicated in cancer progression, with a particular role in tumor angiogenesis. This signaling pathw ay triggers responses in tumor cells and stromal fibroblasts, ECs, and immune cells. In brain tumors, Slit2 signaling via Robol / 2 drives macrophage recruitment, pro-tumoral polarization, and induction of dysmorphic angiogenesis. Slit2 also promotes macrophage chemoattraction and prevents macropinocytosis and cytotoxic polarization, suggesting that this pathway may also play a role in macrophage activation in other pathological conditions. Antibody blockade of Robo 1 inhibits tumor angiogenesis induced by Slit2-expressing malignant melanoma in vivo. However, many cell types such as myeloid cells also express Robo2, calling for pharmacological agents to inhibit both Robol and 2 to prevent Slit signaling in those cells.

[0088] Towards this goal, antibodies were generated recognizing the Robol and 2 ECDs and their biological activity tested in mouse models of ocular neovascular diseases (ONDs). Blindness due to ONDs is a devastating condition that affects preterm infants, as well as elderly and diabetic patients. This group of diseases comprise retinopathy of prematurity (ROP), which is a leading cause of childhood blindness in the USA, diabetic retinopathy (DR) and neovascular / wet age-related macular degeneration (AMD), which are the leading causes of irreversible blindness and visual impairment in the developed world.

[0089] Despite different pathophysiological mechanisms underlying ROP, DR and AMD, Attorney Docket No. 047162-7536W01(02740) excessive ocular neovascularization is a common driver of OND progression, and current therapies focus on inhibiting angiogenesis by targeting vascular endothelial growth factor (VEGF) signaling to inhibit the formation of neovessels and decrease their permeability. VEGF inhibition via intravitreal injections slows vision loss and even improves vision in a subset of patients, but does not halt the visual decline that occurs in the long term in 30% of AMD patients. Current treatments inhibiting VEGF mainly target ECs, which are activated by VEGF binding to an endothelial tyrosine kinase receptor, VEGFR2. However, it is well recognized that additional cell types, notably myeloid cells and pericytes, play important roles in vascular development and in OND progression. Despite the recognized role of these cells, how they cooperate to orchestrate neoangiogenesis is poorly understood, and there are no known pathways that can simultaneously target these different cell types to improve OND progression.

[0090] Here it is shown that antibody-mediated blockade of Slit2 signaling through both Robot and Robo2 prevents pathological retinal neovascularization in OIR and laser-induced choroidal neovascularization (CNV) models, mainly by inhibiting myeloid cell recruitment. Single cell RNA sequencing of OIR retinas revealed the effects of this pathway in the heterogeneous population of myeloid cells, and myeloid cell specific ablation o Robol&2 phenocopied effects of Anti-Robol / 2 blocking antibodies, revealing a new strategy7to combat inflammation in ONDs.

[0091] In one aspect, this disclosure is generally directed to certain monoclonal antibodies, or fragments thereof, that recognize Robol and Robo2 extracellular domains. It is demonstrated herein that the antibodies of the invention recognize and inhibit Robo / Slit mediated signaling.

[0092] In certain embodiments, the antibodies of the invention bind to Robol and / or Robo2 with a dissociation constant Ka equal to or higher than about IO-10M. about IO-9M, about 10’8M, about IO’7M, about 10’6M, about 10'5M, about 10‘4M, or about 10'3M. In other embodiments, the antibodies of the invention bind to Robol and / or Robo2, with a dissociation constant Ka equal to or less than about IO'3M, about 10'4M, about 10'5M, about 10’6M. or about 10'7M. Binding affinities of the antibodies can be determined by using a variety of methods recognized in the art, including methods described elsewhere herein, such as but not limited to isothermal calorimetry, surface plasmon resonance, immunoassays such as ELISA or RIAs, and the like.

[0093] Compositions Comprising Antibodies

[0094] In one aspect, the invention comprises isolated monoclonal antibodies that selectively Attomey Docket No. 047162-7536W01(02740) bind Robol and / or Robo2 extracellular domains. In certain embodiments, the antibody comprises a heavy chain. In other embodiments, the heavy chain comprises three complementary-determining regions (CDR), namely CDR1, CDR2 and CDR3. In yet other embodiments, the light chain comprises three complementary-determining region (CDR), namely CDR1, CDR2 and CDR3.

[0095] In certain embodiments, the monoclonal antibody is derived from a hybridoma with light and heavy variable chains having the sequences shown below:

[0096] Table 1: Light and Heavy Chains

[0097] Table 2: VL Analysis

[0098] Table 3: VH Analysis Attorney Docket No. 047162-7536W01(02740)

[0099] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region (VL) comprising the amino acid sequence of SEQ ID NOs: 1, 2, or 3.

[0100] In certain embodiments, the antibody comprises an immunoglobulin heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NOs: 4, 5, or 6.

[0101] In certain embodiments, the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 1, and a VH comprising SEQ ID NO: 4. In other embodiments, the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 2, and a VH comprising SEQ ID NO: 5. In other embodiments, the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 3, and a VH comprising SEQ ID NO: 6.

[0102] In certain embodiments, the antibody comprises a VL comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 10, 11, or 12.

[0103] In certain embodiments, the antibody comprises a VL comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the antibody comprises a VL comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the antibody comprises a VL comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 12.

[0104] In certain embodiments, the antibody comprises a VH comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 13, 14, or 15; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 16, 17, or 18; and a CDR3 region Attorney Docket No. 047162-7536W01(02740) comprising the amino acid sequence of SEQ ID NOs: 19, 20, or 21.

[0105] In certain embodiments, the antibody comprises a VH comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 13; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 16; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the antibody comprises a VH comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 14; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 17; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the antibody comprises a VH comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 15; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 18; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 21.

[0106] In certain embodiments, the antibody comprises a VL comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 10; and a VH comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 13; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 16; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 19.

[0107] In certain embodiments, the antibody comprises a VL comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 1 1 ; and a VH comprising: a CDR 1 region comprising the amino acid sequence of SEQ ID NO: 14; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 17; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 20.

[0108] In certain embodiments, the antibody comprises a VL comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 12; and a VH comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 15; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 17; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 21.

[0109] The invention further provides isolated polynucleotides (including RNA and / or DNA) encoding the antibodies or antigen binding fragments thereof, for example a nucleic acid encoding for one or more CDRs, or a variable heavy chain or variable light chain region of the a-Syn antibodies of the invention. Nucleic acid includes DNA and RNA.

[0110] In certain embodiments, the invention provides an isolated polynucleotide comprising Attorney Docket No. 047162-7536W01(02740) a nucleic acid encoding the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, or 6. In other embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid encoding the amino acid sequence of SEQ ID NOs: 1 and 4. In yet other embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid encoding the amino acid sequence of SEQ ID NOs: 2 and 5. In yet other embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid encoding the amino acid sequence of SEQ ID NOs: 3 and 6.

[0111] The invention further provides a host cell comprising the expression vector. In certain embodiments, the host cell is isolated. In other embodiments, the host cell is a non-human cell. The expression vector can comprise nucleic acid sequences that direct and / or control expression of the inserted polynucleotide. Such nucleic acid sequences can include regulatory sequence, including promoter sequences, terminator sequences, polyadenylation sequences, and enhancer sequences. Systems for cloning and expression of a polypeptide in a variety of cells are well know n in the art.

[0112] The antibody of the invention can be a mammalian antibody, such as primate, human, rodent, rabbit, ovine, porcine or equine antibody. The antibody can be any class or isotype antibody, for example IgM or IgG. In certain embodiments, the antibody is IgG.

[0113] The invention further provides a kit comprising an antibody of the invention. The antibody may be an intact immunoglobulin molecule or fragment thereof such as Fab, F(ab)2 or Fv fragment. The antibody can be labelled as described elsewhere herein. The kit can be for use in a method of determining whether a subject has a neurodegenerative disease, and / or for treating a subject afflicted or thought to be afflicted with a neurodegenerative disease. The kit can further any other reagent or instrument that is required to implement a method of the invention, such as a buffer, an applicator, and the like.

[0114] The non-limiting generation of these antibodies is illustrated in Example 2. Data showing high affinity binding in FIG. 1A, and these properties are further described in FIGs. 10A-10F. In certain embodiments, the invention comprises pharmaceutical compositions comprising each of these antibodies in combination with one or more pharmaceutically acceptable excipients. In some embodiments the pharmaceutical composition is formulated for parenteral delivery. In some embodiments the pharmaceutical composition is formulated for intraocular delivery'. In other embodiments, the antibodies are humanized.

[0115] Methods of Treating an Ocular Neovascular Disease or a Blinding Eye Disease

[0116] In another aspect, the invention provides a method of treating, ameliorating, and / or Attorney Docket No. 047162-7536W01(02740) preventing an ocular neovascular disease or a blinding eye disease. In certain embodiments, the method comprises administering a therapeutically effective amount of an isolated monoclonal antibody of the invention to a patient. In certain embodiments, the antibody is humanized. In other embodiments, the antibody is administered as a pharmaceutical composition.

[0117] The monoclonal antibodies described above may be used to treat an ocular neovascular disease or a blinding eye disease by binding to Robol and / or Robo2 and reducing angiogenesis and inflammation, as further described in Example 1.

[0118] Neovascularization of the eye refers to new blood vessels forming where and when they should not form. Neovascularization can affect different parts of the eye including, but not limited to the choroid, retina, macula, iris, cornea, and conjunctiva. Symptoms of neovascularization include, but are not limited to seeing distorted or wavy images, empty or dark spots in central vision, eye pain, redness, watery eyes, sensitivity to light, and an inability7to wear contact lenses for longer than a few hours.

[0119] In certain embodiments, the ocular neovascular disease associated with Robol and Robo2 includes but is not limited to diabetic retinopathy, neovascular age-related macular degeneration (nAMD), retinopathy of prematurity (ROP), and other related ocular neovascular diseases. In certain embodiments, the blinding eye diseases associated with Robol and Robo2 include but are not limited to age-related macular degeneration, glaucoma, diabetic retinopathy, retinitis pigmentosa, cataracts. Behcet’s disease. Retinopathy of Prematurity (ROP), Leber hereditary7optic neuropathy, Anophthalmia, and Microphthalmos, and other related blinding eye diseases. In some embodiments, the disease can be classified as both an ocular neovascular disease and a blinding eye disease. The antibody can be administered systemically or directly to the site where angiogenesis are observed or thought to be present. In a non-limiting example, the antibody can be administered by injection into a blood vessel supplying the eye or into the eye itself. The subject can be a mammal, such as a human or a non-human mammal.

[0120] Methods of Treating Cancer

[0121] In another aspect, the invention provides a method of treating, ameliorating, and / or preventing cancer. In certain embodiments, the method comprises administering a therapeutically effective amount of an isolated monoclonal antibody of the invention to a patient. In certain embodiments, the antibody is humanized. In other embodiments, the antibody is administered as a pharmaceutical composition. Attorney Docket No. 047162-7536W01(02740)

[0122] The monoclonal antibodies described above may be used to treat, ameliorate, and / or prevent cancer by binding to Robol and / or Robo2 and reducing tumor angiogenesis and inflammation, as further described in Example 1. Tumor angiogenesis is the process by which tumors create new blood vessels to supply themselves with oxygen and nutrients. Angiogenesis is a requisite not only for continued tumor grow th, but also for metastasis. The monoclonal antibodies described above can be used to treat, ameliorate, and / or prevent additional diseases associated with VEGF and / or ANG2 including, but not limited to rheumatoid arthritis and macular degeneration.

[0123] In certain embodiments, the cancer associated with Robol and Robo2 includes but is not limited to brain cancer (including glioblastoma), bone cancer, retinoblastoma, colorectal cancer, pancreatic cancer, lung cancer, breast cancer, gastric cancer, glioma, and osteosarcoma. The antibody can be administered systemically or directly to the site where angiogenesis are observed or thought to be present. In a non-limiting example, the antibody can be administered by inj ection into a blood vessel supplying the cancer or into the cancer itself. The subject can be a mammal, such as a human or a non-human mammal.

[0124] Methods of Treating, Preventing, and / or Ameliorating Focal Segmental Glomerulosclerosis (FSG)

[0125] In some aspects described herein, it is contemplated that the antibodies described herein can be used to treat, prevent, and / or ameliorate focal segmental glomerulosclerosis (FSG or FSGS). In certain embodiments, the method comprises administering a therapeutically effective amount of an isolated monoclonal antibody of the invention to a patient diagnosed with FSG. In certain embodiments, the antibody is humanized. In other embodiments, the antibody is administered as a pharmaceutical composition.

[0126] The monoclonal antibodies described above may be used to treat focal segmental glomerulosclerosis by binding to Robol and / or Robo2 and reducing angiogenesis and inflammation.

[0127] FSG is a disease in which scar tissue develops on the glomeruli, the small parts of the kidneys that filter waste from the blood. There are four types of FSG: primary FSG, secondary FSG, genetic FSG, and unknown FSG. Symptoms of FSG include swelling in the legs and ankles, around the eyes, and other body parts; weight gain from fluid buildup; and foamy urine from protein building (referred to as proteinuria). The antibody can be administered systemically or directly to the site where angiogenesis are observed or thought to be present. In a non-limiting example, the antibody can be administered by injection into a Attorney Docket No. 047162-7536W01(02740) blood vessel supplying the kidney or into the kidney itself. The subject can be a mammal, such as a human or a non-human mammal.

[0128] Methods of Detection

[0129] In yet another aspect, the invention provides methods of detecting an ocular neovascular disease, a blinding eye disease, or cancer in a patient. In some embodiments, the monoclonal antibodies described above can be used to detect additional diseases associated with VEGF and / or ANG2 including, but not limited to rheumatoid arthritis and macular degeneration. In other embodiments, the antibodies of the invention can be used as diagnostic tools for detecting an ocular neovascular disease, a blinding eye disease, or cancer associated with Robol and / or Robo2. including but not limited to age-related macular degeneration, glaucoma, diabetic retinopathy, retinitis pigmentosa, cataracts, Behcet’s disease, Retinopathy of Prematurity (ROP), Leber hereditary optic neuropathy, Anophthalmia, Microphthalmos, glioblastoma, and other related diseases as described herein.

[0130] In certain embodiments, the method of detecting an ocular neovascular disease, a blinding eye disease, or cancer in a subject comprises the steps of administering a labeled, isolated monoclonal antibody of the invention to the subject, and detecting the presence of absence of a complex between Robol and / or Robo2 in the subject and the antibody. If the complex is present, that indicates that Robol and / or Robo2 exist in the subject. In certain embodiments, if Robol and / or Robo2 are present in the subject, the subject has a an ocular neovascular disease, a blinding eye disease, or cancer. In other embodiments, if Robol and / or Robo2 are not present in the subject, the subject does not have an ocular neovascular disease, a blinding eye disease, or cancer. In yet other embodiments, if the subject has an ocular neovascular disease, a blinding eye disease, or cancer, the individual is counseled to undergo therapy and / or pharmacological treatment for the ocular neovascular disease, a blinding eye disease, or cancer. In yet other embodiments, if the subject has an ocular neovascular disease, a blinding eye disease, or cancer, the individual is provided therapy and / or pharmacological treatment for the ocular neovascular disease, a blinding eye disease, or cancer.

[0131] In certain embodiments, the method further comprises comparing the level of antibody / Robol and / or Robo2 complex formed in the subject with the level of antibody / Robot and / or Robo2 complex formed in a reference subject. The reference subject can be a subject known not to have Robol and / or Robo2, a subject known to have detectable Robol and / or Robo2, and / or a subject known to have a certain level of Robol and / or Robo2. Attorney Docket No. 047162-7536W01(02740)

[0132] The reference subject can further be the same subject being treated or evaluated, but corresponding to an earlier Robol and / or Robo2 detection experiment, as a way to evaluate disease progression and / or treatment efficacy in the subject.

[0133] In yet another aspect, the invention provides methods of detecting Robol and / or Robo2 in a sample. In certain embodiments, the antibodies of the invention can be used as diagnostic tools for detecting the presence of Robol and / or Robo2 in a sample.

[0134] In certain embodiments, the method of detecting Robol and / or Robo2 in a sample (for example, from a subject) comprises the steps of contacting the sample with a labeled, isolated monoclonal antibody of the invention, and detecting the presence or absence of a complex between any Robol and / or Robo2 in the sample and the antibody. If the complex is detected, that indicates the presence of Robol and / or Robo2 in the sample. The sample can be, in nonlimiting examples, eye, blood or tissues from eye, endothelial cells, myeloid cells, and pericytes. In certain embodiments, the sample is an eye sample. In other embodiments, the sample is used as is after being removed from the subject. In other embodiments, the sample is pre-treated being used within the present methods.

[0135] In certain embodiments, the level of Robol and / or Robo2 detected in a subject or in a sample from a subject correlates with severity or progression of an ocular neovascular disease or a blinding eye disease in the subject. In other embodiments, the methods of the invention can be used to monitor severity or progression of an ocular neovascular disease or a blinding eye disease in the subject. In yet other embodiments, the methods of the invention can be used to monitor effectiveness of a therapy and / or pharmacological intervention in a subject afflicted or believed to be afflicted with an ocular neovascular disease or a blinding eye disease.

[0136] Methods for detecting formation of a complex between the antibody and Robol and / or Robo2 comprise, but are not limited to, radioimmunoassay, enzyme-linked immunosorbant assay (ELISA), sandwich immunoassay, fluorescent immunoassay, precipitation reaction, gel immunodiffusion assay, agglutination assay, protein A immunoassay, immunoelectrophoresis assay, electrophoresis, western blotting, or any other technique known in the art.

[0137] The antibodies of the invention can be combined with a label and used to detect Robol and / or Robo2 in a patient or in a sample. Methods of labeling antibodies are know n in the art and a variety of approaches may be employed. In certain embodiments the label is a radiolabel, such as but not limited to F18, 1123, In111, 1131. C14, H3, Tc"m. P32, 1125, Ga68and the like. In other embodiments, the label is a fluorescent label, such as but not limited to Attorney Docket No. 047162-7536W01(02740) fluorescein, rhodamine and the like. In yet other embodiments, the label is a contrast agent, such as but not limited to gadolinium (Gd), dysprosium and iron, magnetic agents, and the like. Other labels include nuclear magnetic resonance active labels, positron emitting isotopes detectable by a PET scanner, chemiluminescent and enzymatic markers. Nonlimiting imaging techniques include electron microscopy, confocal microscopy, light microscopy, positron emission tomography (PET), gamma-scintigraphy, magnetic resonance imaging (MRI), functional magnetic resonance imaging (FMRI), magnetoencephalography (MEG), and single photon emission computerized tomography (SPECT). In yet other embodiments, the label is on a secondary antibody that binds a primary' antibody comprising the above described sequences.

[0138] Administration / Dosage / Formulations

[0139] Administration of the compounds and / or compositions of the present invention to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to perform a therapeutic and / or imaging method contemplated in the invention. An effective amount of the compound necessary' for adequate therapeutic and / or imaging result may' vary according to factors such as the state of a disease or disorder in the patient; the age, sex, and weight of the patient; and the equipment used to detect the compound of the invention. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic and / or imaging compound without undue experimentation.

[0140] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve successful therapeutic and / or imaging results for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0141] In certain embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the invention comprise an effective amount of a compound of the invention and a pharmaceutically acceptable carrier.

[0142] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the Attorney Docket No. 047162-7536W01(02740) required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or poly alcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0143] Compounds of the invention for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0144] In certain embodiments, the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In certain embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5.000 mg, or less than about 3.000 mg, or less than about 2,000 mg, or less than about 1 ,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in certain embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0145] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g, lubricants, preservatives, stabilizers, wetting agents, Attorney Docket No. 047162-7536W01(02740) emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like.

[0146] Routes of administration of any of the compositions of the invention include oral, nasal, rectal, ocular, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the invention may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g, sublingual, lingual, (trans) buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intraocular, intrabronchial, inhalation, and topical administration.

[0147] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry' powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

[0148] Parenteral Administration

[0149] As used herein, '“parenteral administration7’ of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.

[0150] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Formulations for parenteral administration Attorney Docket No. 047162-7536W01(02740) include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In certain embodiments of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.

[0151] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a nontoxic parenterally-acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-admimstrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. Additional Administration Forms

[0152] Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 2003 / 0147952; 2003 / 0104062; 2003 / 0104053; 2003 / 0044466; 2003 / 0039688; and 2002 / 0051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757.

[0153] EXPERIMENTAL EXAMPLES

[0154] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not Attorney Docket No. 047162-7536W01(02740) intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0155] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, practice the claimed methods of the present invention. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0156] Example 1: Monoclonal antibodies blocking Roundabout 1 and 2 signaling target pathological ocular neovascularization via myeloid cells

[0157] Generation of monoclonal antibodies recognizing Robot and Robo2 receptors.

[0158] Screening of a phage-displayed library of human antigen-binding fragments (Fabs) against Fc-fusions of the entire rat and human Robol and Robo2 ECDs yielded three monoclonal antibodies, including one that bound to both Robol and 2 (Anti-Robol / 2), and two that bound specifically to Robol (Anti-Robol) or Robo2 (Anti-Robo2). In the bivalent human IgGl format, all three antibodies bound to human and rodent Robo receptors, with dissociation constants (Ka) in the picomolar range, as determined by BioLayer Interferometry (BLI) assay (FIG. 1 A, FIGs. 10A-10C).

[0159] To determine if these antibodies blocked Slit2-induced signaling, their capacity' w as tested to prevent chemoattraction of RAW267.5 murine macrophage cells towards Slit2 in Boyden chamber assays. Pre-treatment of RAW267.5 cells for 30 minutes with 50 pg / ml of Anti-Robol / 2 antibody abrogated chemotactic migration towards Slit2 added to the bottom chamber, while Anti-Robol or Anti-Robo2 antibody only reduced Slit2-induced migration by 50-60% (FIG. IB). As myeloid cells express approximately equal amounts of Robol and Robo2 protein, Anti-Robol / 2 likely blocked Slit2-induced macrophage migration by inhibiting signaling through both Robol and Robo2. Anti-Robol / 2 antibody also blocked Slit2 induced migration of primary' murine bone marrow-derived macrophages (BMDM, FIG. 1C), primary' murine microglia (FIG. ID), and both murine and human microglial cell lines (FIGs. 10B-10C).

[0160] Anti-Robol / 2 pre-treatment blocked Slit2 -induced Akt and Erkl / 2 phosphorylation in BMDMs (FIGs. 1E-1F, 10D) and prevented Slit2-induced changes in gene expression, such Attorney Docket No. 047162-7536W01(02740) as increases inMrcl, Vegfa and Argl gene expression (FIG. 1G). Anti-Robol / 2 antibody pretreatment specifically blocked Slit2 -induced macrophage migration, while BMDMs still migrated towards other chemoattractants including CSF-1, CCL21 and IL-10 (FIG. 1H).

[0161] It was also tested if the antibodies prevented Slit2-Robol / 2 signaling in human umbilical vein ECs (HUVECs), which express Robol but not Robo2. Anti-Robol or AntiRobo 1 / 2 antibody abrogated Slit2 -induced HUVEC migration, whereas Anti-Robo2 antibody had no effect (FIG. II). Taken together, these results confirm that Anti-Robol / 2 antibody prevents signaling through murine and human Robol and 2 in vitro, in two cell types relevant for angiogenesis (i.e. myeloid and endothelial cells).

[0162] Robo inhibition prevents pathological retinal neovascularization.

[0163] The in vivo effect of Anti-Robol / 2 antibody was investigated on retinal neovascularization using the OIR model. Postnatal day 7 (P7) pups were exposed to 75% oxy gen until P12 to induce vascular obliteration. Upon return to room air, neovascularization and formation of pathological and leaky neovascular tufts (NVTs) occurred by P17. Mice were treated with 10 mg / kg i.p. of Anti-Robol / 2 or isotype control antibody at days P12, P14 and Pl 6, and retinas were analyzed at Pl 7.

[0164] Anti-Robol / 2 treatment significantly increased the avascular area and reduced the retinal NVT area (FIGs. 2A-2C, FIG. 10E), along with a reduction in smooth muscle actin (SMA) coverage of NVTs (FIG. 2D), and reduced Ibal+ and MRC1+ microglia / macrophage infiltration to the neovascular area (FIGs. 2D-2F, FIG. 1 OF). A single intravitreal injection of 50 pg Anti-Robol / 2 antibody was sufficient to reduce NVT area and increase the avascular area (FIGs. 11A-11C), along with a reduction in MRC1+ microglia / macrophage infiltration (FIG. 1 ID) and reduced IgG leakage and bleeding (FIGs. 1 IE-1 IF).

[0165] FACS analysis of P17 OIR retinas demonstrated a 2-fold decrease in the CD45+CD1 lb+ myeloid cell population in Anti-Robol / 2 treated retinas when compared to controls (FIG. 2G and FIG. 12A), which w as due to a significant decrease in the numbers of CD45+CDl lb+Ly6G- microglia / infiltrating macrophages (FIG. 2H). By contrast, the total numbers of neutrophils (CD45+CD l lb+CDl lc-Ly6G+) were similar between treatment groups (FIG. 12B). CD45+CD1 lb- CD3+ T cells and CD45+ CD1 lb- CD19+ B cells were increased in Anti-Robol / 2 treated P17 retinas when compared to controls (FIGs. 2I-J), but the CD4+ / CD8+ ratio of infiltrating T cells remained similar betw een groups (FIG. 12C). These data revealed a major effect of Slit2 -Robo 1 / 2 signaling in regulating immune cell infiltration in the context of OIR, with opposing effects on myeloid and lymphoid cells, as Attorney Docket No. 047162-7536W01(02740) previously demonstrated in other tissue contexts.

[0166] Single cell RNA sequencing of Anti-Robol / 2 treated OIR retinas.

[0167] To understand how Anti-Robol / 2 treatment affected retinal cells during OIR, single cell RNA sequencing (scRNA-seq) was performed with Pl 7 OIR retinas treated with either isotype control IgG or Anti-Robol / 2 antibody. ECs and myeloid cells were enriched amongst all retinal cell subpopulations using CD31- and CD1 Ib-coated magnetic beads and magnetic cell sorting (MACS) columns before sequencing. After fdtering for low-quality cells and doublets, a total of 29,886 cells were sequenced (12,490 in the CTRL IgG group and 17,396 in the Anti-Robol / 2 group) including 5,999 ECs (1,240 in the CTRL IgG group and 4,759 in the Anti-Robol / 2 group) and 3,164 immune cells (1,335 in the CTRL IgG group and 1,829 in the Anti-Robol / 2 group). Uniform manifold approximation and projection (UMAP) plots were used for visualization and performed graph-based clustering and annotation according to gene expression profdes using previously published retinal markers for annotation (FIGs. 3 A, 13 A). No major cluster differences were observed between CTRL and Anti-Robol / 2- treated retinas, and cell cycle analysis demonstrated no major difference in cell proliferation between treatments (FIGs. 13B-13C).

[0168] Differential gene expression and gene set enrichment analyses (GSEA) were used to investigate the changes induced by Anti-Robol / 2 treatment in retinal cells. GSEA analysis of the photoreceptor subpopulation (10,134 cells, 5,618 in the CTRL IgG group and 4,516 in the Anti-Robol / 2 group) showed downregulation of pathways related to apoptosis / cell death, hypoxia and inflammation when compared to CTRL IgG-treated OIR retinas, as well as downregulation of pathw ays related to abnormal retinal electrophysiology', retinal cell dystrophy and Macular Degeneration (FIG. 13D). In contrast, this cluster upregulated pathways related to neurotransmitter signaling, synapse assembly, ligand-gated ion channel signaling and integrin signaling (FIG. 13D), indicating a neuroprotective effect of Anti- Robol / 2 treatment on photoreceptors.

[0169] GSEA of ECs revealed downregulation of Slit-Robo signaling in Anti-Robol / 2- treated OIR retinas, along with known downstream effectors including PI3K-mTORC l and Rac / Rho / Cdc42 GTPases, as well as a reduction in apoptosis, inflammatory IL-2 and cytokine signaling, and decreased activity' of Vegfa-Vegfr2 and Tgfp-Smad signaling (FIG. 3B), which are two main contributors to NVT formation. Furthermore, Anti-Robol / 2 treated retinal ECs upregulated pathways related to visual phototransduction and neuroprotective signaling, along with genes involved in the maintenance of the Blood-Retina Barrier (BRB) Attorney Docket No. 047162-7536W01(02740) and extracellular matrix formation (FIGs. 3B-3C, FIG. 13E), corroborating the reduced pathological neovascularization and leakage observed in histological analysis.

[0170] As Anti-Robol / 2 treated OIR retinas displayed reduced SMA coverage (FIG. 2D), and SMA is known to be upregulated in OIR pericytes, it was queried if pericytes responded to Slit2-Robol / 2 signaling. The scRNA-seq pericyte population (415 cells, 163 in the CTRL IgG group and 252 in the Anti-Robol / 2 group) displayed decreased Slit-Robo signaling, and an increase in genes related to neurotransmitter transporters (FIG. 3D). Anti-Robol / 2 treatment led to a significant reduction in vascular smooth muscle contractile genes and actin cytoskeleton regulators such as Acta2 (FIGs. 3D, 13F), consistent with reduced SMA (Acta2) coverage of NVTs observed in vivo. Furthermore, downregulated PDGF signaling and decreased inflammatory Jak-Stat, IL- 12 and TGFb signaling was also observed (FIG. 3D). Transwell chamber migration assays demonstrated that human brain pericytes (HBPCs) migrated towards a Slit2 gradient and this migration was abrogated by treatment with Anti- Robol / 2 antibody but was only partially affected by Anti-Robol or Anti-Robo2 antibody (FIG. 13F), indicating that pericytes are responsive to Slit2 signaling.

[0171] Anti-Robol / 2 antibody affects myeloid cell activation in OIR retinas.

[0172] To understand the activation of myeloid cells in OIR retinas, and to explore the effects of Anti-Robol / 2 antibody in the immune cell populations, scRNA-seq was also performed in CD1 Ib-enriched cells from control normoxic P17 retinas, and subclustered the immune cell population of all 3 datasets (P17 normoxia, P17 OIR treated w ith CTRL Abs, and P17 OIR treated with Anti-Robol / 2 antibody, hereafter named Normoxia, CTRL OIR and Anti-Robo OIR datasets). Gene expression profiles identified 13 clusters, including 11 clusters of retinal Myeloid cells. Nine different populations of Microglia / Infiltrating Macrophages were identified based on expression on known microglial (Tmeml 19, P2ryl2 and Gpr34) and macrophage markers (Cd68, Mrcl and Lgals3). Monocytes and Neutrophils were also identified, as w ell as a cluster of B and T lymphocytes, and a population that had mixed myeloid and neuronal transcripts (Mixed Myeloid / Neurons) (FIGs. 4A, 14A).

[0173] When comparing the myeloid cell populations between groups, the cluster Microglia / Infiltrating Macrophages 1 was enriched in Normoxia retinas when compared to OIR CTRL retinas, and its abundance was rescued upon Anti-Robol / 2 treatment. The clusters Microglia / Infiltrating Macrophages 2 and 8 were enriched in both CTRL and Anti- Robol / 2 treated OIR retinas when compared to Normoxia. The cluster Microglia / Infiltrating Macrophages 6 was significantly expanded in Anti-Robol / 2 treated OIR retinas when Attorney Docket No. 047162-7536W01(02740) compared to both Normoxia and CTRL OIR retinas, and cluster Microglia / Infiltrating Macrophages 7 was decreased in CTRL OIR and Anti-Robol / 2 treated when compared to normoxia. The remaining clusters display little variation between the different experimental conditions (FIG. 4B, FIG. 14B). When analyzing proliferation of the immune cell clusters, the cluster Microglia / Infiltrating Macrophages 9 predominantly proliferated in the Normoxia retinas, and this proliferation was abrogated upon OIR but partially rescued by Anti-Robol / 2 treatment (FIG. 14C).

[0174] Differential gene expression analysis and GSEA demonstrated that when compared to hypoxia conditions, exposure to OIR led to an almost homogenous upregulation of pathways related to hypoxia, angiogenesis, Slit-Robo signaling and PI3K signaling across all Microglia / Macrophage clusters (FIG. 4C). Interestingly, an upregulation of glycolysis-related genes was also observed in all these clusters, as previously reported in bulk RNA sequencing of macrophages from OIR retinas (FIG. 4C). Contrasting with this homogeneous effect, pathways classically implicated in classical inflammatory “Ml” and alternative antiinflammatory “M2” macrophage polarization profiles were differently regulated between clusters, with no clear trend towards Ml or M2 polarization (FIG. 4C).

[0175] When compared to CTRL OIR retina myeloid cells, Anti-Robol / 2 treatment led to an overall downregulation of pathways normally induced by Slit-Robo signaling, such as CDC42, Racl / 2 and Rho GTPases, and PI3K signaling (FIG. 14G). Anti-Robol / 2 treatment also led to a downregulation of glycolysis-related genes (FIG. 4F), which are implicated in the pathological activation of retinal macrophages. Downregulation of VEGF signaling was observed in myeloid cells from Anti-Robol / 2 treated retinas (FIG. 4F), while Interferon type 1 (a and P) and Interferon y responses were increased upon Anti-Robol / 2 treatment (FIG. 4F). Anti-Robol / 2 treatment led to an overall downregulation of pathways related to alternative anti-inflammatory “M2” polarization associated with an upregulation of classical inflammatory “Ml” pathways in most of the microglia / macrophage clusters (FIG. 4F). Interestingly, the clusters Microglia / Infiltrating Macrophages 3 and 9 had a different behavior, with upregulation of both pro- and anti264 inflammatory pathw ays (FIG. 4F), highlighting the heterogeneity of the myeloid populations and their responses to Sht-Robo signaling inhibition.

[0176] The cluster Microglia / Infiltrating Macrophages 1 w as underrepresented in CTRL OIR retinas when compared to both normoxia and Anti-Robol / 2 treated OIR retinas. Differential expression and GSEA analysis demonstrated that this cluster had reduced Slit-Robo signaling when compared to all other immune cell clusters, as well as a global downregulation of Attorney Docket No. 047162-7536W01(02740)

[0177] PI3Ksignaling and inflammatory pathways (FIG. 4E, FIGs. 14E-14F). Furthermore, increased neuroprotective signaling was observed in this cluster, with increased expression of genes related to Neurexins and Neuroligins, Nephrins and Neuroactive ligands signaling and increased neurotransmitter and phototransduction signaling (FIG. 4E), suggesting a neuroprotective role for this cluster in developing retinas, which is lost upon OIR and recovered after Anti -Robo 1 / 2 treatment.

[0178] The cluster Microgha / Infiltrating Macrophages 6 was significantly enriched in Anti- Robol / 2 treated OIR retinas when compared to both normoxia and CTRL treated OIR retinas (FIG. 4B, FIG. 14B). Comparison against all other myeloid cell clusters showed upregulation of angiogenesis-associated genes, as well as of pathways related to phagocytosis, antigen processing and presentation (such as HLA genes) and pathways classically implicated in classical inflammatory “Ml” macrophage 281 polarization (such as Ill 3, TNFa and Interferons) (FIGs. 4D, 14D).

[0179] Ligand-receptor interaction analysis were performed using CellChatDB to dissect changes in immune cell interactions in the OIR retina following Anti-Robol / 2 treatment. Increased interactions were observed of all immune cells subclusters with ECs upon Anti- Robol / 2 inhibition in OIR retinas (FIGs. 4G, 14H). Interestingly, the cluster Microglia / Infiltrating Macrophages 6 displayed increased interactions with ECs upon Anti- Robol / 2 treatment, including pro-angiogenic VEGF and Semaphorin signaling (FIG. 141). This contrasts with reduced pro-angiogenic signaling (VEGF. Semaphorins, Angiopoietin) observed between ECs and all other Microglia / Infiltrating Macrophages clusters upon Anti- Robol / 2 signaling (FIG. 14J). Hence, the balance of pro-angiogenic signaling in Anti- Robol / 2 treated OIR retinas changes from global production of pro-angiogenic molecules by all OIR myeloid cells to a more directed production of such molecules by one specific cluster of myeloid cells (Microglia / Infiltrating Macrophages 6).

[0180] Finally, it was observed that neutrophils also had decreased activation of pathways related to Slit-Robo signaling (FIGs. 14K-14L). Furthermore, in this cluster, decreased activation and degranulation markers were observed, as well as decreased IL-4, IL-13, IL-6 and TGF[3 signaling (FIGs. 14K-14L).

[0181] Robo inhibition enhances Anti-Vegfa effects in pathological neovascularization.

[0182] The efficacy of Anti -Robo 1 / 2 antibodies was compared in preventing retinal pathological neovascularization to that of Anti-Vegfaa antibodies, the current standard of care for patients with ONDs. As clinically approved LUCENTIS and AVASTIN antibodies do not Attorney Docket No. 047162-7536W01(02740) recognize mouse Vegfa and are thus not suitable for murine oxygen-induced retinopathy (OIR), phage display selections were used followed by screening with human and mouse Vegfa to identify a novel Anti -Vegfa mAb that bound to human VEGF 121, to human and mouse VEGF165, but not to human VEGFB, VEGFC, or VEGFD (FIG. 15A). Moreover, Anti -Vegfa mAh competed with VEGFR-1 for binding to VEGF, and consequently, antagonized VEGF signaling (FIG. 15B).

[0183] Mice were submitted to OIR and treated with 10 mg / kg i.p. of Anti-Robol / 2 and / or Anti -Vegfa or isotype control antibody at days P12, P14 and Pl 6, and retinas were analyzed at Pl 7. Compared to CTRL IgG treated retinas, Anti-Robol / 2 and Anti-Vegfa mAbs both reduced NVT area and increased the avascular area (FIGs. 5A-5C, FIG. 15C). Combined treatment with Anti-Robol / 2 and Anti-Vegfa further reduced retinal NVT area (FIGs. 5A-5C, FIG. 15C). Anti-Vegfa treatment was associated with an increased infiltration of Ibal+ microglia / macrophages (FIG. 5D), as previously reported after VEGFa inhibition with a ligand trap. The increase in microglia / macrophage recruitment was reversed by treatment with Anti-Robol / 2 mAbs (FIG. 5D), demonstrating the benefit of Slit-Robo signaling inhibition in reducing pathological microglia / macrophage recruitment in the retina.

[0184] Myeloid cell-specific Robol / 2 knockout prevents ocular neovascularization.

[0185] To evaluate the function of Slit2 -Robol / 2 signaling inhibition in macrophages during retinal neovascularization. CSFlRcreERT2mice were crossed with Robol- / -Robo2flox / fl°xmice to generate inducible macrophage Robol / 2 knockout mice (iRoboMacKO) that were subjected to OIR.

[0186] Macrophage specific Robo deletion was induced by i.p. treatment with 1 mg of tamoxifen at P12, P13 and P14, and mice were analyzed at P17. Compared to littermate controls, iRoboMacKO mice exhibited a significant reduction in the NVT area and an increase in the avascular area (FIGs. 6A-6C), along with reduced Ibal+ and MRC1+ microglia / macrophage infiltration (FIGs. 6D-6E), as observed in mice treated with Anti- Robol / 2 antibodies.

[0187] PI3Ky mediates macrophage recruitment during ocular neovascularization.

[0188] Expression of PIK3CG, a previously identified mediator of Slit2-Robo signaling in macrophages, was highly restricted to the immune cell cluster of developing retinas (FIGs. 16B-16C). To confirm that Slit2-induced macrophage migration occurred in a PI3Ky dependent manner, BMDMs from PIK.3CG KO mice were used. In Transwell chamber Attorney Docket No. 047162-7536W01(02740) assays, PIK3CG KO BMDMs lost their capacity to migrate towards Slit2 and other chemoattractants such as CSF-1. CCL21 and VEGFA, while wildtype littermate cells migrated (FIG. 16A).

[0189] PIK3CG KO OIR retinas had significantly reduced NVT area and increased avascular area (FIGs. 16D-16F), along with reduced MRC1+ and Ibal+ microglia / macrophage infiltration in the neovascularized retinas (FIGs. 16G-16H).

[0190] FACS analysis of PIK3CG KO retinas demonstrated a reduction in the numbers of CD45+CD1 Ib+CDl 1c- microglia / infiltrating macrophages (FIGs. 16I-16K) and no change in the numbers of CD45+CDl lb+CDl lc+ type 1 or CD45+CDl lb-CDl 1c- type 2 DCs (FIGs. 16L-16M). Interestingly, there were no changes in the number of infiltrating CD45+CDl lb- CD19+ B cells or CD45+CDl lb- CD3+ T cells when comparing the retinas of PIK3CG WT and PIK3CG KO mice (FIGs. 16N-16O).

[0191] Slit2 induces a “M2-like” macrophage transcriptome

[0192] Given the importance of Slit2-Robol / 2 signaling in OIR microglia / infiltrating macrophages, the effect of Slit2 in macrophage activation was further investigated in vitro. Isolated BMDMs were treated with 6 nM Slit2 for 16 hours and analyzed by bulk RNA sequencing. Upon Slit2 treatment, significant downregulation and upregulation was observed for 715 and 638 genes, respectively, compared to cells in medium without Slit2 (FIGs. 7A- 7B). As expected, Slit2 induced pathways related to immune cell migration / activation and PI3K signaling (FIG. 7C). Furthermore, Slit2 activated matrix metalloproteases and several inflammatory pathways related to M2 macrophage polarization, including IL- 10, IL-20, IL- 23, IL-2 and Interferon y (FIG. 7C), demonstrating the importance of Slit2 in inducing macrophage activation. Among genes related to Ml macrophage polarization, Gprl8, Fpr2, Nos2, Ccr7, Tnfa, Inhba, 1112b, Cd86, 116, Cxcl9, Ptgs2, Cxcl3, Illa, Lcn2, Ppap2a, Ptges exhibited no significant changes after Slit2 treatment, nor did GSEA pathway analysis of IL- 1, Interferon alpha, TNF, Toll-like receptor (TLR), CLEC7A and Myc signaling pathways (FIG. 17B). However, Slit2 activated IL-6 signaling and led to increased expression oflllfi and Cd38 by BMDMs (FIGs. 7C and 17A). Altogether, these data demonstrate that Slit2 induces a predominant M2-like signature in BMDMs, but with some characteristics of Ml polarized macrophages, such as increased IL-6 signaling and IL-1 production.

[0193] Robol / 2 inhibition decreased laser-induced choroidal neovascularization.

[0194] To test effects of Anti-Robo treatment in a model for AMD, laser-induced choroidal Attorney Docket No. 047162-7536W01(02740) neovascularization (CNV) was used. Four laser injuries were induced around the optic nerve of adult mice, disrupting the Bruch’s membrane between the choroid and the retina, and leading to the formation of aneovascular lesion. Mice were then treated i.p. with 10 mg / kg of Anti-Robol / 2 or isotype control antibody at days 0, 2, 4 and 6 post-injuries, and eyes were analyzed at days 3 and 7 post-injury.

[0195] Optical coherence tomography (OCT) imaging confirmed the induction of choroidal neovascularization in both groups, but lesion size in mice treated with Anti-Robol / 2 antibody was reduced when compared to controls (FIGs. 8A-8C). Fundus fluorescein angiography at D7 confirmed the reduction in lesion size (FIGs. 8D-8E) and revealed significantly reduced leakage from the CNV lesions (FIG. 8F) upon Anti-Robol / 2 treatment.

[0196] Staining of whole-mount choroids confirmed that Anti-Robol / 2 treatment reduced neovascular lesion size (FIG. 8G), which was accompanied by a significant reduction in the infiltration of Ibal+ macrophages in these lesions (FIG. 8H), confirming the potential of Slit2-Robol / 2 signaling inhibition for the treatment of ONDs.

[0197] To address the role of macrophage Slit-Robo and PI3Ky signaling, laser-induced CNV was performed in iMacRoboKO and PIK3CGKO mice. Robo deletion was induced by treating mice i.p. with 2 mg of tamoxifen at days 1, 2 and 3 after injury. Fundus fluorescein angiography at D7 demonstrated reduced lesion size and leakage in both iRoboMacKO and PIK3CGKO mice when compared to littermate controls (FIGs. 9A-9E). Whole-mount imaging of the choroids of these mice confirmed the reduction in CNV lesion size (FIGs. 9F- 9H) and demonstrated reduced infiltration of Ibal+ macrophages in both iRoboMacKO and PIK3CGKO mice (FIGs. 9I-9J).

[0198] Anti-Robol / 2 antibody impairs retinal angiogenesis in vivo

[0199] Lastly, the effect of Anti-Robol / 2 antibody in postnatal retinal angiogenesis was tested. Neonatal mice expressing a macrophage reporter Cx3crl-YFP at postnatal days (P) 0, 2 and 4 intraperitoneally (i.p.) were treated with 10 mg / kg of antibody and analyzed retinas at P5 (FIG. 18A). Treatment with Anti-Robol / 2 antibody reduced vascular outgrowth, sprouting and number of branchpoints in treated retinas compared with isotype control IgG (FIGs. 18B-18F).

[0200] Anti-Robol / 2 treatment also reduced the number of CX3CR1+ macrophages in both the vascular front and plexus (FIG. 18G), suggesting that macrophage Slit2-Robol / 2 signaling contributed to retinal vasculature development. Anti-Robol / 2 treated mice had fewer macrophage-tip cell contacts at the vascular front (FIG. 18H) and a smaller percentage Attorney Docket No. 047162-7536W01(02740) of macrophages contacting tip cells (FIG. 181). Furthermore, the number of sprouts contacted by at least one CX3CR1+ macrophage was also reduced upon Anti-Robol / 2 treatment (FIG. 18J), suggesting a role for Slit2-Robol / 2 signaling in mediating macrophage-tip cell interactions.

[0201] To understand if cell-autonomous Robol / 2 signaling in macrophages affects the developing retina vasculature, developing retinas of iRoboMacKO and PIK3CGKO mice were analyzed. Robol / 2 deletion was induced by 100 pg tamoxifen injection postnatally at P0, Pl and P2, and retinas were analyzed at P6. Interestingly, these mice had a reduced retinal vascular outgrowth (FIGs. 19A-19B). PIK3CG KO retinas were analyzed at P5, and when compared to wild-type retinas, had a similar phenotype, displaying a minor but significant vascular outgrowth delay (FIGs. 19A-19C).

[0202] Both iRoboMacKO and PIK3CGKO mice also displayed reduced numbers of sprouts in the vascular front (FIGs. 19D-19F), and a reduction in the number of Ibal+ macrophages in the vascular front and plexus (FIGs. 19G-19H). Fewer macrophage-tip cell interactions were observed at the vascular front (FIGs. 19I-19N) with a smaller percentage of macrophages contacting tip cells and a reduced number of sprouts contacted by at least one Ibal+ macrophage both in iRoboMacKO and PIK3CGKO mice. This reduction in tip cell macrophage interactions is similar to that observed upon Anti-Robol / 2 treatment (FIGs. 19G- 191), suggesting that these interactions depend on Slit-Robol / 2 signaling in macrophages to drive sprouting angiogenesis.

[0203] Non-limiting Comments

[0204] Chronic inflammation, activation of retinal microglial cells, and recruitment of peripheral immune cells to the retina play an important role in the progression of ONDs. Murine models of ONDs and human data have demonstrated increased recruitment of pro- angiogenic M2 macrophages to the retina. Abnormal activation of monocyte derived macrophages and retinal microglial cells exacerbate neovascularization via different signaling pathways, such as IL-6, IL-17A, Tie2, and TGFb, and disease severity can be reduced by reducing myeloid cell recruitment and activation. Despite this important role of retinal microglia and infiltrating macrophages in the development and progression of ONDs, no therapeutic strategy focusing on these cells has been developed to date.

[0205] This documents describes in one aspect generation and characterization of a functionblocking mAb against both Robol and 2 receptors, and reveals the biological activity associated with pathway blockade in mouse models of OND. In vitro assays demonstrated Attorney Docket No. 047162-7536W01(02740) that the Anti-Robol / 2 antibody blocked Slit2 induced migration and signaling in myeloid cells, endothelial cells and pericytes in vitro. Systemic or intraocular treatment with AntiRobo 1 / 2 antibodies led to regression of NVTs in OIR and reduced CNV, accompanied by an important reduction in myeloid cell infiltration in both pathological models. Mechanistically, inhibition of PI3Ky signaling and genetic inhibition of myeloid Robol&2 signaling recapitulated reduced NVT formation along with myeloid cell recruitment, demonstrating the importance of the Slit2-Robol / 2-PI3Ky axis in myeloid cells during ocular neovascularization. scRNAseq from OIR retinas confirmed downregulaled Slit-Robol / 2 signaling in three cell ty pes associated with the vasculature that are targeted by Slit2-Robol / 2 signaling inhibition. Among those, ECs display decreased activity’ of Vegfa-Vegfr2 and Tgfb-Smad signaling and increased expression of BRB genes, which contribute to the observed amelioration of neovascularization. However, these EC signaling changes alone cannot mediate protection against OIR, as EC-specific deletion of Robol and Robo2 does not block retinal neovascularization. Inhibition of pericyte Slit-Robol / 2 signaling prevented pericyte pathological activation and aSMA expression, thereby reducing contractility and ameliorating NVT bleeding. Concurrently, a significant upregulation of neuroprotective pathways was observed in ECs, pericytes and immune cells in OIR retinas treated with Anti-Robol / 2 mAbs, associated with a neuroprotective signature in photoreceptors, demonstrating protection from degeneration in this model.

[0206] Comparison with an Anti-Vegfa antibody revealed that Anti-Robol / 2 mAbs reduced inflammation and cooperated with Vegfa inhibition in reducing NVT formation. Hence, Anti- Robol / 2 antibodies appear as an attractive novel alternative to be used alone or in combination with Anti-Vegfa therapy for the treatment of ONDs.

[0207] A surprising finding of the single cell RNA sequencing was the heterogeneity of microglial and macrophage subpopulations observed in the OIR retinas. Bulk RNA sequencing had previously demonstrated a distinct activation profile of myeloid cells in OIR and laser-induced CNV, but the scRNAseq data details how different populations of retinal microglia and macrophages changed in response to OIR and to Slit-Robol / 2 signaling inhibition, unveiling a network of pathological retinal myeloid cell activation. OIR induces upregulation of glycolysis in all myeloid cell clusters. Glycolysis drives polarization into pro- angiogenic pathological macrophages, which express genes related to both Ml and M2 activation and promote retinal neovascularization. While glycolytic metabolism was originally correlated with pro-inflammatory Ml polarization, data demonstrate that M2- Attorney Docket No. 047162-7536W01(02740) polarized macrophages can be induced by lactic acid under pathological conditions and are highly glycolytic. The data suggest that in OIR, glycolysis is associated with a particular microglia / macrophage activation phenotype where both Ml and M2 pathways are induced. Interestingly, Anti-Robol / 2 mAb treatment inhibited glycolysis, proangiogenic signaling and some M2 pathways (such as IL-10, TGFb and Wnt) while supporting the increase of Ml pathways, particularly interferon signaling. Overall, the data indicate that Anti-Robol / 2 mAbs can be used to prevent the pro-angiogenic pathological activation of retinal myeloid cells in patients with ONDs.

[0208] Interestingly, OIR retinas lost cluster Microglia / Infiltrating Macrophages 1, which was present in the healthy retina and characterized by the production of neurotrophic factors. Blocking Slit-Robol / 2 signaling during OIR led to the recovery of this population, which may thus confer a neuroprotective effect. The Microglia / Infiltrating Macrophages 6 cluster was enriched in retinas treated with Anti-Robol / 2 mAbs when compared to both Control IgG-treated and Normoxia retinas. Unlike the other myeloid cell clusters, the Microglia / Infiltrating Macrophages 6 cluster displayed increased pro-angiogenic signaling upon Anti-Robol / 2 mAb treatment. Anti-Robol / 2 treatment therefore restricts the production of pro-angiogenic molecules to one cluster of OIR myeloid cells, which may facilitate retinal revascularization. No significant changes were observed in the relative frequency of the other myeloid cell clusters. However, all myeloid cell clusters display significant changes in their activation profiles following Anti-Robol / 2 mAb treatment, underlining the importance of myeloid Slit-Robo signaling in pathological angiogenesis and the therapeutic potential of Anti-Robol / 2 mAbs.

[0209] Changes were also observed in several signaling pathways important for OND pathogenesis in Anti-Robol / 2 treated retinas. In addition to downregulation of VEGF signaling in most myeloid cells, dow nregulation of pro-angiogenic IL- 10 and IL- 12 signaling was observed, while type I interferon (a and ) and interferon y signaling, which inhibit retinal neovascularization, increased under Anti-Robol / 2 treatment. This indicates in certain embodiments that blocking Slit-Robo 1 / 2 signaling could simultaneously inhibit pathological myeloid cell activation and promote the activation of protective pathways in the context of ONDs.

[0210] In vitro bulk RNA sequencing of macrophages treated with Slit2 confirmed that inflammatory pathways that were decreased upon Anti-Robol / 2 treatment in OIR retinal macrophages were increased in vitro by Slit2, treatment, further corroborating the notion that the effects observed in the OIR retinas are due to direct inhibition of Slit-Robo 1 / 2 signaling Attorney Docket No. 047162-7536W01(02740) in myeloid cells.

[0211] Anti-Robol / 2 treatment also affected developmental retinal angiogenesis and mimicked the phenotype of global inducible Slit2 ox Robo 1 and Robo2 knockouts, further demonstrating the specificity of these antibodies. Retinal macrophages are know n to act as cellular chaperones that promote tip cell anastomosis at the vascular front via yet unknow n molecular mechanisms. Both iRoboMacKO and PI3Ky knockout affected macrophage numbers and their interaction with tip cells in the angiogenic front the same way as Anti- Robol / 2 treatment, indicating that Slit2-Robol / 2 signaling via PI3Kgamma is key for macrophage tip-cell interactions in the developing retinal vasculature.

[0212] Altogether, the data show7that Slit2-Robol / 2 signaling in retinal myeloid cells drives physiological and pathological retinal angiogenesis. During development, myeloid cell Slit2- Robol / 2 signaling promotes endothelial tip cell-macrophage interactions and vascular remodeling. During pathological neovascularization, this signaling induces activation of glycolysis and pro-angiogenic inflammatory pathways related to neovascularization, such as IL- 10 and IL- 12. Inhibition of Slit2-Robol / 2 signaling using a human monoclonal antibody reduced activation of these pathways and increased activation of protective pathways, such as interferon y and type I interferons, thereby reducing NVT formation and vascular leakage.

[0213] Example 2: MATERIALS AND METHODS

[0214] Antibody development

[0215] Antibodies targeting Robol Robo2 and Vegfa were selected from a previously described phage-displayed human Fab library (H. Persson et al., CDR-H3 diversity' is not required for antigen recognition by synthetic antibodies. J. Mol. Biol. 425 (2013), doi: 10. 1016 / j.jmb.2012. 11.037). Antigens were immobilized on 96-well Maxisorp plates from a 2 pg / ml solution in PBS and remaining protein binding sites were blocked with BSA. Phage library' pools were precleared for 1 hour at ambient temperature before transfer to a positive selection well for a further 1-hour incubation. Fab-phage that remained bound to the positive selection wells after extensive washing were eluted using 0.1 MHC1 and amplified to produce an enriched phage library pool. This process was repeated for four rounds of selections, after which 95 clonal phage populations were isolated and subjected to DNA sequencing. DNA encoding the VH or VL variable domain regions for selected Fab phage were amplified and cloned into the pSCSTa mammalian expression vectors in frame with the constant regions of either human IgGl heavy chain or human kappa light chains. IgG expression was performed by transiently co-transfecting both the heavy’ and light chain DNA Attorney Docket No. 047162-7536W01(02740) constructs for the desired IgG into Expi293F cells using FectoPro transfection reagent (Polyplus Transfection). After 5 days of expression, antibodies were purified from the conditioned media using recombinant Protein A Sepharose (GE Healthcare) and buffer exchanged into PBS using an Amicon Ultra- 15 Centrifugal Filter Device (Millipore) for storage. Antibody concentration was determined by the measuring the absorbance at 280 nm.

[0216] Biolayer interferometry (BLI) analysis for Anti-Robo and Anti-Vegfa mAbs

[0217] The binding kinetics of the Anti-Robo antibodies binding to Fc-fused Robo ECDs were determined by biolayer interferometry (BLI) using an Octex HTX Instrument (Forte Bio). Fc-fused Robo proteins were immobilized on AR2G biosensors via amino-coupling to achieve a signal of 1.4-1.7 nm. Empty sites were quenched and the sensors were equilibrated in assay buffer (PBS supplemented with 1% BSA and 0.05% Tween20). Sensors were then dipped into wells containing 3-fold serial dilutions of the antibodies diluted in assay buffer. Association was monitored at 25 °C for 600 s, before transferring the sensors to wells containing solely assay buffer to monitor disassociation for an additional 600 s at 25 °C. Data were fitted using the Octex HTX analysis software (v9.0) to determine apparent affinity (KD).

[0218] Binding of the Anti-Vegfa antibody to VEGFA or other proteins was assessed in a similar manner. Briefly, AR2G Biosensors coated with protein were dipped into 200 nM Anti-Vegfa mAb diluted in assay buffer and steady state signals were recorded at 300 s. Competition of the Anti-Vegfa antibody with VEGFR-1 for binding to VEGF121 was determined by immobilizing VEGF121 or human IgGl Fc (non-specific control protein) on AR2G Biosensors. Biosensors were preblocked with 200 nM VEGFRl-Fc or a non-binding control protein (trastuzumab) diluted in assay buffer for 10 minutes. Sensors were then probed in wells containing 200 nM Anti-Vegfa antibody and steady state signals were recorded at 300 s.

[0219] Reagents and antibodies

[0220] Recombinant Robo ectodomains for phage display antibody library screening were purchased as Fc-fusion proteins from R&D Biosystems: rat Robol (1749-RB-050), human Robol (8975-RB-050), mice Robo2 (3147-RB-050), human Robo2 (8366-RB-050). Importantly, rat and mouse Robol have 100% homology, therefore rat Robol-Fc fusion protein was used to predict binding to mouse Robol. Control human IgGl was purchased from BioXCell (InVivoMAb human IgGl isotype control, BE0297). Antibodies used were as follows: anti-MRCl (AF2535. R&D Systems. 1: 100). anti-Ibal (019-19741, Wako, 1 :200), anti-PDGFRP (1 / 100, AF1042, R&D Systems), anti-NG2 (1 / 200, AB5320, Millipore), anti- Attorney Docket No. 047162-7536W01(02740) collagen IV (1 / 300, AB769, Millipore), anti-a-smooth muscle actin CY3 (1 / 200, a-SMA, C6198. Sigma), anti-TER119 eFluor 450 (1 / 50, 48-5921-82, eBioscience-ThermoFisher Scientific), anti-CD31 (1 / 100, 550274, BD Biosciences), anti-p44 / 42 MAP kinase (1 / 1000, phospho-ERK, #9106, Cell Signaling), anti-44 / 42 MAP kinase (1 / 1000, total ERK, #9102, Cell Signaling), anti-pAKT (1 / 1000, #4060, Cell Signaling), and anti-panAKT (1 / 1000, total AKT, #4685. Cell Signaling). Appropriate secondary antibodies were conjugated to horseradish peroxidase (Vector Laboratories) or fluorescently labeled (Life Technologies). IB4 was purchased from Life Technologies.

[0221] Cells

[0222] Bone-marrow derived macrophages (BMDMs) were isolated from C57BL / 6 mice as previously described (L. H. Geraldo et al.. SLIT2 / ROBO signaling in tumor associated microglia and macrophages drives glioblastoma immunosuppression and vascular dysmorphia. J. Clin. Invest. 131, el41083 (2021)). Briefly, BM was obtained by flushing the femur and tibia with PBS. The bone marrow cells were resuspended in DMEM GlutaMax (Gibco) containing 1% Pen / Strep (Gibco). 20% FBS (Gibco) and 100 ng / mL M-CSF (R&D Systems). Cells were incubated for 2 days at 37 °C and 5% CO2 in non-treated bacterial dishes for adhesion of bone-marrow resident macrophages, and then changed for treated plastic dishes and culture for 6 days with medium change every' 2 days. Microglial cells were isolated from postnatal day 0 mice brains as previously described (L. H. Geraldo et al., SLIT2 / ROBO signaling in tumor associated microglia and macrophages drives glioblastoma immunosuppression and vascular dysmorphia. .1 Clin. Invest. 131, el 41083 (2021). Before experiments, cells were starved in serum- and CSF-free medium overnight. RAW264.7 mouse macrophages, EOC2 murine microglia and HMC3 human microglia were cultured in DMEM Gluta-MAX (Gibco) supplemented with 10% FBS (Gibco), 1% penicillin / streptomycin (Gibco) until a maximum of 10 passages. HUVECs and HBPCs were obtained from Lonza and cultured on 0.1% gelatin-coated plates in EGM2 medium (Lonza).

[0223] Transwell migration Assay

[0224] For chemotactic migration assays with 8.0pm Polycarbonate Membrane Transwell inserts (Coming Inc), 20.000 primary cells were plated in 125 pL of serum-free DMEM medium on the top chambers. In experiments involving antibody treatment, cells were pretreated with 50ug / mL of Anti-Robo antibodies or iso t pe control IgG for 30 minutes at 37°C before being plated in the top well. Then, bottom chambers were filled with 500 pL of serum-free DMEM with chemoattractants (R&D Systems). Cells were cultured overnight at 37°C and 5% CO2, then incubated for 30 minutes with Calcein AM (Invitrogen) to stain live Attorney Docket No. 047162-7536W01(02740) cells. Then the wells were washed and 10 pictures per well were acquired at lOx magnification using a Leica DMIRB inverted epifluorescence microscope. Migrated cells per field were counted using ImageJ software.

[0225] Western blot analysis

[0226] Cells were lysed in RIPA lysis buffer including phosphatase and protease inhibitors (Invitrogen). Equal amounts of proteins were separated on 4-15% Criterion precast gel (Biorad) and transferred on nitrocellulose membrane with Transblot Turbo (Bio-rad). Then membranes were blocked in 5% non-fat milk in TBS-T for 30 minutes at room temperature and incubated with primary antibodies overnight at 4°C under agitation. After washing with TBS-T membranes were incubated with proper HRP-conjugated secondary antibodies for 3 hours at room temperature under agitation. Western blots were developed with chemiluminescence HRP substrate (Bio-rad) on a Luminescent image analyser, ChemiDoc XRS+ (Bio-rad).

[0227] Mice

[0228] Mice were housed at 20-24°C. with 30-70% humidity under a 12 h light-dark cycle. To generate macrophage-specific Robol / 2 knockouts, Robol- / -Robo2fl / fl mice were bred with CSFl-R-CreERT2 knock-in mice. CX3CXR1-YFP and C57 / B16J mice were purchased from The Jackson Laboratory . PI3Kgamma knockout mice were a kind gift from Dr. Carrie Lucas (A. J. Takeda et al., Human PI3Ky deficiency and its microbiota-dependent mouse model reveal immunodeficiency and tissue immunopathology. Nat. Commun. 10 (2019). doi : 10. 1038 / s41467-019-1231 1-5). Gene deletion was induced by injection of tamoxifen (Sigma T5648) diluted in com oil (Sigma C8267). Postnatal gene deletion was induced by 3 injections of lOOug of tamoxifen at P0, Pl and P2.

[0229] In OIR experiments, gene deletion was induced by 3 injections of Img of tamoxifen at P12, P13 and P14; whereas adult gene deletion in the CNV model was induced by 3 injections of 2 mg of tamoxifen. Animals from both sexes were used.

[0230] Oxygen-induced retinopathy (OIR)

[0231] For the induction of OIR, the mother and P7 pups were placed in 75% 02 until P12. Upon return to room air, the pups were placed for adoption with a nursing mother and treated. Eyes were collected at Pl 7, retinas were stained, and avascular area, tufts and macrophage infiltration were quantified.

[0232] For antibody treatment, lOmg / kg of antibodies or isotype control IgG were diluted in 200uL of sterile 0.9% NaCl and given i.p. at P12, P14 and P16. For IPI-549 treatment, 15mg / kg of the inhibitor was diluted in 200uL of sterile vehicle (0.9% NaCl 2%DMSO) and Attorney Docket No. 047162-7536W01(02740) given i.p. daily from P12 to P16; and control mice were treated i.p. with 200uL of vehicle.

[0233] Immunohistochemistry

[0234] The eyes of pups from different ages were prefixed in 4% paraformaldehyde for 20 min at room temperature (RT). The retinas were dissected out and blocked during 30 min at RT in TNBT buffer (0.1 M Tris pH 7.4; NaCl 150 mM; 0.5% blocking reagent from Perkin Elmer, 0.5% Triton X-100). Retinas were then incubated with primary antibodies in TNBT overnight. After washing, the retinas were incubated with IB4 and the corresponding secondary antibody for 3h at RT. Then, the retinas were mounted in fluorescent mounting medium (Dako, Carpinteria, CA, USA). High-resolution pictures were acquired using a Leica SP8 confocal microscope. Quantification of staining and retinal vascular development was done using the ImageJ software. For macrophage-tip cell interaction quantifications, CX3CR1+ or Ibal+ macrophages were considered as interacting with a tip cell if at least one of its protrusions was in direct contact with a CD31+ or IB-4+ tip cell sprout. The number of macrophages contacting tip cell sprouts and number of sprouts contacted by at least one macrophage were counted and normalized by the total number of macrophages, total number of sprouts or by area.

[0235] Flow-cytometric staining of OIR retinas

[0236] After euthanasia, retinas were dissected from the eyes of P17 mice in ice-could DMEM. Retinas were incubated with DMEM containing 2.5 mg / ml collagenase D, and 5 U / ml DNase I for 20 min at 37°C. The digested tissue was passed through a 40pm nylon cell strainer (Falcon).

[0237] After blocking with mouse FcR Blocking Reagent (MACS Miltenyi Biotec) Single cell suspensions were stained with the following monoclonal antibodies: anti-CD45 BB700 or APC (Clone 30-F11, BD), anti-CDl lb BV510 or PE / Cy7 (Clone MI / 70, BD), anti-Ly6G APC / Cy7 (Clone 1 A8, BD), anti-CD3e PE / Cy7 or BV421 (Clone 145-2C11, BD), anti-CD19 APC or PE / CF594 (Clone 1D3, BD), anti-CD4 AF700 (Clone GK1.5, BD), and anti-CD8 BV650 (Clone 53-6.7, BD). As a control, cells were stained with the appropriate isoty pe control. Data acquisition as performed on BD Symphony and analysis was performed with FlowJo VlO.

[0238] Tissue dissociation and sample preparation for single cell RNA sequencing

[0239] 8 retinas from P17 pups subjected to OIR and treated with either Anti-Robol / 2 antibody or isotype control IgG were prepared as previously described (Z. Liu et al., Glycolysis links reciprocal activation of myeloid cells and endothelial cells in the retinal angiogenic niche. Sci. Transl. Med. 12 (2020), doi:10.1126 / SCITRANSLMED.AAY137I). Attorney Docket No. 047162-7536W01(02740)

[0240] Briefly, 8 retinas per group were dissected and dissociated with the Neural Tissue Dissociation kit (P) (Miltenyi Biotec, Cat# 130- 092-628). The digestion was stopped by adding BSA (5% final) and the cells were immediately filtered through a 70mm cell strainer. After centrifugation (600g for 5 min), cells were resuspended in PBS supplemented with 2mM EDTA and 0.5% BSA. The single cell suspension was enriched for ECs and immune cells using CD31 and CD45 MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions.

[0241] Single cell suspensions of retinal cells were resuspended in PBS containing 0.04% ultra-pure BSA. scRNAseq libraries were prepared using the Chromium Single Cell 30 Reagent Kits v3.1 (lOx Genomics: Pleasanton, CA, USA) according to the manufacturer’s instructions. The target cell recovery for each library was 10,000. Generated libraries were sequenced on an Illumina HiSeq4000, followed by de-multiplexing and mapping to the mouse genome (mml0-3.0.0) using CellRanger (lOx Genomics).

[0242] Single cell RNA sequencing Data processing

[0243] After generation of gene expression matrices using CellRanger (lOx Genomics), processing and analysis of the data were performed using the Seurat R-package (v4) (C. Baer, M. L. Squadrito, M. L. Iruela-Arispe, M. De Palma, Reciprocal interactions between endothelial cells and macrophages in angiogenic vascular niches, Exp. Cell Res. 319 (2013), doi:10.1016 / j.yexcr.2013.03.026).

[0244] First, quality control steps filtered out genes that were expressed in less than 10 cells and cells that expressed fewer than 100 genes. Lastly, only cells which had fewer than 60000 counts, between 500 and 7500 genes and less than 20% of the unique molecular identifiers (UMIs) originating from mitochondrial genes were retained for further analysis. Normalized gene expression data was used for differential gene expression analysis.

[0245] For clustering and visualization, individual samples were normalized using the SCTransform function. Seurat SCTransform dataset integration was performed to merge P17 Control and Anti-Robol / 2 datasets to correct for batch effects. Dimensional reduction was performed using Uniform Manifold Approximation and Projection (UMAP) as implemented in the RunUMAP function, where 15 dimensions were used for the dims parameter and all other settings were default. Louvain graph-based clustering was performed on a shared nearest neighbor graph, and retinal cell clusters were annotated based on previously published retinal single cell RNA sequencing data (G. Zarkada, J. P. Howard, X. Xiao, H. Park. M. Bizou, S. Leclerc, S. E. Kunzel, B. Boisseau, J. Li, G. Cagnone. J. S. Joyal. G. Andelfinger, A. Eichmann, A. Dubrac, Specialized endothelial tip cells guide neuroretina Attorney Docket No. 047162-7536W01(02740) vascularization and blood-retina-barrier formation. Dev. Cell 56 (2021), doi: 10.1016 / j.devcel.2021.06.021), using well-known gene expression markers.

[0246] Finally, UMAP and graph-based clustering were repeated on the subclustered immune cell dataset, as previously described. In this case, cell cluster annotation was based both in well known marker gene expression combined with the use of the CellTypist package (C. Dominguez et al., Cross-tissue immune cell analysis reveals tissue-specific features in humans. Science (80-.). 376 (2022), doi: 10.1126 / science.abl5197). For comparisons between clusters, differential expression was analyzed using FindMarkers function with and Gene set enrichment analyses were performed with fgsea vl.16.0 using log2 fold change estimates from DESeq2 and gene sets from MSigDB or internal data as indicated.

[0247] Bulk mRNA-sequencing on BMDMs

[0248] RNA isolation was performed on frozen cell pellets on DNA / RNA Shield (Zymo research) using Direct-zol RNA Miniprep kit (Zymo Research). RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific) and RNA integrity was determined using Bioanalyzer (Agilent). Library construction of 300 ng total RNA for each sample was made using KAPA Stranded mRNA-Seq Kit (Illumina Platforms; Kapa Biosystems) using 10 cycles of PCR amplification. Libraries were purified using AxyPrep Mag PCR Clean-up kit (AxygenTM). Each library' was quantified using a Qubit fluorometer (Life Technologies) and the size distribution assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Sequencing was performed on an IlluminaR Hiseq 2500 (Illumina, San Diego, CA. USA) instrument using the TruSeq PE Cluster Kit V4-cBot-HS (IlluminaR) to generate lOlbp Paired-end reads sequencing with v4 chemistry'. Quality control of RNA-Seq reads was performed using FastQC vO.11.9, samtools vl.7 and 729 Picard v2.23.8. Transcipts were quantified with Salmon vl.4.01 and reads aligned using STAR v2.7.62 to the mouse genome (mml0 / GRCm38, gencode vM24). Dow nstream analysis was performed in R, differential expression was analysed with DESeq2 vl.30.14 with standard models and normal shrinkage estimators. Gene set enrichment analyses were performed with fgsea vl. 16.0 using log2 fold change estimates from DESeq2 and gene sets from MSigDB or internal data as indicated.

[0249] Laser-induced choroidal neovascularization (CNV)

[0250] Male 10-16-week-old mice laser injury was done as previously described (V. Lambert, J. Lecomte, S. Hansen, S. Blacher, M. L. A. Gonzalez, I. Struman, N. E. Sounni, E. Rozet, P. De Tullio, J. M. Foidart, J. M. Rakic, A. Noel, Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice. Nat. Protoc. 8 (2013), doi: 10.1038 / nprot.2013.135.. Briefly, mice were anesthetized with ketamine / xylazine Attorney Docket No. 047162-7536W01(02740) and pain control and hydration were achieved with subcutaneous injection of Mel oxicam and intraperitoneal injection of Buprenorphine. Eyes were dilated, and a drop of GenTeal was applied to the eyes. Four focal bums (at the 3, 6 and 9 and 12 o'clock positions at 300 mW, 100 ms) were administered in each eye using a 532 nm argon ophthalmic laser via a slit lamp.

[0251] For fluorescein angiography imaging and optical coherence tomography (OCT), mice were anesthetized with a mixture of xylazine and ketamine, and pupils were dilated. For angiography, mice were intraperitoneally injected with fluorescein (Sigma) and images were taken 10 minutes after fluorescein injection at 7 days post laser induction (7dpi). OCT images were acquired at day 3 and 7 after laser lesion using a Phoenix Micron IV.

[0252] At 7dpi, mice were euthanized and enucleated, and choroids were dissected for whole mount staining as described for retinas.

[0253] Example 3: Robo Alignments

[0254] Table 4: Robo Alignments

[0255] Enumerated Embodiments

[0256] The following exemplary' embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

[0257] Embodiment 1 provides an isolated monoclonal antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 10, 11. or 12, and wherein the VH comprises a CDR1 region comprising the amino acid sequence of Attorney Docket No. 047162-7536W01(02740)

[0258] SEQ ID NOs: 13, 14, or 15; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 16, 17, or 18; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 19, 20, or 21.

[0259] Embodiment 2 provides the monoclonal antibody of embodiment 1, wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 11 or 12. and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 14 or 15; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 17 or 18; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 20 or 21.

[0260] Embodiment 3 provides the monoclonal antibody of embodiment 1, wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 10; and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 13; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 16; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 19.

[0261] Embodiment 4 provides the monoclonal antibody of embodiment 1, wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9 and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 1 1 , and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 14; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 17; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 20.

[0262] Embodiment 5 provides the monoclonal antibody of embodiment 1, wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 12, and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 15; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 18; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 21.

[0263] Embodiment 6 provides the monoclonal antibody of embodiment 1. wherein the VL comprises the amino acid sequence of SEQ ID NOs: 1, 2. or 3, and wherein the VH comprises the amino acid sequence of SEQ ID NOs: 4, 5, or 6. Attorney Docket No. 047162-7536W01(02740)

[0264] Embodiment 7 provides the monoclonal antibody of embodiment 1, wherein the VL comprises the amino acid sequence of SEQ ID NOs: 2 or 3. and wherein the VH comprises the amino acid sequence of SEQ ID NOs: 5 or 6.

[0265] Embodiment 8 provides the monoclonal antibody of embodiment 1, wherein:

[0266] (a) the VL comprises the amino acid sequence of SEQ ID NO: 1, and the VH comprises SEQ ID NO: 4;

[0267] (b) the VL comprises the amino acid sequence of SEQ ID NO: 2, and the VH comprises SEQ ID NO: 5; or

[0268] (c) the VL comprises the amino acid sequence of SEQ ID NO: 3, and the VH comprises SEQ ID NO: 6

[0269] Embodiment 9 provides the monoclonal antibody of embodiment 1, which is humanized.

[0270] Embodiment 10 provides the monoclonal antibody of embodiment 1, which is labeled.

[0271] Embodiment 11 provides a pharmaceutical composition comprising the monoclonal antibody of embodiment 1 and at least one pharmaceutical excipient.

[0272] Embodiment 12 provides an isolated polynucleotide comprising a first nucleic acid encoding a light chain variable region (VL) and a second nucleic acid encoding a heavy chain variable region (VH), wherein the first nucleic acid comprises a first segment encoding the amino acid sequence of SEQ ID NOs: 8; a second segment encoding the amino acid sequence of SEQ ID NOs: 9; and a third segment encoding the amino acid sequence of SEQ ID NOs: 10, 11, or 12, and wherein the second nucleic acid comprises a first segment encoding the amino acid sequence of SEQ ID NOs: 13, 14, or 15; a second segment encoding the amino acid sequence of SEQ ID NOs: 16, 17, or 18; and a third segment encoding the amino acid sequence of SEQ ID NOs : 19, 20, or 21.

[0273] Embodiment 13 provides a method of treating, ameliorating, and / or preventing an ocular neovascular disease in a subject in need thereof, the method comprising administering to the subj ect a therapeutically effective amount of an agent comprising at least one of the following:

[0274] (i) a monoclonal antibody that binds to Robot and Robo2 extracellular domains and blocks Robo-l / Robo-2-mediated Slit signaling;

[0275] (ii) a monoclonal antibody that binds to Robol extracellular domain and a Attorney Docket No. 047162-7536W01(02740) monoclonal antibody that binds to Robo2 extracelular domain; whereby binding of the antibody (ies) to Robol and Robo2 blocks Slit signaling.

[0276] Embodiment 14 provides the method of embodiment 13, wherein the agent in (i) comprises the monoclonal antibody of embodiment 3 and / or embodiment 8(a).

[0277] Embodiment 15 provides the method of embodiment 13, wherein the agent in (ii) comprises: the monoclonal antibody of embodiment 4 and / or embodiment 8(b); and the monoclonal antibody of embodiment 5 and / or embodiment 8(c).

[0278] Embodiment 16 provides the method of embodiment 13, wherein the ocular neovascular disease is at least one from the group consisting of diabetic retinopathy, neovascular age-related macular degeneration (nAMD). and retinopathy of prematurity (ROP).

[0279] Embodiment 17 provides the method of embodiment 13, wherein the agent is provided to the subject as a pharmaceutical composition.

[0280] Embodiment 18 provides the method of embodiment 13, wherein the agent is administered parenterally and / or intraocularly to the subject.

[0281] Embodiment 19 provides a method of detecting an ocular neovascular disease in a subject, the method comprising administering to the subject a labeled isolated monoclonal antibody of claim 1, and detecting presence or absence of a complex of the labeled isolated monoclonal antibody with any Robol and / or Robo2 present in the subject, wherein, if the complex is detected, the subject has an ocular neovascular disease.

[0282] Embodiment 20 provides a method of treating a blinding eye disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent comprising at least one of the following:

[0283] (i) a monoclonal antibody that binds to Robol and Robo2 extracellular domains and blocks Robo-l / Robo-2-mediated Slit signaling;

[0284] (ii) a monoclonal antibody that binds to Robol extracellular domain and a monoclonal antibody that binds to Robo2 extracelular domain; whereby binding of the antibody (ies) to Robo l and Robo2 blocks Slit signaling.

[0285] Embodiment 21 provides the method of embodiment 20, wherein the agent in (i) comprises the monoclonal antibody of embodiment 3 and / or embodiment 8(a).

[0286] Embodiment 22 provides the method of embodiment 20, wherein the agent in (ii) comprises: the monoclonal antibody of embodiment 4 and / or embodiment 8(b); and Attorney Docket No. 047162-7536W01(02740) the monoclonal antibody of embodiment 5 and / or embodiment 8(c).

[0287] Embodiment 23 provides the method of embodiment 20, wherein the blinding eye disease is at least one from the group consisting of age-related macular degeneration, glaucoma, diabetic retinopathy, retinitis pigmentosa, cataracts, Behcet’s disease, Retinopathy of Prematurity (ROP), Leber hereditary7optic neuropathy, Anophthalmia, and Microphthalmos.

[0288] Embodiment 24 provides the method of embodiment 20, wherein the agent is provided to the subject as a pharmaceutical composition.

[0289] Embodiment 25 provides the method of embodiment 24, wherein the agent is administered parenterally and / or intraocularly to the subject.

[0290] Embodiment 26 provides a method of detecting a blinding eye disease in a subject, the method comprising administering to the subject a labeled isolated monoclonal antibody of claim 1, and detecting presence or absence of a complex of the labeled isolated monoclonal antibody with any Robot and / or Robo2 present in the subject, wherein, if the complex is detected, the subject has a blinding eye disease.

[0291] Embodiment 27 provides a method of treating, ameliorating, and / or preventing cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent comprising at least one of the following:

[0292] (i) a monoclonal antibody that binds to Robol and Robo2 extracellular domains and blocks Robo-l / Robo-2-mediated Slit signaling;

[0293] (ii) a monoclonal antibody that binds to Robol extracellular domain and a monoclonal antibody that binds to Robo2 extracelular domain; whereby binding of the antibody (ies) to Robol and Robo2 blocks Slit signaling.

[0294] Embodiment 28 provides the method of embodiment 27, wherein the agent in (i) comprises the monoclonal antibody of embodiment 3 and / or embodiment 8(a).

[0295] Embodiment 29 provides the method of embodiment 27, wherein the agent in (ii) comprises: the monoclonal antibody of embodiment 4 and / or embodiment 8(b); and the monoclonal antibody of embodiment 5 and / or embodiment 8(c).

[0296] Embodiment 30 provides the method of embodiment 27, wherein the cancer is at least one from the group consisting of glioblastoma, bone cancer, retinoblastoma, colorectal cancer, pancreatic cancer, lung cancer, breast cancer, gastric cancer, glioma, and osteosarcoma.

[0297] Embodiment 31 provides the method of embodiment 27, wherein the agent is provided to the subject as a pharmaceutical composition. Attorney Docket No. 047162-7536W01(02740)

[0298] Embodiment 32 provides the method of embodiment 27, wherein the agent is administered parenterally and / or intraocularly to the subject.

[0299] Embodiment 33 provides a method of treating, preventing, and / or ameliorating focal segmental glomerulosclerosis (FSG or FSGS) in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent comprising at least one of the following:

[0300] (i) a monoclonal antibody that binds to Robot and Robo2 extracellular domains and blocks Robo-l / Robo-2-mediated Slit signaling;

[0301] (ii) a monoclonal antibody that binds to Robot extracellular domain and a monoclonal antibody that binds to Robo2 extracelular domain; whereby binding of the antibody (ies) to Robol and Robo2 blocks Slit signaling.

[0302] Embodiment 34 provides the method of embodiment 33, wherein the agent in (i) comprises the monoclonal antibody of claim 3 and / or claim 8(a).

[0303] Embodiment 35 provides the method of any one of embodiments 33-34, wherein the agent in (ii) comprises the monoclonal antibody of claim 4 and / or claim 8(b); and the monoclonal antibody of claim 5 and / or claim 8(c).

[0304] Embodiment 36 provides the method of any one of embodiments 33-35, wherein the agent is provided to the subject as a pharmaceutical composition.

[0305] Embodiment 37 provides the method of any one of embodiments 33-36, wherein the agent is administered into a blood vessel supplying a kidney of the subject or into the kidney itself.

[0306] Embodiment 38 provides a method of detecting cancer in a subject, the method comprising administering to the subject a labeled isolated monoclonal antibody of embodiment 1. and detecting presence or absence of a complex of the labeled isolated monoclonal antibody with any Robol and / or Robo2 present in the subject, wherein, if the complex is detected, the subject has cancer.

[0307] Embodiment 39 provides a method of detecting Robol and / or Robo2 in a sample, the method comprising contacting the sample with a labeled isolated monoclonal antibody of claim 1, and detecting presence or absence of a complex of the labeled isolated monoclonal antibody with any Robol and / or Robo2 present in the sample, wherein, if the complex is detected, Robol and / or Robo2 are present in the sample.

[0308] The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features show n and described or portions thereof, but it is recognized Attomey Docket No. 047162-7536W01(02740) that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application.

[0309] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

[0310] While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

Attomey Docket No. 047162-7536W01(02740)CLAIMSWhat is claimed is:

1. An isolated monoclonal antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 10, 1 1, or 12, and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 13, 14, or 15; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 16, 17, or 18; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 19, 20, or 21.

2. The monoclonal antibody of claim 1 , wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 11 or 12, and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 14 or 15; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 17 or 18; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 20 or 21.

3. The monoclonal antibody of claim 1, wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 10; and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 13; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 16; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 19.

4. The monoclonal antibody of claim 1 , wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9; and aAttomey Docket No. 047162-7536W01(02740)CDR3 region comprising the amino acid sequence of SEQ ID NO: 11, and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 14; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 17; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 20.

5. The monoclonal antibody of claim 1. wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 12, and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 15; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 18; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 21.

6. The monoclonal antibody of claim 1, wherein the VL comprises the amino acid sequence of SEQ ID NOs: 1, 2, or 3, and wherein the VH comprises the amino acid sequence of SEQ ID NOs: 4, 5, or 6.

7. The monoclonal antibody of claim 1, wherein the VL comprises the amino acid sequence of SEQ ID NOs: 2 or 3, and wherein the VH comprises the amino acid sequence of SEQ ID NOs: 5 or 6.

8. The monoclonal antibody of claim 1, wherein:(a) the VL comprises the amino acid sequence of SEQ ID NO: 1, and the VH comprises SEQ ID NO: 4;(b) the VL comprises the amino acid sequence of SEQ ID NO: 2, and the VH comprises SEQ ID NO: 5; or(c) the VL comprises the amino acid sequence of SEQ ID NO: 3, and the VH comprises SEQ ID NO: 6.

9. The monoclonal antibody of claim 1, which is humanized.

10. The monoclonal antibody of claim 1, which is labeled.

11. A pharmaceutical composition comprising the monoclonal antibody of claim 1Attomey Docket No. 047162-7536W01(02740) and at least one pharmaceutical excipient.

12. An isolated polynucleotide comprising a first nucleic acid encoding a light chain variable region (VL) and a second nucleic acid encoding a heavy chain variable region (VH), wherein the first nucleic acid comprises a first segment encoding the amino acid sequence of SEQ ID NOs: 8; a second segment encoding the amino acid sequence of SEQ ID NOs: 9; and a third segment encoding the amino acid sequence of SEQ ID NOs: 10, 11, or 12, and wherein the second nucleic acid comprises a first segment encoding the amino acid sequence of SEQ ID NOs: 13, 14, or 15; a second segment encoding the amino acid sequence of SEQ ID NOs: 16, 17, or 18; and a third segment encoding the amino acid sequence of SEQ ID NOs: 19, 20, or 21.

13. A method of treating, ameliorating, and / or preventing an ocular neovascular disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent comprising at least one of the following:(i) a monoclonal antibody that binds to Robot and Robo2 extracellular domains and blocks Robo-l / Robo-2-mediated Slit signaling;(ii) a monoclonal antibody that binds to Robot extracellular domain and a monoclonal antibody that binds to Robo2 extracelular domain; whereby binding of the antibody (ies) to Robol and Robo2 blocks Slit signaling.

14. The method of claim 13, wherein the agent in (i) comprises the monoclonal antibody of claim 3 and / or claim 8(a).

15. The method of claim 13, wherein the agent in (ii) comprises: the monoclonal antibody of claim 4 and / or claim 8(b); and the monoclonal antibody of claim 5 and / or claim 8(c).

16. The method of claim 13, wherein the ocular neovascular disease is at least one from the group consisting of diabetic retinopathy, neovascular age-related macular degeneration (nAMD). and retinopathy of prematurity (ROP).Attomey Docket No. 047162-7536W01(02740)17. The method of claim 13, wherein the agent is provided to the subject as a pharmaceutical composition.

18. The method of claim 13, wherein the agent is administered parenterally and / or intraocularly to the subject.

19. A method of detecting an ocular neovascular disease in a subject, the method comprising administering to the subject a labeled isolated monoclonal antibody of claim 1, and detecting presence or absence of a complex of the labeled isolated monoclonal antibody with any Robol and / or Robo2 present in the subject, wherein, if the complex is detected, the subject has an ocular neovascular disease.

20. A method of treating a blinding eye disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent comprising at least one of the following:(i) a monoclonal antibody that binds to Robol and Robo2 extracellular domains and blocks Robo-l / Robo-2-mediated Slit signaling;(ii) a monoclonal antibody that binds to Robol extracellular domain and a monoclonal antibody that binds to Robo2 extracelular domain; whereby binding of the antibody (ies) to Robol and Robo2 blocks Slit signaling.

21. The method of claim 20, wherein the agent in (i) comprises the monoclonal antibody of claim 3 and / or claim 8(a).

22. The method of claim 20, wherein the agent in (ii) comprises: the monoclonal antibody of claim 4 and / or claim 8(b); and the monoclonal antibody of claim 5 and / or claim 8(c).

23. The method of claim 20, wherein the blinding eye disease is at least one from the group consisting of age-related macular degeneration, glaucoma, diabetic retinopathy, retinitis pigmentosa, cataracts, Behcet' s disease, Retinopathy of Prematurity (ROP), Leber hereditary' optic neuropathy, Anophthalmia, and Microphthalmos.

24. The method of claim 20, wherein the agent is provided to the subject as aAttomey Docket No. 047162-7536W01(02740) pharmaceutical composition.

25. The method of claim 24, wherein the agent is administered parenterally and / or intraocularly to the subject.

26. A method of detecting a blinding eye disease in a subject, the method comprising administering to the subject a labeled isolated monoclonal antibody of claim 1, and detecting presence or absence of a complex of the labeled isolated monoclonal antibody with any Robot and / or Robo2 present in the subject, wherein, if the complex is detected, the subject has a blinding eye disease.

27. A method of treating, ameliorating, and / or preventing cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent comprising at least one of the following:(i) a monoclonal antibody that binds to Robol and Robo2 extracellular domains and blocks Robo-l / Robo-2-mediated Slit signaling;(ii) a monoclonal antibody that binds to Robol extracellular domain and a monoclonal antibody that binds to Robo2 extracelular domain; whereby binding of the antibody (ies) to Robol and Robo2 blocks Slit signaling.

28. The method of claim 27, wherein the agent in (i) comprises the monoclonal antibody of claim 3 and / or claim 8(a).

29. The method of claim 27, wherein the agent in (ii) comprises: the monoclonal antibody of claim 4 and / or claim 8(b); and the monoclonal antibody of claim 5 and / or claim 8(c).

30. The method of claim 27, wherein the cancer is at least one from the group consisting of glioblastoma, bone cancer, retinoblastoma, colorectal cancer, pancreatic cancer, lung cancer, breast cancer, gastric cancer, glioma, and osteosarcoma.

31. The method of claim 27, wherein the agent is provided to the subject as a pharmaceutical composition.Attomey Docket No. 047162-7536W01(02740)32. The method of claim 27, wherein the agent is administered parenterally and / or intraocularly to the subject.

33. A method of treating, preventing, and / or ameliorating focal segmental glomerulosclerosis (FSG or FSGS) in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent comprising at least one of the following:(i) a monoclonal antibody that binds to Robot and Robo2 extracellular domains and blocks Robo-l / Robo-2-mediated Slit signaling;(ii) a monoclonal antibody that binds to Robot extracellular domain and a monoclonal antibody that binds to Robo2 extracelular domain; whereby binding of the antibody(ies) to Robot and Robo2 blocks Slit signaling.

34. The method of claim 33, wherein the agent in (i) comprises the monoclonal antibody of claim 3 and / or claim 8(a).

35. The method of claim 33, wherein the agent in (ii) comprises: the monoclonal antibody of claim 4 and / or claim 8(b); and the monoclonal antibody of claim 5 and / or claim 8(c).

36. The method of claim 33, wherein the agent is provided to the subject as a pharmaceutical composition.

37. The method of claim 24, wherein the agent is administered into a blood vessel supplying a kidney of the subject or into the kidney itself.

38. A method of detecting cancer in a subject, the method comprising administering to the subject a labeled isolated monoclonal antibody of claim 1, and detecting presence or absence of a complex of the labeled isolated monoclonal antibody with any Robol and / or Robo2 present in the subject, wherein, if the complex is detected, the subject has cancer.

39. A method of detecting Robol and / or Robo2 in a sample, the method comprising contacting the sample with a labeled isolated monoclonal antibody of claim 1,Attomey Docket No. 047162-7536W01(02740) and detecting presence or absence of a complex of the labeled isolated monoclonal antibody with any Robol and / or Robo2 present in the sample, wherein, if the complex is detected, Robol and / or Robo2 are present in the sample.

Images