Methods of using tie-2 activators

Abstract

The invention features methods of treating a subject having a disease by administering to the subject a Tie-2 activator. Diseases that may be treated by the Tie-2 activator include ocular diseases and vascular diseases.

Description

[0001] METHODS OF USING TIE-2 ACTIVATORS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U.S. Patent Application Serial No. 63 / 608,526, filed on December 1 1 , 2023, and U.S. Patent Application Serial No. 63 / 561 ,276, filed on March 4, 2024. The disclosure of the prior applications is considered part of (and is incorporated by reference in) the disclosure of this application.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under K12EY016335 and K23EY035342 awarded by the National Eye Institute of the National Institute of Health. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] This application relates to treating disorders (e.g., ocular diseases and vascular diseases) related to regulating angiogenesis, vessel maturation, and vascular health involving angiopoietin-Tie signaling; particularly, angiopoietin-2 (Ang-2) / Tie 2 signaling.

[0008] For example, central serous chorioretinopathy (CSC) is a maculopathy involving serious retinal detachments associated with a thickened and dilated choroidal vasculature, pigment epithelial detachments and subretinal fluid (SRF). For some patients the SRF resolves spontaneously and quickly, but for others the SRF persists or recurs leading to photoreceptor damage and visual decline. CSC is relatively common worldwide, with estimates ranging from 1 in 4000 to up to 1 .7% in India (van Rijssen et al., Prog Retin Eye Res 2019;73:100770; Sahoo et al., Nepal J Ophthalmol 2019;1 1 :5-10).

[0009] Treatment options for CSC are currently limited. A minority of patients are candidates for photodynamic therapy (PDT), which helps to shorten the duration of SRF, but the long-term visual benefits of PDT have not been definitively shown. Anti-VEGF injections have not been shown to be helpful unless patients have developed a choroidal neovascular membrane (CNV), which is the minority of patients and furthermore which will only treat the CNV component rather than the SRF related to CSR. Spironolactone was once thought to be helpful but a randomized clinical trial unfortunately showed no benefit.

[0010] There is a need in the art for effective treatment options for ocular diseases (e.g., CSC) and vascular diseases.

[0011] SUMMARY OF THE INVENTION

[0012] In one aspect, the invention features a method of treating a subject having an ocular disease, the method comprising administering to the subject a therapeutically effective amount of a Tie-2 activator.

[0013] In some embodiments, the ocular disease is central serous chorioretinopathy, venous overload choroidopathy, or separation of retinal layers.

[0014] In some embodiments, the Tie-2 activator is an Ang-2 inhibitor. In some embodiments, the Ang-2 inhibitor binds to both Ang-2 and VEGF. In some embodiments, the Ang-2 inhibitor is an anti-Ang-2 antibody or antigen-binding fragment thereof. In some embodiments, the anti-Ang-2 antibody has a heavy chain sequence having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 1 , 2, or 5. In some embodiments, the anti-Ang-2 antibody has a light chain sequence having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 3, 4, or 6. In some embodiments, the anti-Ang-2 antibody is faricimab. In some embodiments, the anti-Ang-2 antibody is nesvacumab.

[0015] In some embodiments, the Tie-2 activator is an agent that oligomerizes Tie-2. In some embodiments, the agent that oligomerizes Tie-2 is Tie2.1 -hexamer.

[0016] In some embodiments, the Tie-2 activator is an anti-Tie-2 antibody or antigen-binding fragment thereof. In some embodiments, the anti-Tie-2 antibody is ASP4021 .

[0017] In some embodiments, the Tie-2 activator is administered at a dose of about 1 mg to about 10 mg. In some embodiments, the Tie-2 activator is administered at a dose of about 6 mg.

[0018] In some embodiments, the Tie-2 activator is administered in 1 or 2 doses.

[0019] In some embodiments, the Tie-2 activator is administered intravitreally or intravenously.

[0020] In another aspect, the invention features a method of treating a subject having a vascular disease, the method comprising administering to the subject a therapeutically effective amount of a Tie-2 activator.

[0021] In some embodiments, the vascular disease is a lymphatic disease, a venous disease, pleural effusion, hemangioma, capillary nevus, hemorrhoids, or pulmonary embolism. In some embodiments, the lymphatic disease is lymphangioma. In some embodiments, the venous disease is varicose veins, venous thromboembolism, chronic peripheral venous insufficiency, or deep vein thrombosis.

[0022] In some embodiments, the Tie-2 activator is an Ang-2 inhibitor. In some embodiments, the Ang-2 inhibitor binds to both Ang-2 and VEGF. In some embodiments, the Ang-2 inhibitor is an anti-Ang-2 antibody or antigen-binding fragment thereof. In some embodiments, the anti-Ang-2 antibody has a heavy chain sequence having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 1 , 2, or 5. In some embodiments, the anti-Ang-2 antibody has a light chain sequence having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 3, 4, or 6. In some embodiments, the anti-Ang-2 antibody is faricimab. In some embodiments, the anti-Ang-2 antibody is nesvacumab.

[0023] In some embodiments, the Tie-2 activator is an agent that oligomerizes Tie-2. In some embodiments, the agent that oligomerizes Tie-2 is Tie2.1 -hexamer.

[0024] In some embodiments, the Tie-2 activator is an anti-Tie-2 antibody or antigen-binding fragment thereof. In some embodiments, the anti-Tie-2 antibody is ASP4021 .

[0025] In some embodiments, the Tie-2 activator is administered at a dose of about 5 mg / kg to about 15 mg / kg. In some embodiments, the Tie-2 activator is administered every two weeks or every three weeks.

[0026] In some embodiments, the Tie-2 activator is administered intravenously.

[0027] As described herein, CSC is readily treated with a Tie-2 activator (for example, an anti-Ang-2 antibody such as faricimab). Because CSC is a disease of serous retinal detachment, treatment of CSC with such Tie-2 activators is unexpected, as retinal detachments such as CSC are typically not treated with injections of medication. CSC has no proven medical therapies, and even anti-VEGF injections have been tried and shown to fail in CSC. Furthermore, CSC patients do not share any characteristics with the patients typically treated with anti-Ang-2 antibodies; for example, CSC patients are younger than patients treated for age-related macular degeneration (AMD). Furthermore, CSC patients do not have vascular etiologies as is seen in AMD, diabetic macular edema (DME), and retinal vein occlusion (RVO). In fact, their choroid is thick, while the choroid of AMD patients is thin. Moreover, CSC patients have no correlation with diabetes, neovascularization is absent, and vein occlusions are not present to explain the serous detachment. It is accordingly surprising that a Tie-2 activator was found to advantageously treat an ocular disease such as CSC.

[0028] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0029] Definitions

[0030] As used herein, the term “about” refers to a value that is within 10% above or below the value being described.

[0031] As used herein, the term “percent (%) sequence identity” refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In some embodiments, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence (which can alternatively be phrased as a given candidate sequence that has or includes a certain percent amino acid (or nucleic acid) sequence identity to, with, or against a given reference sequence) is calculated as follows:

[0032] 100 x (fraction of A / B) where A is the number of amino acid (or nucleic acid) residues scored as identical in the alignment of the candidate sequence and the reference sequence, and where B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments where the length of the candidate sequence does not equal to the length of the reference sequence, the percent amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence would not equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0033] As used herein, the term “Tie-2 activator” refers to an agent that upregulates, directly or indirectly, the Tie-2 signaling pathway. In some embodiments, a Tie-2 activator may directly contact a positive regulator of the Tie-2 signaling pathway to upregulate said pathway. For example, a Tie-2 activator may bind to the Tie-2 receptor and upregulate the Tie-2 signaling pathway. In other embodiments, a Tie-2 activator may directly contact and block a negative regulator of the Tie-2 signaling pathway to upregulate the Tie-2 pathway (i.e. , act as an inhibitor of an inhibitor of the Tie-2 signaling pathway). For example, a Tie-2 activator may be an “Ang-2 inhibitor”, which is an agent that binds to and inhibits the function of the Tie-2 negative regulator Ang-2. Ang-2 inhibitors include anti-Ang-2 antibodies and antigen-binding fragments thereof, such as faricimab or nesvacumab. In some embodiments, a Tie-2 activator may be “an agent that oligomerizes Tie-2”, such as Tie2.1 -hexamer (described in Agard et al., Transl Vis Sci Technol. 2022 Oct 3;11 (10):27). Agents that oligomerize Tie-2 drive Tie-2 receptor activation. In other embodiments, a Tie-2 activator may be an anti-Tie-2 antibody or antigen-binding fragment thereof, which have an agonistic effect that activates Tie-2. An exemplary anti-Tie-2 antibody is ASP4021 (described in Koya et al., Sci Rep. 2021 Jul 7;11 (1 ):14021 ).

[0034] The term “antibody” is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

[0035] “Antigen-binding fragments” include a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al. Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments.

[0036] “Disease” is used in this disclosure to mean, and is used interchangeably with, the terms condition, disorder, or illness, unless otherwise indicated.

[0037] As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a disease. The disease may be an ocular disease or a vascular disease. Treatment may be administered to a subject who does not exhibit signs of the disease and / or to a subject who exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0038] As used herein, the terms “subject” and “patient” are interchangeable and refer to a subject (e.g., a mammalian subject, e.g., a human subject) that receives treatment or diagnosis for a disorder as described herein.

[0039] As used herein, “therapeutically effective amount” refers to an amount of a Tie-2 activator described herein sufficient to treat a disorder as described herein in a subject (e.g., a human).

[0040] As used herein, the term “ocular disease” refers to conditions or diseases involving the eye, including central serous chorioretinopathy, venous overload choroidopathy, or separation of retinal layers. In some embodiments, an ocular disease may be central serous chorioretinopathy.

[0041] As used herein, the terms “central serous chorioretinopathy”, “central serous retinopathy”, “central serous choroidopathy”, “central serous chorioretinitis”, “CSC”, “CSR”, and “CSCR” are used interchangeably and refer to a maculopathy characterized by serous detachment of the neurosensory retina, often with the presence of pigment epithelial detachments and a thickened choroid. As used herein, the term “vascular disease” refers to conditions or diseases involving the blood vessels, including a lymphatic disease, a venous disease, pleural effusion, hemangioma, capillary nevus, hemorrhoids, or pulmonary embolism.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. For any term present in the art which is identical to any term expressly defined in this disclosure, the term's definition presented in this disclosure will control in all respects. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are described herein.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0045] FIG. 1 shows Rs113791087 and risk of central serous chorioretinopathy in different study populations.

[0046] FIG. 2 shows a genome-wide association study of central serous chorioretinopathy in FinnGen.

[0047] FIG. 3 shows OCT progression of study patients.

[0048] FIG. 4 shows an image of the eye of a 41 year old male engineer with blurry vision in the right eye caused by acute CSCR.

[0049] FIG. 5 shows an autofluorescent image of the right and left eye of a patient with chronic CSCR. Vision loss can become severe and permanent if CSCR becomes chronic.

[0050] FIGS. 6A-6D are a series of images of eyes showing that CSCR has very characteristic findings in the eye.

[0051] FIG. 7 shows a genome-wide association study of central serous chorioretinopathy in FinnGen. A genome-wide association study of central serous chorioretinopathy was conducted including 1 ,477 patients with central serous chorioretinopathy and 455,449 controls from the FinnGen study. Each genomic variant is plotted as a data point, with P-values shown on the y-axis on a logarithmic scale and chromosomal position shown on the x-axis. The genome-wide significance threshold (P = 5x10-8) is shown with a dashed line. In each of three loci reaching genome-wide significance, the nearest proteincoding gene to the lead variant is labeled (blue = previously reported loci, red = novel locus).

[0052] FIG. 8 shows associations of variants in the PTPRB locus with central serous chorioretinopathy in FinnGen. A genome-wide association study of central serous chorioretinopathy was conducted including 1 ,477 patients with central serous chorioretinopathy and 455,449 controls from the FinnGen study. Results are shown for the genome-wide significant locus containing the PTPRB gene on chromosome 12. Each genomic variant is plotted as a data point, with P-values shown on the y-axis on a logarithmic scale. Linkage disequilibrium between the lead variant (rs113791087, purple) and other variants in the region are shown on a color scale.

[0053] FIG. 9 shows that rs113791087 replicates in 3 cohorts and has a strong effect size in all: OR 2.21 - 8.28. FIG. 10 shows that increased abnormalities on OCT were observed in those with the risk allele in the UK Biobank.

[0054] FIG. 11 shows rs113791087 is associated to risk for disease of the venous and lymphatic systems and to protection from glaucoma in FinnGen. A phenome-wide association study of the PTPRB missense variant rs113791087 in FinnGen was conducted. To identify potential pleiotropic associations of the rs113791087 variant, a phenome-wide association study was conducted including 2,469 phenotypes. Data are shown for all phenotypes that were at least nominally associated (P < 0.05) with rs 113791087. The negative common logarithm of each P-value is shown on the y-axis. The genome-wide significance threshold (P = 5x10-8) is shown with a dashed line. The direction of association with the risk of each disease is denoted by symbols (arrow up = increased risk, arrow down = decreased risk).

[0055] FIG. 12 shows a meta-analysis across several cohorts for associated phenotypes.

[0056] FIG. 13 shows that Rs113791087 is also associated with increased risk of venous thromboembolism, pulmonary embolism, deep vein thrombosis and pleural effusion, and with reduced risk of glaucoma in the meta-analysis.

[0057] FIG. 14 shows images of an eye of a 66 year old male with chronic diffuse CSR for over 6 years whose macular fluid went away completely 2 weeks after injection with faricimab.

[0058] FIG. 15 shows subretinal fluid (highlighted in yellow) in a patient with CSR.

[0059] FIG. 16 shows that post-injection subretinal fluid decreases with Faricimab but not anti-VEGF alone.

[0060] FIG. 17 shows quantile-quantile plot of the genome-wide association study of central serous chorioretinopathy in FinnGen. A genome-wide association study of central serous chorioretinopathy was conducted including 1 ,477 patients with central serous chorioretinopathy and 455,449 controls from the FinnGen study. The expected distribution (x-axis) and observed distribution (y-axis) of P-values is shown; the red line corresponds to the line of expectation. The genomic inflation factor was 1 .018.

[0061] FIGS. 18A-18C show genotype cluster plots for the PTPRB variant 12:70559589:T:G in FinnGen. FIG. 18A shows signal intensities and assigned genotype clusters based on chip data for 12:70559589:T:G (rs113791087) for 7,918 individuals from the FinnGen study. FIG. 18B shows imputed genotypes for the same individuals overlaid on the signal intensities from chip data. FIG. 18C shows exome sequencing calls in a subset of individuals demonstrating concordance with chip data.

[0062] FIG. 19 shows associations of the PTPRB missense variant rs 113791087 with central serous chorioretinopathy in 4 studies. The association of rs113791087 with central serous chorioretinopathy (CSC) was examined in 4 different studies. In all biobank-based studies (FinnGen, Million Veteran Program [MVP] and All of Us) included in the meta-analysis, patients with CSC were identified based on International Statistical Classification of Diseases codes, and all participants with age-related macular degeneration were excluded from patients and controls following a harmonized study protocol. In the chronic CSC (cCSC) cohort, patients were identified from ophthalmological clinics based on expert review. For the All of Us cohort, the upper range of the confidence interval is truncated (arrow). An inverse-weighted fixed-effects meta-analysis was conducted to combine data from all studies. No statistically significant heterogeneity was observed for rs 113791087 in the meta-analysis (I2= 0.48, Cistatistic = 5.8, Q-statistic P-value = 0.12). FIG. 20 shows associations of variants in the PTPRB locus with central serous chorioretinopathy in a meta-analysis of four study cohorts. Variants in the chromosome 12 locus containing PTPRB were evaluated in a meta-analysis of genomic association studies of central serous chorioretinopathy. Data from a total of 2,452 patients and 881 ,210 controls from FinnGen, Million Veteran Program, All of Us, and a European chronic CSC cohort were included in the meta-analysis. Each genomic variant is plotted as a data point, with P-values shown on the y-axis on a logarithmic scale. The genome-wide significance threshold (P = 5e-8) is shown with a dashed line. Linkage disequilibrium (LD) between the lead variant (rs1 13791087) and other variants in the loci are shown on a color scale. The figure was generated with LocusZoom using the European-ancestry reference panel.

[0063] FIG. 21 shows associations of variants in the PTPRB locus with central serous chorioretinopathy in All of Us. A case-control genomic association study was performed in All of Us, including 133 CSC patients and 1 19043 controls. Association results are shown for the region of the PTPRB gene, including all variants with a minor allele count over 40.

[0064] FIG. 22 shows retinal abnormalities in optical coherence tomography images by PTPRB rs1 13791087 genotype status in UK Biobank. Optical coherence tomography (OCT) images of 266 participants with the rs1 13791087 G allele (GG or GT genotype) and 442 age-matched participants lacking the G allele (TT genotype) were obtained from UKB. Images were independently evaluated by 3 retina specialists who were blinded to genotype. Each grader was tasked with identifying and categorizing retinal pigment epithelium (RPE) abnormalities according to the following categories: rare drusen (1 -5), drusen (>5), pattern dystrophy, pigment epithelial detachment (PED) or nonspecific RPE irregularity, and other rare findings (subretinal fluid, pachychoroid pigment epitheliopathy, atrophy, intraretinal fluid, or evidence of central serous chorioretinopathy). Panel a depicts a B-scan superior to the fovea showing an area of RPE irregularity and pigment migration (arrowhead) from a randomly selected participant, who had the TT genotype. Panel b depicts a B-scan inferior to the fovea showing a second area of RPE irregularity and pigment migration (arrowhead) in the same participant. Panel c shows the prevalence of abnormalities for participants with the GG or GT genotype (red) and TT genotype (blue). Statistical significance was evaluated using linear regression including age, sex, examiner and the first 10 PCs as covariates. Binomial proportion 95% confidence intervals were calculated using the Agresti-Coull method. Optical coherence tomography images are reproduced by kind permission from UK Biobank.

[0065] FIG. 23 shows associations of variants in the PTPRB locus with varicose veins in FinnGen. A genome-wide association study of varicose veins was conducted including 38,467 patients with varicose veins and 432,223 controls from the FinnGen study. Results are shown for the genome-wide significant locus containing the PTPRB gene on chromosome 12. Each genomic variant is plotted as a data point, with P-values shown on the y-axis on a logarithmic scale. Linkage disequilibrium between the lead variant (rs772508190, purple) and other variants in the region are shown on a color scale.

[0066] FIG. 24 shows a cross-study meta-analyses of other diseases most significantly associated with the PTPRB missense variant rs 1 13791087. The distinct diseases most significantly associated with rs 1 13791087 in the phenome-wide association study of rs 1 13791087 in FinnGen were carried forward for multi-study meta-analyses including available data from FinnGen, UK Biobank (UKB), Million Veteran Program (MVP), and All of Us (AoU). Pulmonary embolism and deep vein thrombosis were examined separately as the major subtypes of venous thromboembolism, and primary open-angle glaucoma was examined separately as a major subtype of glaucoma. Association results from contributing studies were combined in an inverse variance weighted meta-analysis.

[0067] FIG. 25 shows disease associations of predicted loss-of-function variants in PTPRB among UK Biobank and All of Us participants. The risk of ocular and vascular disease of interest was evaluated for participants with a predicted loss-of-function (pLOF) variant in PTPRB in All of Us and UK Biobank. Due to lack of ophthalmological outpatient clinic data in UK Biobank participants, patients with central serous chorioretinopathy were only evaluated in All of Us. Examined diseases were selected based on the most significant associations observed for rs113791087 in FinnGen. Pulmonary embolism and deep vein thrombosis were examined separately as the major subtypes of venous thromboembolism, and primary open-angle glaucoma was examined separately as a major subtype of glaucoma. Association results from UK Biobank and All of Us were combined in an inverse variance weighted meta-analysis.

[0068] FIGS. 26A-26D shows a study design. FIG. 26A depicts the study flowchart. A multi-center retrospective chart review was conducted to identify patients with chronic CSC with sub-retinal fluid who received at least one faricimab 6mg injection. Study sites included Massachusetts Eye and Ear and University of California San Francisco. Patients with evidence of a choroidal neovascular membrane on color photos, optical coherence tomography (OCT) and / or fluorescein angiography were excluded. Macular thickness was quantified from OCT images taken 52 weeks before to 52 weeks after the first faricimab injection. FIG. 26B demonstrates the confirmation of proper segmentation from Bruch's membrane to the ILM. FIGS. 26C-26D demonstrate extraction of automated macular thickness measurements (in microns) of the ETDRS central subfield and pericentral ring (inside the red circle in panel D), the median of which yielded a single thickness value per scan. ETDRS, Early Treatment of Diabetic Retinopathy Study.

[0069] FIG. 27 shows characteristics of the study participants. ‘Duration of SRF or IRF is based on the last consecutive OCT with fluid (moving backward in time). “Duration of CSC is best of our knowledge according to electronic medical records, however often the true CSC consent preceded the formal diagnosis. ***Data prior to first faricimab injection was collected up to 1 year. ““VA is defined as the best VA during a visit, either uncorrected, corrected or pinhole. AF: autofluorescence; CNVM: Choroidal neovascular membrane; FA: fluorescein angiography; FAF: fundus autofluorescence; SRF: subretinal fluid; VA: visual acuity.

[0070] FIG. 28 shows trajectories of individual patients before and after the first injection with faricimab. Time up to 52 weeks before and up to 52 weeks after the first treatment with faricimab is shown on the x- axis, with the time of the first faricimab treatment for each patient set to 0. Median macular thickness measurement (in microns) across inner ETDRS regions is shown on the y-axis for each patient treated with at least one dose of faricimab. The y-axis scale varies between patients depending on the maximum and minimum values observed for each patient. Visits during which the patients received any form of treatment are denoted with colored circles (blue, faricimab; red, bevacizumab; green, aflibercept; pink, photodynamic therapy [PDT]), and visits during which the patients did not receive treatment are denoted with black circles. Patient 8 had two treated eyes. ‘Patient 13 had SRF far from the fovea and thus the superior and nasal outer ETRDS regions were used. OD, right eye; OS, left eye. ETDRS, Early Treatment of Diabetic Retinopathy Study. SRF, subretinal fluid.

[0071] FIG. 29 shows distributions of macular thickness before and after the first treatment with faricimab. The distribution of macular thickness across visits (in pm) is shown on the y-axis for each patient before and after faricimab initiation. Macular thickness at each visit is defined as the median of the ETDRS central subfield and inner pericentral ETDRS subfields, as shown in FIGS. 26A-26D. Values are shown separately for measurements taken before (red) and after the first treatment with faricimab (blue). Data were included from a period ranging from 52 weeks before to 52 weeks after the first treatment with faricimab. The box and whisker plots denote the median (black line), 25th percentile (bottom of box), 75th percentile (top of box), minimum value (lower whisker) and maximum value (upper whisker) across visits for each patient. ETDRS, Early Treatment of Diabetic Retinopathy Study; OD, right eye; OS, left eye.

[0072] FIGS. 30A-30C show optical coherence tomography images of patients 1 , 6 and 8 before and after treatment with faricimab. FIG. 30A depicts optical coherence tomography (OCT) images from Patient

[0073] 1 who had a 6.5 year history of SRF. Faricimab was administered on day 0 and the SRF resolved by day 27. FIG. 30B depicts Patient 6 who had a 1 .5 year history of SRF. Faricimab was given on day 0 (first day in our system) and the SRF improved at day 14 but returned at day 29. FIG. 30C depicts OCT images from Patient 8 who had a 6 year history of both subretinal fluid and intraretinal fluid. 18 days after faricimab injection, SRF and IRF were dramatically improved.

[0074] FIG. 31 shows an updated meta-analysis identified 10 loci in 2,319 patients and 750,000 controls.

[0075] FIG. 32 shows macular thickness decreased in the majority of CSCR patients with Faricimab when comparing measurements during 1 year prior to after injection.

[0076] DETAILED DESCRIPTION OF THE INVENTION

[0077] The invention provides methods for treatment of disorders (e.g., an ocular disease or a vascular disease), involving administering to the subject a therapeutically effective amount of a Tie-2 activator.

[0078] I. Tie-2 activators

[0079] Provided herein are Tie-2 activators that may be used in any of the methods described herein.

[0080] The Ang / Tie-2 signaling pathway is essential for vascular and lymphatic development during embryogenesis; in the developed eye, Tie signaling also regulates angiogenesis and the stability and permeability of blood vessels. The transmembrane Tie complex is composed of Tie-2 and Tie-1 , the latter of which may be an orphan receptor that cooperates with Tie-2. Increased Tie-2 activity can support endothelial cell junction integrity, stability of the actin cytoskeleton, and cell survival-effects. Ang-1 and Tie-2 can also support adherens junction stability in the context of increased levels of VEGF or inflammation mediators.

[0081] Among the two well characterized Tie-2 ligands, Ang-1 is a strong agonist of Tie-2, whereas Ang-

[0082] 2 may act either as a weak Tie-2 agonist or antagonist; in the presence of high levels of Tie-1 or in an inflammatory context, Ang-2 may function primarily as an antagonist of Tie-2. In these settings, inhibition of Ang-2 can potentiate the effects of Ang-1 , increasing Tie-2 signaling activity and thus stabilizing endothelial adherens junctions, which may otherwise be compromised in ocular diseases and vascular diseases.

[0083] A Tie-2 activator is an agent that upregulates, directly or indirectly, the Tie-2 signaling pathway. In some embodiments, a Tie-2 activator may directly contact a positive regulator of the Tie-2 signaling pathway to upregulate said pathway. For example, a Tie-2 activator may bind to the Tie-2 receptor and upregulate the Tie-2 signaling pathway.

[0084] In other embodiments, a Tie-2 activator may directly contact and block a negative regulator of the Tie-2 signaling pathway to upregulate the Tie-2 pathway (i.e., act as an inhibitor of an inhibitor of the Tie- 2 signaling pathway).

[0085] In some embodiments, the Tie-2 activator is an Ang-2 inhibitor. An Ang-2 inhibitor may bind to Ang-2, or to both Ang-2 and VEGF. An Ang-2 inhibitor may be an anti-Ang-2 antibody or antigen-binding fragment thereof. In some embodiments, the anti-Ang-2 antibody or antigen-binding fragment thereof may be a bispecific antibody that binds to both Ang-2 and VEGF. In some embodiments, the anti-Ang-2 antibody or antigen-binding fragment thereof binds to Ang-2 only.

[0086] An exemplary anti-Ang-2 antibody is faricimab. Faricimab is a humanized bispecific antibody that targets both vascular endothelial growth factor A (VEGF-A) and angiopoietin-2 (Ang-2). Faricimab has a first heavy chain having a sequence of SEQ ID NO: 1 , a second heavy chain having a sequence of SEQ ID NO: 2, a first light chain having a sequence of SEQ ID NO: 3, and a second light chain having a sequence of SEQ ID NO: 4.

[0087] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQG RVTMTRDTSISTAYMELSRLRSDDTAVYYCARSPNPYYYDSSGYYYPGAFDIWGQGTMVTVSSASVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMASRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNK ALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK (SEQ ID NO: 1 )

[0088] EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRR FTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALGAPIEKTISK AKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK (SEQ ID NO: 2)

[0089] SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNT ATLTISRVEAGDEADYYCQVWDSSSDHWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSC (SEQ ID NO: 3) DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 4)

[0090] In some embodiments, the anti-Ang-2 antibody is nesvacumab (also known as REGN910). Nesvacumab is a human monoclonal antibody that targets angiopoietin-2 (Ang-2). Nesvacumab is described in Daly et al., Cancer Res. 2013 Jan 1 ;73(1 ) :108-18. Nesvacumab has a heavy chain having a sequence of SEQ ID NO: 5 and a light chain having a sequence of SEQ ID NO: 6.

[0091] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDIHWVRQATGKGLEWVSAIGPAGDTYYPGSVKGRFTI SRENAKNSLYLQMNSLRAGDTAVYYCARGLITFGGLIAPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5)

[0092] EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQHYDNSQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC (SEQ ID NO: 6)

[0093] CDRs for faricimab and nesvacumab are known in the art. In some embodiments, the anti-Ang-2 antibody has a heavy chain having at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the heavy chain sequence of faricimab (SEQ ID NO: 1 or 2) or to the heavy chain sequence of nesvacumab (SEQ ID NO: 5). In some embodiments, the anti-Ang-2 antibody has a light chain having at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the light chain sequence of faricimab (SEQ ID NO: 3 or 4) or to the light chain sequence of nesvacumab (SEQ ID NO: 6). Sequence identity is assessed according to standard methods.

[0094] In some embodiments, the Tie-2 activator is an agent that oligomerizes Tie-2 and drives receptor activation. For example, the Tie-2 activator may be a multivalent PEG-Fab conjugate, such as Tie2.1 - hexamer. Tie2.1 -hexamer (described in Agard et al., Transl Vis Sci Technol. 2022 Oct 3;1 1 (10):27) is a 6- kDa PEG hexamer conjugated with Tie2.1 Fab (an anti-Tie-2 Fab antibody fragment), resulting in a 295- kDa conjugate.

[0095] In some embodiments, the Tie-2 activator is an anti-Tie-2 antibody or antigen-binding fragment thereof. Anti-Tie-2 antibodies or antigen-binding fragments thereof bind to Tie-2 and act as an agonist, activating Tie-2. An exemplary anti-Tie-2 antibody is ASP4021 , which is an engineered tetra-valent agonistic antibody that activates Tie-2. ASP4021 is described in Koya et al., Sci Rep. 2021 Jul 7;11 (1 ):14021 .

[0096] II. Methods of Treatment

[0097] 1. Ocular Diseases

[0098] In some embodiments, the invention provides methods of treating a subject having an ocular disease, the method including administering to the subject a therapeutically effective amount of a Tie-2 activator. The Tie-2 activator may be any Tie-2 activator described herein, such as an anti-Ang-2 antibody or antigen-binding fragment thereof (e.g., faricimab or nesvacumab), an agent that oligomerizes Tie-2 (e.g., Tie2.1 -hexamer), or an anti-Tie-2 antibody or antigen-binding fragment thereof (e.g., ASP4021 ). In some embodiments, the Tie-2 activator is faricimab.

[0099] An ocular disease is a condition or disorder involving the eye. Exemplary ocular diseases that can be treated by the methods of the disclosure include central serous chorioretinopathy, venous overload choroidopathy, or separation of retinal layers.

[0100] In some embodiments, the ocular disease is central serous chorioretinopathy (CSC). CSC is a maculopathy associated with a thickened and dilated choroidal vasculature, pigment epithelial detachments and subretinal fluid (SRF). In some cases the SRF resolves spontaneously; in others it either persists or recurs, leading to outer retinal damage and loss of vision. Treatment options are currently limited. A minority of patients are candidates for photodynamic therapy (PDT), which helps to shorten the duration of SRF, but the long term visual benefits of PDT have not been definitively shown. Anti-VEGF injections have not been shown to be helpful unless patients have developed a choroidal neovascular membrane (CNV), which is the minority of patients and furthermore which will only treat the CNV component rather than the SRF related to CSR. Spironolactone was once thought to be helpful but a randomized clinical trial unfortunately showed no benefit.

[0101] In the Examples below, the inventors describe genetic findings implicating Tie-2 signaling in the pathophysiology of central serous chorioretinopathy. In a retrospective case series of patients with chronic central serous chorioretinopathy and longstanding subretinal fluid, improvements in macular thickness were observed after intravitreal injection with faricimab (an activator of Tie-2 signaling). These findings show that Tie-2 pathway modulation (e.g., faricimab) is useful in the management central serous chorioretinopathy.

[0102] Patients having CSC who would benefit from the treatments described herein are identified as follows. Any patient with subretinal fluid from CSC is a candidate for treatment.

[0103] 2. Vascular Diseases

[0104] In some embodiments, the invention provides methods of treating a subject having a vascular disease, the method including administering to the subject a therapeutically effective amount of a Tie-2 activator. The Tie-2 activator may be any Tie-2 activator described herein, such as an anti-Ang-2 antibody or antigen-binding fragment thereof (e.g., faricimab or nesvacumab), an agent that oligomerizes Tie-2 (e.g., Tie2.1 -hexamer), or an anti-Tie-2 antibody or antigen-binding fragment thereof (e.g., ASP4021 ). In some embodiments, the Tie-2 activator is faricimab. A vascular disease is a condition or disorder involving the blood vessels. Exemplary vascular diseases that can be treated by the methods of the disclosure include a lymphatic disease, a venous disease, pleural effusion, hemangioma, capillary nevus, hemorrhoids, or pulmonary embolism.

[0105] In some embodiments, the vascular disease is a lymphatic disease, which is condition or disorder involving the lymph, lymph vessels, or lymph nodes. An exemplary lymphatic disease is lymphangioma.

[0106] In some embodiments, the vascular disease is a venous disease, which is condition or disorder involving the veins. An exemplary venous disease is varicose veins, venous thromboembolism, chronic peripheral venous insufficiency, or deep vein thrombosis.

[0107] VV is a common disease affecting roughly 22% of the population (females more than males) and represents a weakening of the vessel wall with resultant vascular dilation in the lower extremity (typically in the greater and lesser saphenous veins).

[0108] III. Dosing and Administration

[0109] 1. Ocular disease

[0110] To treat an ocular disease, a Tie-2 activator may be administered at a dose of about 1 mg to about 10 mg (e.g., about 1 mg to about 2 mg, about 2 mg to about 3 mg, about 3 mg to about 4 mg, about 4 mg to about 5 mg, about 5 mg to about 6 mg, about 6 mg to about 7 mg, about 7 mg to about 8 mg, about 8 mg to about 9 mg, or about 9 mg to about 10 mg). In some embodiments, the Tie-2 activator is administered at a dose of about 6 mg.

[0111] In some embodiments, the Tie-2 activator is administered in 1 dose. In some embodiments, the Tie-2 activator is administered in 2 doses.

[0112] In some embodiments, the Tie-2 activator is administered intravitreally or systemically (for example, intravenously).

[0113] In some embodiments, faricimab may be administered at a dose of about 1 mg to about 10 mg (e.g., about 1 mg to about 2 mg, about 2 mg to about 3 mg, about 3 mg to about 4 mg, about 4 mg to about 5 mg, about 5 mg to about 6 mg, about 6 mg to about 7 mg, about 7 mg to about 8 mg, about 8 mg to about 9 mg, or about 9 mg to about 10 mg). In some embodiments, faricimab is administered at a dose of about 6 mg. In some embodiments, faricimab is administered in 1 dose. In some embodiments, faricimab is administered in 2 doses. In some embodiments, faricimab is administered intravitreally or systemically (for example, intravenously).

[0114] 2. Vascular disease

[0115] To treat a vascular disease, the Tie-2 activator may be administered at a dose of about 5 mg / kg to about 15 mg / kg (e.g., about 5 mg / kg to about 6 mg / kg, about 6 mg / kg to about 7 mg / kg, about 7 mg / kg to about 8 mg / kg, about 8 mg / kg to about 9 mg / kg, about 9 mg / kg to about 10 mg / kg, about 10 mg / kg to about 11 mg / kg, about 11 mg / kg to about 12 mg / kg, about 12 mg / kg to about 13 mg / kg, about 13 mg / kg to about 14 mg / kg, or about 14 mg / kg to about 15 mg / kg).

[0116] In some embodiments, the Tie-2 activator is administered every two weeks. In some embodiments, the Tie-2 activator is administered every three weeks. In some embodiments, the Tie-2 activator is administered systemically, for example, intravenously.

[0117] In some embodiments, faricimab may be administered at a dose of about 5 mg / kg to about 15 mg / kg (e.g., about 5 mg / kg to about 6 mg / kg, about 6 mg / kg to about 7 mg / kg, about 7 mg / kg to about 8 mg / kg, about 8 mg / kg to about 9 mg / kg, about 9 mg / kg to about 10 mg / kg, about 10 mg / kg to about 11 mg / kg, about 11 mg / kg to about 12 mg / kg, about 12 mg / kg to about 13 mg / kg, about 13 mg / kg to about 14 mg / kg, or about 14 mg / kg to about 15 mg / kg). In some embodiments, faricimab may be administered at a dose of about 10 mg / kg. In some embodiments, faricimab is administered every two weeks. In some embodiments, the Tie-2 activator is administered every three weeks. In some embodiments, faricimab is administered systemically, for example, intravenously.

[0118] EXAMPLES

[0119] Table of Contents

[0120] Example 1. Shared genetic risk locus for central serous chorioretinopathy and varicose veins

[0121] In a prior genome-wide association study (GWAS) meta-analysis for central serous choroidopathy (CSC), a nominally significant rare variant (P = 8.4e-07) stood out because of a potential genetic risk overlap with varicose veins (VV). Here, this variant was interrogated in a larger sample (over double the size) for central serous chorioretinopathy (CSC) to test for definitive evidence of association to both CSC and VV, thus underscoring the mechanistic theory of CSC as a venous overload choroidopathy.

[0122] Results

[0123] In the discovery cohort, the lead variant rs113791087 was associated with an almost 3-fold increase in the risk of CSC (OR = 2.85; P = 4.5e-9). The effect size was robust to the exclusion of cases and controls with 37 ICD codes reflecting potential confounding causes of fluid maculopathy (OR=3.15, P=1 .8e-6). Rs113791087 had similar effect estimates in all replication cohorts (ORs = 2.21-2.86, P = 0.02-3.0E-9) (FIG. 1) and a cross-cohort meta- analysis (OR=2.74, P=4.3e-19). In a phenome-wide association study of 2,469 diverse traits in FinnGen, rs 113791087 was most significantly associated with varicose veins (38,467 cases and 432,223 controls; OR = 1 .39; P = 3.1 e-10), and this association replicated in a single-variant analysis of varicose veins in the UK Biobank (OR = 1 .22, P = 6.2e-3).

[0124] Conclusion

[0125] Here, a novel genetic locus with an unusually high odds ratio linking CSC and varicose veins was identified, supporting CSC as a venous overload choroidopathy.

[0126] The above results were obtained using the following materials and methods.

[0127] Methods

[0128] A larger genome-wide association study of CSC was first conducted including 1 ,477 patients and 455,449 controls identified using ICD-10 codes from the FinnGen study. These results confirmed a strong association of the rare variant rs 113791087 (minor allele frequency = 0.5%) on chromosome 12 which was the lead variant in the region and lacked convincing nearby tag SNPs. Directed replication was conducted in participants of European ancestry in the Million Veteran Program (MVP; 706 cases and 273,198 controls) and AllOfUs cohorts (128 cases and 114,069 controls), as well as a previously published European dataset of patients with chronic CSC.

[0129] Example 2. A Missense Variant in PTPRB is Associated with Central Serous Chorioretinopathy and Varicose Veins

[0130] Further to Example 1 , above, this Example provides additional results showing that a missense variant in PTPRB is associated with disease. This Example describes a rare missense variant in the gene PTPRB that is strongly associated not only to CSC, but also to VV, providing an intriguing window into shared underlying pathophysiology.

[0131] A genome-wide association study of CSC in FinnGen identified a missense variant in PTPRB

[0132] A total of 1 ,477 central serous chorioretinopathy cases and 455,449 controls without retinal and choroidal diseases were identified in FinnGen based on hospital discharge and outpatient ICD-10 codes.

[0133] A case-control genome-wide association study identified three loci at genome-wide significance (P<5e-8) (FIG. 2), two of which have been previously reported and are marked by common noncoding variants at CFH and CD46. In the third locus on chromosome, the lead variant was a well-imputed (INFO = 0.997) low-frequency (AF = 0.5%) missense variant in the PTPRB gene (rs113791087, 12:70559589:T :G) that was associated with increased risk of CSC (OR = 2.85; P = 4.5e-9). Fine-mapping with SUSIE assigned a 0.995 probability for rs113791087 being the causal variant in the locus.

[0134] To ensure that this association was not confounded by AMD — which has certain overlapping phenotypic and genotypic features with CSC — a sensitivity analysis was conducted by only excluding patients with AMD symmetrically from both CSC cases and controls and observed that the association of rs 113791087 with CSC remained consistent (1092 cases and 485392 controls; OR = 2.95; P = 1 .8e-7). The effect size also remained robust even after excluding 37 diagnosis codes that reflect potentially confounding causes of fluid maculopathy from both cases and controls (769 cases and 452038 controls; OR = 3.15; P = 1.8e-6).

[0135] A phenome-wide association study of PTPRB reveals a co-association with varicose veins

[0136] A phenome-wide association study was conducted in FinnGen to identify potential pleiotropic associations with the PTPRB rs 1 13791087 variant. A genome-wide significant association was observed with varicose veins (38,467 cases and 432,223 controls; OR = 1 .39, P = 3.1 e-10), as well as nominally significant associations with venous thromboembolism (a combined phenotype of deep venous thrombosis and pulmonary embolism) (OR = 1 .29, P = 1 ,8e-5), pleural effusion (OR = 1 .55, P = 9.5-e5), and a combined outcome of “diseases of veins, lymphatic vessels and lymph nodes not elsewhere classified” (OR = 1 .24, P = 1 ,5e-7) after correction for multiple testing.

[0137] No difference was observed in the risk of CSC among participants with varicose veins in FinnGen (OR = 1 .0, P = 0.4), suggesting that the shared association of rs 1 13791087 with both outcomes is reflective of a true causal association with both phenotypes rather than population-wide correlation between the phenotypes.

[0138] The association of PTPRB rs113791087 with OCT abnormalities

[0139] Genetic variation may manifest in subclinical differences in individuals who are not diagnosed with disease. To evaluate the potential endophenotypes of carriers of the rs1 13791087, manual reviews were conducted of OCT images from 600 carriers and 600 non-carriers of rs 1 13791087 in UKB.

[0140] Conclusions

[0141] PTPRB was identified as a likely novel risk gene for CSC and varicose veins. PTPRB encodes the vascular endothelial protein tyrosine phosphatase protein, which affects vascular stability and permeability by negatively regulating the angiopoietin receptor Tie-2. These findings implicate the angiopoietin / Tie-2 signaling pathway as a potential therapeutic target in CSC.

[0142] The above-described results were obtained using the following materials and methods.

[0143] Methods

[0144] Study design

[0145] FinnGen is a public-private partnership research project that combines genotype data from newly collected and legacy samples administered by Finnish biobanks (finngen.fi / en) to provide novel insight into human diseases. This study includes genotype data from 500,348 individuals from FinnGen Data Freeze 12. The data were linked by unique national personal identification numbers to the national hospital discharge registry (available from 1968) and the specialist outpatient registry (1998-).

[0146] Ethics statement Study subjects in FinnGen provided informed consent for biobank research, based on the Finnish Biobank Act. Alternatively, separate research cohorts, collected prior the Finnish Biobank Act came into effect (in September 2013) and the start of FinnGen (August 2017), were collected.

[0147] Phenotype ascertainment

[0148] For the discovery GWAS, CSC patients were identified based on the presence of at least one instance of the International Classification of Diseases 10th revision (ICD-10) diagnosis code H35.7. Additionally, controls with retinal or choroidal disorders were excluded using the ICD-10 codes H30-H36 or any related endpoint described in the publicly available definition H7_CHOROIDRETINA (risteys.finregistry.fi / endpoints / H7_CHOROIDRETINA).

[0149] Additional sensitivity analyses were conducted to evaluate for bias due to potential confounders. In these analyses, instead of control-specific exclusion criteria, the following exclusion criteria were applied to both cases and controls: 1 ) at least one instance of a code corresponding to age-related macular degeneration (H35.30 or H35.31 ), and 2) at least one instance of a larger set of ICD-10 codes corresponding to common and rare causes of fluid maculopathy.

[0150] Genome-wide association studies and meta-analysis

[0151] FinnGen GWAS were conducted using REGENIE v 2.2.4, with sex, age at death or end of followup, principal components 1-10, genotyping array, and genotyping batch as fixed-effect covariates. An approximate Firth correction was used for variants reaching nominal significance (P<0.01 ) in an initial test, and standard errors were computed based on the Firth beta estimate and Firth P-value.

[0152] Epidemiological analyses

[0153] The association of CSC and VV was evaluated in R version 4.3.2 using logistic regression with CSC as the outcome and varicose veins, sex, and age at death or end of follow-up as independent predictors.

[0154] Data availability

[0155] Individual-level genotypes and register data from FinnGen participants can be accessed by approved researchers via the Fingenious portal (site.fingenious.fi / en / ) hosted by the Finnish Biobank Cooperative FinBB (finbb.fi / en / ).

[0156] Example 3. Case series of patients with central serous chorioretinopathy treated with faricimab

[0157] The above Examples herein describe the identification of a rare coding variant in the gene PTPRB that is highly associated to CSR with a strong effect. PTPRB is a protein tyrosine phosphatase that has an inhibitory effect on the Tie- 2 receptor. Tie-2 is negatively regulated by Ang-2, which is the target of one of the components of the bi-specific monoclonal antibody faricimab.

[0158] Given that PTPRB and Ang-2 have similar effects on Tie-2, and assuming that PTPRB is in the causal pathway of CSR, it was hypothesized that faricimab may influence the SRF seen in CSR patients. This Example reports a retrospective case series of patients with CSR with active SRF treated with faricimab and describe the immediate post-injection outcomes.

[0159] Results

[0160] All 4 patients in this study had improvement in SRF and IRF (if originally present) with 1 or 2 intravitreal injections of faricimab. 1 out of 4 patients experienced an associated improvement in vision, while the remaining 3 patients experienced stable vision. There were no complications.

[0161] Patient 1

[0162] A 60 year old male presented with a 30 year history of CSR OU. Imaging showed a thickened and dilated choroid OU with diffuse staining consistent with CSR. Though initially he only had evidence of prior (but not active) SRF OS, at year 2 he developed foveal and nasal SRF that did not resolve for 6.5 years. At this time he received a 6mg faricimab intravitreal injection in his left eye. Two weeks later, the SRF and IRF had completely resolved (FIG. 3).

[0163] Patient 2

[0164] A 74 year old female presented with CSR OS. Imaging showed a thickened choroid, RPE irregularity and outer retinal loss consistent with CSR without active SRF. She subsequently developed SRF that was treated with Aflibercept to no effect for 2 years (11 injections). She was switched to faricimab and after 2 injections the SRF had resolved (FIG. 3).

[0165] Patient 3

[0166] A 65 year old female presented with CSR OS. Imaging showed a thickened choroid and SRF associated outer retinal thinning suggestive of chronicity. After 1 year of observation, Avastin was tried (3 injections) to no effect. She was switched to faricimab and at 4 weeks had no foveal SRF (FIG. 3)

[0167] Patient 4

[0168] A 52 year old female presented with CSR OD. Imaging showed a thickened choroid and SRF with outer retinal thinning suggestive of chronicity. Intravitreal faricimab was given and 2 weeks later the SRF had resolved. She returned at 4 weeks and the SRF had begun to return (FIG. 3).

[0169] Table 1. Patient demographics and outcome details

[0170] Conclusion

[0171] Intravitreal faricimab is useful in treating patients with chronic CSR.

[0172] The above-results were obtained using the following materials and methods.

[0173] Methods

[0174] 4 patients (age 52 to 74) diagnosed with CSR were treated with one or two doses of intravitreal faricimab 6mg. 2 of the 4 received anti-VEGF medications previously and no patients underwent PDT. No patients were on spironolactone. Optical coherence tomography (OCT) images were taken pre- and posttreatment. Chronicity of disease, pre- and post-injection visual acuity, prior injections, effect of prior injections, and pre- and post-injection OCT findings were recorded.

[0175] Ethics

[0176] As this is a retrospective case series, consent for the case series was not obtained. However, patients were consented fully at the time of injection, including the off-label nature of giving anti-VEGF and anti-Ang-2 in the setting of CSR. Institutional review board approval for the retrospective case series was submitted.

[0177] Example 4. Discovery of a rare coding variant conferring risk for central serous chorioretinopathy and varicose veins.

[0178] Further to Examples 1 -3, above, this Example provides additional results showing that a missense variant in PTPRB is associated with disease and that administration of a Tie-2 activator (e.g., faricimab) is useful in treating disease.

[0179] Central serous chorioretinopathy (CSR) is a disease of the retina, specifically the macula. The main feature of CSR is fluid buildup under the retina (see, e.g., FIG. 4, showing an image of the eye of a 41 year old male engineer with blurry vision in the right eye). Vision loss can become severe and permanent if CSR becomes chronic (FIG. 5). CSR has very characteristic findings in the eye but lacks pathophysiologic understanding and thus effective treatment (FIG. 6). CSR affects approximately 1 -3 in 1000 (1 in 50 patients in clinic). The age at onset is 40-50 year olds, and it is more common in men (5:1 ). Risk factors include steroids, Type A personality, and pregnancy. Treatment is limited, and includes PDT, micropulse laser, and anti-VEGF (if CNV).

[0180] A recent meta-analysis identified 5 loci in 1 ,176 cases and 500K controls from FinnGen, Estonia Biobank and a Dutch cohort (Ramo et al., JAMA Ophthalmology 2023). Prioritized genes pointed to vascular cells as the pathophysiologic tissue - this answered an important question in CSR. When FinnGen reached 500K individuals (1 ,477 cases), a rare coding variant appeared to rise to significance (FIG. 7).

[0181] The variant corresponded to rs1 13791087, which is a missense ILE to LEU change in PTPRB (FIG. 8). The rs1 13791087 association remains robust when excluding confounding phenotypes (Table 2).

[0182] Table 2. rs1 13791087 association remains robust when excluding confounding phenotypes

[0183] Three independent cohorts were assessed for replication of the rs1 13791087 association: Million Veteran Program (706 cases, 273198 controls), All of Us Research Program (133 cases, 1 19043 controls), and European chronic CSC cohort (521 cases, 3577 controls), rs 1 13791087 replicated in 3 cohorts and had a strong effect size in all: OR 2.21 - 8.28 (FIG. 9). Furthermore, increased abnormalities on OCT were observed in those with the risk allele in the UK Biobank (FIG. 10).

[0184] Next, it was determined that rs 1 13791087 is also associated to risk for disease of the venous and lymphatic systems and to protection from glaucoma in FinnGen (FIG. 11 ). Varicose veins, separation of retinal layers, lymphatic and venous disease, venous thromboembolism, and pleural effusion can be treated with anti-Ang-2 medications or other Tie-2 activators. Glaucoma has a different direction of effect - inhibition of VE-PTP is useful.

[0185] Additionally, in ICD-based analyses of 500,348 participants in FinnGen, rs 1 13791087 is associated with increased risk of other vascular diseases including lymphangioma, hemangioma, capillary nevus and haemorrhoids (Table 3). These represent additional potential treatment targets for Anti-Ang2 medications or other Tie-2 activators. Table 3. In ICD-based analyses of 500,348 participants in FinnGen, rs 1 13791087 is associated with increased risk of other vascular diseases including lymphangioma, hemangioma, capillary nevus and haemorrhoids CSR experts have always likened CSR to varicose veins as a venous overload choroidopathy

[0186] (Spaide et al., Prog. Petin. Eye Res. 86, 100973, 2022). FIG. 12 shows a meta-analysis across several cohorts for associated phenotypes. Rs1 13791087 is also associated with increased risk of venous thromboembolism, pulmonary embolism, deep vein thrombosis and pleural effusion, and with reduced risk of glaucoma in the meta-analysis (FIG. 13). Furthermore, higher circulating Angiopoietin-2 levels are associated with varicose veins, pulmonary embolism, deep vein thrombosis, pleural effusion, chronic peripheral venous insufficiency, capillary nevus and primary open-angle glaucoma among 50,000 UK Biobank participants (Table 4). This provides further support for therapeutic inhibition of Angiopoietin-2 in these diseases. Table 4. Higher circulating Angiopoietin-2 levels are associated with varicose veins, pulmonary embolism, deep vein thrombosis, pleural effusion, chronic peripheral venous insufficiency, capillary nevus, and primary open-angle glaucoma among 50,000 UK Biobank participants.

[0187] VE-PTP (PTPRB) has complex interactions with VE-cadherin and Tie-2, both of which are involved in endothelial cell tight junctions. Upregulation of Tie-2 has been shown to stabilize tight junctions in the vascular endothelium (Gal et al. Sec. Evo. and Pop. Gen. 2020). Faricimab is an FDA-approved bispecific antibody against VEGF and Ang-2 which is used in the clinic for wet AMD that upregulates Tie-2. Anything that activates Tie-2 (e.g., Ang-2 inhibition) could be a useful therapeutic target for these diseases.

[0188] Six subsequent patients with CSR who received Faricimab quantitatively were reviewed (see, e.g., FIGS. 14 and 15). Post-injection subretinal fluid decreased with Faricimab but not anti-VEGF alone (FIG. 16).

[0189] In summary, this Example describes the identification of a rare coding variant in PTPRB that confers risk for CSCR with an OR of 3.06. This variant is strongly associated to risk for varicose veins, while protective against glaucoma. PTPRB is in an important gene in endothelial cell tight junctions. Targeting this pathway is useful for management of CSCR.

[0190] Example 5. Rare genetic variation in VE-PTP is associated with central serous chorioretinopathy, venous dysfunction and glaucoma

[0191] Further to Examples 1 , 2, and 4, above, this Example provides additional results showing that a missense variant in PTPRB is associated with disease.

[0192] To identify potential mechanisms involved in CSC, genotype data from 1 ,477 CSC patients and 455,449 controls in FinnGen was analyzed. An association for a low frequency (AF=0.5%) missense variant (rs113791087) in the gene encoding vascular endothelial protein tyrosine phosphatase (VE-PTP) (OR=2.85, P=4.5x109) was identified. This was confirmed in a meta-analysis of 2,452 CSC patients and 865,767 controls from 4 studies (OR=3.06, P=7.4x10-15). Rs113791087 was associated with a 56% higher prevalence of retinal abnormalities (35.3% vs 22.6%, P=8.0x10-4) in 708 UK Biobank participants and, surprisingly, with varicose veins (OR=1 .31 , P=2.3x10-11) and glaucoma (OR=0.82, P=6.9x10-9). Predicted loss-of-function variants in VEPTP, though rare in number, were associated with CSC in All of Us (OR=17.10, P=0.018). These findings described in this Example highlight the significance of VEPTP in diverse ocular and systemic vascular diseases.

[0193] Further, in this Example, a genome-wide association study of CSC in new data from the FinnGen study was conducted. The associations of a newly identified locus with CSC and co-associated traits across 5 studies including over 1 .3 million individuals were further evaluated. These findings described below provide insight into shared underlying pathophysiology between ocular diseases and systemic vascular dysfunction.

[0194] Results

[0195] A genome-wide association study of CSC in FinnGen identifies a missense variant in PTPRB First, a case-control GWAS including 1 ,477 patients with CSC and 455,449 controls in FinnGen was conducted (FIG. 17, Table 5).

[0196] Table 5. Central serous chorioretinopathy studies and subcohorts. CSC = central serous chorioretinopathy, AMD = age-related macular degeneration

[0197] The prevalence of other ocular diseases among patients with CSC was low (Table 6). This analysis identified 3 loci at genome-wide significance (FIGS. 7 and 8), 2 of which have been previously reported and are marked by common noncoding variants at CFH (Complement Factor H) and CD46 (Membrane cofactor protein). At the 12q15 locus, the lead variant was a low frequency (AF = 0.5%) missense variant (rs113791087, 12:70559589:T:G) in the PTPRB (Protein Tyrosine Phosphatase Receptor Type B; NCBI Gene ID: 5787) gene that encodes the VE-PTP protein (UniProt ID: P23467) (FIG. 18). Rs113791087 was associated with increased risk of CSC rs113791087 (OR = 2.85 [2.01 -4.05] per G allele, P = 4.5x1 O'9). Fine-mapping with the sum of single effects (SuSiE) approach nominated rs 113791087 as the most likely causal variant in the locus, with a posterior inclusion probability of 0.995 in its credible set.

[0198] Table 6. Characteristics of the central serous chorioretinopathy patients and controls in newly analyzed biobank-based studies included in the meta-analysis

[0199] Participants with central serous chorioretinopathy or varicose veins were identified using ICD-10 and ICD-9 codes in biobank-based studies. Data are shown after excluding participants with age-related macular degeneration from patients and controls following a harmonized study protocol. APPME, acute posterior multifocal placoid pigment epitheliopathy; CSC, central serous chorioretinopathy; DNA, Deoxyribonucleic acid; N / A, not available; SD, standard deviation. * >20 individuals in All of Us did not have recorded age at DNA sampling, t Counts for these diseases are reported in aggregate due to restrictions on reporting small sample sizes in participating studies. Any disease either before or after diagnosis of CSC was included.

[0200] To ensure that this association was not confounded by AMD — which has certain overlapping phenotypic and genotypic features with CSC — a sensitivity analysis was conducted by excluding patients with AMD symmetrically from both CSC patients and controls and observed that the association of rs 113791087 with CSC remained consistent (1092 patients and 485392 controls, OR = 2.95 [1 .97-4.43], P = 1 .8x10-7) (Table 8). The effect size remained robust even after excluding 37 diagnosis codes that reflect potentially confounding causes of fluid maculopathy from patients and controls (769 patients and 452038 controls, OR = 3.15 [1 .97-5.05], P = 1 .8x106) (Tables 7 and 8) (van Dijk et al., Prog. Retin. Eye Res. 84, 100955, 2021 ). Table 7. ICD codes used as exclusion criteria in additional sensitivity analyses in FinnGen corresponding to potentially confounding causes of fluid maculopathy

[0201] Table 8. The associations of rs113791087 with CSC in different study cohorts. CSC = central serous chorioretinopathy, AMD = age-related macular degeneration

[0202] Replication and meta-analysis of the association of PTPRB rs113791087 with CSC

[0203] Replication of variants in the PTPRB locus in 3 independent studies were carried out, including newly analyzed data from Million Veteran Program (MVP) participants of European ancestry (706 patients with CSC and 273,198 controls, Table 5), newly analyzed data from All of Us participants of European ancestry (133 patients with CSC and 103,600 controls, Table 5), and data from a previously reported European chronic CSC cohort (521 patients with chronic CSC and 3,577 population controls) (Schellevis et al., JAMA Ophthalmol. 136, 1128-1136, 2018). Patients with AMD were excluded from all newly analyzed studies contributing to the meta-analysis and had been previously excluded from analyses of the chronic CSC cohort (Schellevis et al., JAMA Ophthalmol. 136, 1128-1136, 2018). Consistently significant associations were observed between rs113791087 and CSC in all studies (FIG. 19 and Table 8) and in a cross-study meta-analysis of the locus (OR = 3.06 [95% Cl 2.31 -4.06], P = 7.3x1015; 2452 patients with CSC and 865767 controls) (FIG. 20). Additionally, rs 113791087 was the lead variant in its locus in the All of Us study even when considering all variants with a minor allele count > 40 uncovered by whole genome sequencing (FIG. 21). The point estimate was higher in All Of Us (OR = 8.28 [95% Cl 3.37-20.33]; 133 patients with CSC and 103600 controls) compared with studies that included more patients with CSC, but confidence intervals were overlapping between all studies. All 133 patients with CSC in All of Us were unrelated. In addition to the broad sensitivity analysis conducted in FinnGen, the granularity of ICD-10- CM and ICD-9-CM codes in MVP allowed us to examine the association of rs113791087 with dystrophies primarily involving the RPE (H35.54 and 362.76, respectively), an endpoint that includes pattern dystrophy which can pose diagnostic challenge with respect to CSC. No significant association of rs113791087 was observed with this outcome (OR = 1 .20 [0.78- 1 .86], P = 0.41 ; 2228 patients and 311134 controls).

[0204] Another missense variant in the PTPRB gene was also evaluated (rs61758735, 12:70555234:G>A, AF = 0.69%), which has previously been suggestively associated with CSC in 2 Dutch families in an exome sequencing study and which is in linkage equilibrium (D’ = 1 .0, R2= 0.0) with rs 113791087 according to 1000 Genomes European subpopulation reference panel (Schellevis et al., Mol Genet Genomic Med7, e00576, 2019; 1000 Genomes Project Consortium et al., Nature 526, 68-74, 2015). In the meta-analysis of 4 studies, rs61758735 was associated with increased risk of CSC at nominal significance although with a smaller effect size than rs 113791087 (OR = 1 .63 [1 .15-2.32], P = 0.006142).

[0205] Rs113791087 is associated with retinal abnormalities on optical coherence tomography

[0206] Genetic risk may manifest in subclinical differences even in the absence of diagnosed disease. To identify retinal characteristics potentially associated with the rs113791087 variant, optical coherence tomography (OCT) images were extracted from 266 participants who were heterozygous or homozygous for the G allele of rs113791087 and 442 age-matched participants with the TT allele in UK Biobank (UKB). These images were graded by 3 ophthalmologists blinded to genotype status. An increased overall prevalence of retinal abnormalities at the level of the RPE in participants with the GT or GG genotype was observed compared to participants with the TT genotype (35.3% vs 22.6%, P=0.00078 corrected for age, sex, examiner and first 10 PCs) (FIG. 22 and Table 9).

[0207] Table 9. Retinal abnormalities in optical coherence tomography images by rs113791087 genotype status in UK Biobank. Optical coherence tomography images of 266 participants with the rs113791087 G allele (GG or GT genotype) and 442 age-matched participants lacking the G allele (TT genotype) were obtained from UKB. Images were independently evaluated by 3 retina specialists who were blinded to genotype. Each grader was tasked with identifying and categorizing retinal pigment epithelium (RPE) abnormalities according to the following categories: Rare drusen (1 -5), drusen (>5), pattern dystrophy, pigment epithelial detachment (PED) or nonspecific RPE irregularity, and other rare findings (subretinal fluid, pachychoroid pigment epitheliopathy, atrophy, intraretinal fluid, or evidence of central serous chorioretinopathy). Binomial proportion 95% confidence intervals were calculated using the

[0208] Agresti-Coull method.

[0209] Co-associations of PTPRB rs13791087 with varicose veins and glaucoma revealed by a phenome-wide association study in FinnGen

[0210] A phenome-wide association study of 2,469 phenotypes in FinnGen was conducted to identify potential pleiotropic effects of the PTPRB rs 1 13791087 variant. A genome-wide significant association with varicose veins of the lower extremity was observed (38,467 patients and 432,223 controls, OR = 1 .39 [1 .25-1 .54], P = 3.1 x10-10) (FIG. 11 and 23). Among the most significant suggestive-level associations, an increased risk of venous thromboembolism (OR = 1 .29 [1 .15-1 .45], P = 1 .8x10-5), an increased risk of pleural effusion (OR = 1 .55 [1 .24-1 .94], P = 9.5x10-5), and a reduced risk of glaucoma (OR = 0.79 [0.69-0.90], P = 5.1 x104) were noted.

[0211] By contrast, rs1 13791087 was not significantly associated with common eye diseases including wet AMD (OR = 1 .05 [0.81 -1 .35], P = 0.72), dry AMD (OR = 1 .03 [0.82-1 .29], P = 0.79), or diabetic retinopathy (OR = 0.98 [0.82-1 .18], P = 0.59).

[0212] Because VE-PTP has been investigated as a target for diabetic macular edema (Campochiaro et al., Ophthalmology 123, 1722-1730, 2016), further association analyses were conducted limited to participants with type 1 or type 2 diabetes, and observed no significant associations of rs1 13791087 with diabetic retinopathy among type 1 (OR = 2.14 [95% Cl 0.88-5.20], P = 0.092; 2712 patients and 1 1 16 controls) or type 2 diabetic patients (OR = 0.88 [95% Cl 0.66-1 .16], P = 0.355; 5443 patients and 77435 controls), or with diabetic maculopathy among type 1 (OR = 1 .40 [95% Cl 0.53-3.71 ], P = 0.50; 715 patients and 31 13 controls) or type 2 diabetic patients (OR = 0.79 [95% Cl 0.49- 1 .29], P = 0.352; 1724 patients and 81 154 controls).

[0213] To better understand the co-association of rs1 13791087 with CSC and varicose veins of the lower extremity, the association of these diseases in the general FinnGen population was evaluated. No difference in the risk of CSC among all participants with varicose veins was observed (OR = 0.99 [95% Cl 0.98-1 .01 ], P = 0.43), suggesting that the shared association of rs 1 13791087 with both outcomes may be reflective of true causal associations with both phenotypes rather than simple population-level correlation between the phenotypes.

[0214] The clinical manifestations of varicose veins of the lower extremity are broad-ranging and include, among others, asymptomatic venous dilation, edema, and ulceration. In analyses of ICD code based varicose vein subtypes in FinnGen, rs1 13791087 was associated with uncomplicated varicose veins of the lower extremity (OR = 1 .39 [1 .25-1 .53], P = 4.3x10-10; 38,025 patients and 455,679 controls), but not with ulcerated varicose veins of the lower extremity (OR = 0.93 [0.68-1 .28], P = 0.67; 4,075 patients and 455,679 controls).

[0215] Cross-study meta-analyses of most significant disease associations for rs13791087

[0216] The robustness of associations between rs113791087 and the top 5 distinct diseases from the FinnGen phenome-wide association study was evaluated by conducting single-variant meta-analyses including data from FinnGen, MVP, UKB, and All of Us (FIG. 24 and Tables 10 and 11). In this meta analysis, rs113791087 was associated with increased risk of uncomplicated varicose veins (OR = 1 .31 [95% Cl 1.21-1.42], P = 2.3x10-11; 62621 patients and 1341326 controls) (FIG. 24 and Table 11). An association between rs113791087 and reduced risk of glaucoma at genome-wide significance was observed (OR = 0.82 [95% Cl 0.76-0.88], P = 6.9x10-9; 125075 patients and 1290261 controls); the effect estimate was concordant for primary open-angle glaucoma (OR = 0.82 [95% Cl 0.72-0.93], P = 0.0014; 35557 patients and 1366253 controls). Consistent with this observation, rs 113791087 was also associated with 0.51 mmHg [95% Cl 0.26-0.76mmHg] lower intraocular pressure (P = 5.2x105) among 77,449 UKB participants.

[0217] Table 10. Patient and control participant characteristics for traits highlighted by the phenome- wide association study for rs113791087 in FinnGen. Data are only shown for participants of genetically inferred European ancestry. For Million Veteran Program, custom analyses were only conducted for uncomplicated varicose veins among the shown traits. Characteristics are not shown for the traits previously reported by the genome-wide PheWAS (gwPheWAS) project. >20 individuals in All of

[0218] Us did not have recorded age at DNA sampling. SD = standard deviation.

[0219]

[0220] Table 11. Replication and cross-cohort meta-analysis of associations highlighted by the phenome- wide association study of rs113791087. Traits that were most significantly associated with rs 1 13791087 in FinnGen were carried forward for replication in UK Biobank (UKB), Million Veteran Program (MVP) and All of Us (AoU). The results were combined in inverse variance weighted metaanalyses.

[0221] In addition to the genome-wide significant associations, in the cross-study meta-analyses a suggestive association between rs1 13791087 and increased risk of venous thromboembolism was observed (OR = 1 .21 [95% 1 .12-1 .32], P = 4.6x10-6; 59887 patients and 1237762 controls). Association estimates for the constituent endpoints of pulmonary embolism (OR = 1 .24 [95% Cl 1 .12-1 .38], P = 2.9x1 O'5; 36456 patients and 1395022 controls) and deep vein thrombosis (OR = 1 .15 [95% Cl 1 .00- 1 .31 ], P = 0.044; 23316 patients and 894706 controls) were directionally concordant.

[0222] Disease associations of predicted rare loss-of-function variants in PTPRB To identify other genetic variation in PTPRB that might be associated with ocular or vascular systemic diseases, and to inform whether rs1 13791087 might act in a loss-of-function or gain-of-function manner, participants who had rare pLOF PTPRB variants in UKB (n = 120 participants with pLOF variant) and All of Us (n = 75 participants with pLOF variant) were identified. While statistical power was limited, among traits highlighted by the rs1 13791087 phenome-wide association study, participants with a PTPRB pLOF variant had suggestively increased risk of CSC (OR = 17.10 [95% Cl 1 .64-178.00], P = 0.018), venous thromboembolism (OR = 2.22 [95% Cl 1 .29-3.83], P = 0.0039), pulmonary embolism (OR = 2.90 [95% 1 .51-5.57], P = 0.0014), and deep vein thrombosis (OR = 2.15 [95% Cl 1 .08- 4.27], P = 0.029)

[0223] (FIG. 25 and Table 12).

[0224] Table 12. Disease associations of rare predicted loss-of-function variants in PTPRB

[0225] Summary

[0226] Despite progress in characterizing common variant genetic risk loci for CSC, pointing to specific genes and mechanisms that explain pathophysiology remains challenging due to the inherent hurdles faced when interpreting noncoding lead variants in GWAS. In contrast, this Example describes the identification of a low-frequency missense variant (rs 1 13791087) in PTPRB, the gene encoding VE-PTP, that was associated with a markedly increased risk of CSC and did not appear to be complicated by large stretches of linkage disequilibrium. Unexpectedly, rs1 13791087 was also associated with an increased risk of varicose veins and with a reduced risk of glaucoma. These previously uncharacterized genetic associations point directly to VE-PTP and demonstrate its relevance in ocular and systemic vascular diseases, support a role for vascular dysfunction in CSC, and in the future may inform therapeutic development.

[0227] In summary, this Example described the identification of a low-frequency missense variant in the gene encoding VE-PTP that was associated with a significantly increased risk of CSC and varicose veins and with a reduced risk of glaucoma. Our findings provide support for a central role of venous dysfunction in CSC and implicate the interconnected vascular endothelial function regulators VE-PTP, Tie-2 and VE- cadherin as potential therapeutic targets in diverse ocular and systemic vascular diseases.

[0228] The above-described results were obtained using the following materials and methods.

[0229] Data availability

[0230] Individual-level genotypes and register data from FinnGen participants can be accessed by approved researchers via the Fingenious portal (site.fingenious.fi / en / ) hosted by the Finnish Biobank Cooperative FinBB (finbb.fi / en / ). Access to individual-level UKB data may be requested by researchers in academic, commercial, and charitable organizations. This study used data from the All of Us Research Program’s Controlled Tier Dataset v7, available to authorized users on the Researcher Workbench. Summary-level association data in MVP are available through dbGaP, with accession phs001672.v1 1 ,p1 .

[0231] Study design

[0232] FinnGen is a public-private partnership research project that combines genotype data from newly collected and legacy samples administered by Finnish biobanks (www.finngen.fi / en) to provide novel insight into human diseases. This study includes genotype data from 500,348 individuals from FinnGen Data Freeze 12. The data were linked by unique national personal identification numbers to the national hospital discharge registry (available from 1968) and the specialist outpatient registry (1998-).

[0233] The Million Veteran Program is a population-scale biobank within the Department of Veterans Affairs. Voluntary enrollment of veterans receiving care in the VA began in 201 1 (Gaziano et al., J. Clin. Epidemiol. 70, 214-223, 2016). All samples were scanned on the MVP 1 .0 Axiom array (ThermoFisher); details on the design and QC are provided elsewhere (Hunter-Zinck et al., Am. J. Hum. Genet. 106, 535- 548, 2020). Samples were phased using SHAPEIT4 and imputed to the TOPMed reference panel (version r2; N= 97,256 samples) using Minimac4. Phenotypes were based on electronic health record data with follow-up through the end of 2022.

[0234] The All of Us Research Program opened for enrollment in May 2018 and plans to enroll at least 1 million persons in the United States, collecting electronic health record data and biospecimens to advance the prevention and treatment of diseases (All of Us Research Program Investigators et al., N. Engl. J. Med. 381, 668-676, 2019). In the current project, genetic data from 245,394 short-read whole genome-sequencing samples in the v7 release were used, and excluded participants who did not have any electronic health record data.

[0235] Details for the European chronic CSC cohort have been reported previously (Schellevis et al., JAMA Ophthalmol. 136, 1128-1136, 2018). For the genomic study, 521 European patients were recruited from the outpatient clinics of the Radboud University Medical Center (Netherlands), University Hospital of Cologne (Germany), and Leiden University Medical Center (Netherlands). Controls included 3,577 participants in the Nijmegen Biomedical Study. The patients with chronic CSC had subretinal fluid in at least one eye, retinal pigment epithelium irregularities with characteristic leakage on fluorescein angiography and corresponding hyperfluorescence on ICG.

[0236] UK Biobank (UKB) is a deeply phenotyped and genotyped prospective population-level cohort which recruited approximately 500,000 participants aged 40-69 in the UK between 2006-2010 (Bycroft et al., Nature 562, 203-209, 2018). UKB participants were genotyped on the Affymetrix Applied Biosystems UK BiLEVE Axiom Array and the Affymetrix Applied Biosystems UKB Axiom Array. Sample and variant QC are described in detail by Bycroft et al (Bycroft et al., Nature 562, 203-209, 2018). rs113791087 was directly genotyped with high quality statistics (missingness rate of 0.00198). PCA was performed using fastPCA as described and the first 10 PCs were downloaded and used as covariates in further analysis (Bycroft et al., Nature 562, 203-209, 2018). OCT imaging was performed for a subset of UKB participants (Keane et al., PLoS One 11 , e0164095, 2016). For this study, UKB data was accessed under applications #50211 and #17488.

[0237] Ethics statement

[0238] Study subjects in FinnGen provided informed consent for biobank research, based on the Finnish Biobank Act. Alternatively, separate research cohorts, collected between the Finnish Biobank Act coming into effect (in September 2013) and the start of FinnGen (August 2017), were collected.

[0239] The MVP024 study protocol was approved by the Veterans Affairs (VA) central Institutional Review Board (I RB) . MVP participants provided written informed consent.

[0240] Use of All of Us data was approved under a data use agreement between the Massachusetts General Hospital and the All of Us research program. Study participants provided written informed consent.

[0241] The study of European patients with chronic CSC was carried out in accordance with the tenets of the Declaration of Helsinki and was approved by the local ethics committees of the Radboudumc, Leiden University Medical Center, and University Hospital of Cologne. Written informed consent was obtained for all participants.

[0242] The UKB OCT substudy was approved by the North West Multi-centre Research Ethics Committee in accordance with the principles of the Declaration of Helsinki. Written, informed consent was obtained for all UKB participants.

[0243] Phenotype ascertainment

[0244] For the discovery GWAS, patients with CSC were identified based on the presence of at least one instance of the Finnish version of the International Classification of Diseases 10th revision (ICD-10) diagnosis code H35.7. Additionally, controls with retinal or choroidal disorders were excluded using the ICD-10 codes H30-H36 or any related endpoint as described previously (risteys.finregistry.fi / endpoints / H7_RETINASEPAR).

[0245] Additional sensitivity analyses were conducted to evaluate bias due to potential confounders. In these analyses, instead of control-specific exclusion criteria, the following exclusion criteria were applied to both patients and controls: 1 ) at least one instance of an ICD-10 code corresponding to age-related macular degeneration (H35.30 or H35.31 ), and 2) at least one instance of any ICD-10 code corresponding to a larger set of potentially confounding causes of fluid maculopathy (Table 7).

[0246] In MVP and All of Us, patients with CSC were identified based on at least one instance of the ICD-10-CM code H35.71 * or ICD-9-CM code 362.41 , and all participants with AMD (ICD10: H35.1 *, H35.2*, H35.3*; ICD-9: 362.5, 362.51 , 362.52) were excluded. Case definitions for the European chronic CSC cohort have been described previously (Schellevis et al., JAMA Ophthalmol. 136, 1128-1136, 2018). Ages of individuals in All of Us were calculated at 2022 / 7 / 1 or date of death.

[0247] Genome-wide association study and regional meta-analysis of CSC

[0248] All FinnGen GWAS were conducted using Regenie v 2.2.4 (Mbatchou et al., Nat. Genet. 53, 1097-1103, 2021 ), with sex, age at death or end of follow-up, principal components (PCs) 1-10, genotyping array, and genotyping batch as fixed-effect covariates. An approximate Firth correction was used for variants reaching nominal significance (p<0.01 ) in initial tests, and standard errors were computed based on the Firth beta estimate and Firth P-value.

[0249] Genomic analysis of CSC in MVP was performed using SAIGE v1 .3.0 on the set of samples classified as European ancestry using the HARE (Harmonized Ancestry and Race / Ethnicity) method (Zhou et al., Nat. Genet. 50, 1335-1341 , 2018). L leave-one-chromosome-out (LOCO) model fitting was used and enabled Firth effect size estimation for variants with P<0.05. Sex, age at enrollment, meancentered age-squared, and the first ten within-ancestry PCs were included as covariates.

[0250] Genomic analysis of CSC in All of Us was performed using Regenie v3.2.2 with age at death or end of follow-up, (age at death or end of follow-up) (Feenstra et al., Prog. Petin. Eye Res. 101236, 2024), sex, and PCs 1 -5 and 15 (based on association with CSC at p < 0.05 in a separate association test).

[0251] To evaluate whether rs113791087 was the lead variant in its locus on chromosome 12 even when combining data from 4 different studies, an inverse variance weighted meta-analysis of CSC for all genomic variants in the region of rs113791087 with GWAMA (v2.2.2) was performed. LocusZoom was used to generate locus plots for the meta-analysis using the European ancestry reference panel (Boughton et al., Bioinformatics 37 , 3017-3018, 2021 ).

[0252] Analysis of Optical Coherence Tomography images in UKB

[0253] Deidentified OCT images of patients with the rs 113791087 G allele (GG or GT) along with age- matched controls lacking the G allele (TT) were obtained from UKB (642 GG / GT OCTs, 1 ,058 TT OCTs; 10 age-matched participants with the TT genotype were pulled for every 1 participant with the GT or GG genotype, of which -16% had an OCT) (Keane et al., PLoS One 11 , e0164095, 2016). Repeat OCTs from the same patient were removed. Images were randomly chosen and independently evaluated by 3 retina specialists who were blinded to genotype. Because the scope of manual OCT review has practical limitations, only the left eye of each patient underwent assessment and 708 OCTs were reviewed. Each grader was tasked with identifying and categorizing various types of RPE abnormalities (drusen, pattern dystrophy, pigment epithelial detachment or nonspecific retinal pigment epithelium irregularity, subretinal fluid, pachychoroid pigment epitheliopathy, atrophy, intraretinal fluid and / or evidence of CSC). Statistical significance was evaluated using linear regression in R including age, sex, examiner and the first 10 PCs as covariates.

[0254] Phenome-wide association study, replication and meta-analyses

[0255] The association of rs113791087 with 2,469 traits in FinnGen was evaluated using Regenie v2.2.4 similarly to the association analyses for CSC (Mbatchou et al., Nat. Genet. 53, 1097-1103, 2021 ). Phenotype definitions and the characteristics of study participants for these traits are publicly available on risteys.finregistry.fi / .

[0256] For top traits identified in this phenome-wide association study, single-variant replication and meta-analyses were pursued including data from FinnGen, UKB, MVP and All of Us. Where possible, custom disease definitions to match these outcomes were created in UKB, All of Us, and Million Veteran Program.

[0257] The association of rs113791087 with each outcome in unrelated participants of genetically inferred European ancestry (excluding second degree or closer relatives in UK Biobank and Million Veteran programs, and based on a kinship score cutoff of 0.1 in All of Us) was evaluated with Firth logistic regression. Covariates in UK Biobank included genotyped sex, age, the first 10 PCs and genotyping array. Covariates in All of Us included age, age squared, sex, first 5 PCs, and additional outcome-related PCs (P<0.05 in separated models). Covariates for analyses of uncomplicated varicose veins in MVP included age, age2, sex and the first 10 PCs. For other phenotypes in MVP, European- ancestry (HARE) summary statistics generated by the genome-wide PheWAS (gwPheWAS) project (Verma et al., medRxiv (2023) doi:10.1101 / 2023.06.28.23291975) were used. Briefly, outcomes were derived from phecodes following standard definitions (Denny et al., Bioinformatics 26, 1205-1210, 2010), surveys distributed to all MVP enrollees (Gaziano et al., J. Clin. Epidemiol. 70, 214-223, 2016), and clinical laboratory and vital signs measurements. A linear or logistic regression GWAS was performed on each phenotype in a modified version of SAIGE using sex, age, age2, and the first 10 PCs as covariates.

[0258] Results from different studies were combined in an inverse variance weighted meta-analysis as implemented in the metagen function of the meta package (v6.5-0) in R (v4.2.0).

[0259] Additionally, the association of rs113791087 with the mean of the left and right eye corneal- compensated intraocular pressure (IOP) measurements in UKB participants was evaluated. Participants with a history of surgery for glaucoma were excluded and IOP readings less than 5 or greater than 60 were excluded. Linear regression analyses were adjusted by genotyped sex, age, the first 10 PCs and genotyping array.

[0260] Lastly, rare predicted loss-of-function (pLOF) variants in PTPRB were identified among UKB and All of Us participants by using the LOFTEE algorithm, excluding low-confidence or flagged pLOF variants and variants present at a frequency above 0.1% in either biobank or across gnomAD v2 super- populations (Karczewski et al., Nature 581 , 434-443, 2020). Associations with disease outcomes were evaluated with Firth logistic regression using similar analysis designs as for rs 113791087, except all individuals were included regardless of genetically inferred ancestry.

[0261] Additional epidemiological analyses

[0262] The association of CSC and varicose veins was evaluated in all available FinnGen participants using logistic regression as implemented in the glm function in R (v4.3.2) with CSC as the outcome and varicose veins, sex, and age at death or end of follow-up as independent predictors.

[0263] FinnGen genotyping and imputation

[0264] Newly collected FinnGen samples were genotyped using a FinnGen ThermoFisher Axiom custom array (Thermo Fisher Scientific, San Diego, CA, USA), and legacy cohorts were genotyped using Illumina and Affymetrix arrays (Illumina Inc., San Diego, and Thermo Fisher Scientific, Santa Clara, CA, USA) as detailed previously (Kurki et al., Nature 613, 508-518, 2023). Principal component analysis was used to remove samples who were not of genotype-inferred Finnish ancestry. Genotype imputation was performed using a population-specific SISu v4 imputation reference panel comprised of 8,557 whole genomes based on the protocol available at: dx.doi.org / 10.17504 / protocols.io.xbgfijw. For rs113791087, genotype calls were based on genotyping in the majority of individuals and imputation with high quality (INFO score = 0.997) in the remaining individuals. Whole exome sequencing based calls in a subset of participants had good concordance with genotyping (FIG. 18).

[0265] Genomic association analyses for additional custom outcomes in the Million Veteran

[0266] Program Participants with uncomplicated varicose veins were identified based on at least two instances of the ICD-10 codes I83.9* or ICD-9 code 454.9*. The association of rs113791087 with uncomplicated varicose veins was evaluated in participants of European ancestry who were unrelated within 2 degrees of relatedness and had a history of visiting eye clinics within the VA system. Independent predictors included rs 113791087, age, age 2, sex and the first 10 genomic principal components. Participants with dystrophies primarily involving the retinal pigment epithelium were identified in MVP using the ICD-9-CM code 362.76 and ICD-10-CM code 35.54. The association of rs 113791087 with dystrophies primarily involving the retinal pigment epithelium was examined using logistic regression in participants of European ancestry who were unrelated within 2 degrees of relatedness and had a history of visiting eye clinics within the VA system. Independent predictors included rs 113791087, age, age 2 , sex and the first 10 genomic principal components.

[0267] Genomic data quality control and analyses in All of Us

[0268] The All of Us Research Program started enrollment in May 2018, planning to enroll over 1 million people from diverse groups in the United States (All of Us Research Program Investigators et al., N. Engl. J. Med. 381, 668-676, 2019). Information collected includes health questionnaires, electronic health records (EHR), and physical measurements. Participants also provided biospecimens for genomic and other laboratory assessments. In this current project, genetic data from 245,394 short-read whole genome-sequencing samples in the v7 release was used. Variants were removed that 1 ) had GQ < 20, 2) did not pass genotype filter (FT), 3) had call rate < 95%, or 4) were monomorphic. Individuals were kept if they were 1 ) not included in the flag file (sample outlier QC failed), 2) European descent from genetic prediction, and 3) had EHR data available. Regenie v3.2.2 was used to test the association of each variant in the PTPRB locus with CSC. In step 1 , a smaller pruned dataset was used for model fitting and prediction. Two rounds of pruning were performed in all individuals who passed variant QC by Plink, and 460,525 variants were included in our final pruned dataset (round 1 : -indep-pairwise 100 5 0.1 -maf 0.005 -mac 100 -geno 0.01 -mind 0.01 ; round 2: -indep-pairwise 100 10 0.03). In step 2, the genomic predictions from step 1 were used to assess variant-outcome associations. Models were adjusted for age, age squared, sex, first 5 principal components (PC), and any additional outcome-associated PCs (p< 40 from summary statistics. Single-variant replication and analyses of predicted loss-of-function variants were carried out using Firth logistic regression. Related individuals were excluded based on the centrally provided table of related pairs (using a kinship score cutoff of 0.1 ), prioritizing the inclusion of patients over references.

[0269] Exome sequencing and quality control in UK Biobank

[0270] Exomes were captured in UK Biobank using the revised version of the IDT xGen Exome Research Panel v1 .0 on Illumina NovaSeq 6000 machines (ukbiobank.ac.uk / media / najcnoaz / access_064-uk-biobank-exome-release-faq_v 11 -1_final-002.pdf). Alignment using BWA-MEM, calling using DeepVariant, and joint genotyping using GLNexus have been described in detail elsewhere (biobank.ndph.ox.ac.uk / showcase / ukb / docs / UKB_WES_Protocol.pdf). For analyses of rare predicted loss-of-function variation in the present study, the OQFE exome call set was used and closely followed a previously published pipeline to perform stringent additional quality-control (QC), including genotype QC, variant QC and sample QC (Jurgens et al., Nat. Genet. 54, 240-250, 2022). UK Biobank exome sequencing data analyses were performed under application number 17488 and were approved by the Mass General Brigham Institutional Review Board.

[0271] Predicted loss-of-function variant annotation in UK Biobank and All of Us

[0272] Variants in UK Biobank and All of Us were annotated using the Loss-of-Function Transcript Effect Estimator (LOFTEE) plug-in implemented in the Variant Effect Predictor (VEP; v.105) (github.com / konradjk / loftee) (Karczewski et al., Nature 581 , 434-443, 2020). Variants annotated as high- confidence loss-of-function variants by LOFTEE include frameshift indels, stop-gain variants and splice site disrupting variants. LOFs flagged for caution by LOFTEE were excluded. Variants were additionally annotated with the highest continental allele frequency based on gnomAD v2 exomes (including allele frequencies for each the European, East Asian, South Asian, African / African American and Latino / Admixed American super-populations).

[0273] Sources of diagnosis codes in FinnGen

[0274] The setting in which patients in FinnGen had received the ICD-10 diagnosis code H35.7 was evaluated. Among 1 ,477 patients, 1 ,471 (99.6%) had received at least one instance of the H35.7 diagnosis code in an ophthalmological specialty setting; 1048 (71 .2%) of these patients had received at least one diagnosis code in a university hospital and 386 (26.2%) had received at least one diagnosis code in a central hospital within the Finnish healthcare system. Consistent with the classically selfresolving nature of CSC, only 45 (3.0%) of the patients had been included in the Finnish Register of Visual Impairment, which requires a corrected visual acuity permanently less than 0.3 in the better eye of the patient or a similar degree of permanent visual impairment.

[0275] Prevalence of potentially confounding diagnoses in FinnGen and All of Us

[0276] The prevalence of potentially confounding diagnoses including diabetic retinopathy, retinal vein occlusion, pattern dystrophy, acute posterior multifocal placoid pigment epitheliopathy, myopic degeneration, toxic maculopathy, uveitis, Vogt-Koyanagi-Harada disease, inherited retinal disease or choroidal hemangioma among patients with CSC in FinnGen and All of Us was evaluated. Any diagnosis of the aforementioned diseases was counted separately for patients with CSC and controls, counting both diagnoses before and after CSC diagnosis. Due to restrictions with reporting data from small sample numbers in the biobank-based studies, these diagnoses were evaluated in aggregate. In total, 66 (6.0%) of patients with CSC and 16017 (3.3%) controls in FinnGen had any of the aforementioned diagnoses. In the Million Veteran Program, the aforementioned diagnoses were registered in fewer than 121 (17.1%) patients with CSC and 17363 (6.4%) controls. In All of Us, the aforementioned diagnoses were registered in fewer than 20 (<15%) patients with CSC and 1522 (1 .47%) controls. No CSC patients in the Million Veteran Program of All of Us had diagnoses of Vogt-Koyanagi-Harada disease; no precise ICD code for Vogt-Koyanagi-Harada was available in FinnGen.

[0277] Example 6. Targeting the Tie-2 Receptor with Faricimab in Central Serous Chorioretinopathy: A Case Series Motivated by a Genetic Finding

[0278] Further to Examples 3 and 4, above, this Example provides additional results showing that administration of a Tie-2 activator (e.g., faricimab) is useful in treating disease.

[0279] Introduction

[0280] To investigate the effects of faricimab, a bispecific antibody targeting VEGF and Ang-2 (thus increasing Tie-2 activity), in patients with CSC based on the genetic studies described in Examples 1 , 2, 4, and 5 that implicated Tie-2 signaling in CSC pathophysiology. This Example reports the macular thickness captured at visits during a time range from up to 52 weeks prior to up to 52 weeks post injection.

[0281] Results

[0282] 20 eyes (19 patients) were identified with a diagnosis of chronic CSC with sub-retinal fluid (some also with intra-retinal fluid) who received faricimab, of which 16 eyes (15 patients) met our inclusion criteria (FIG. 26). Four eyes were excluded because of angiographic evidence of choroidal neovascular membrane. Male patients comprised 75%, and the average age was 62.6 years (range 52-74 ). All patients had a diagnosis of chronic CSC (persistent or recurrent subretinal fluid for ~6 months). Prior to treatment with faricimab, CSC had been diagnosed a median of 4.05 years (range 0.9 to 8) earlier and SRF (and intraretinal fluid [IRF] in a subset) had been continuously present for a median of 30 weeks (range 9 to 257). See FIG. 27.

[0283] During the 1 -year pre-faricimab window, treatments included intravitreal bevacizumab 1.25 mg, aflibercept 2.0 mg and PDT (FIG. 28). Fifty-six percent of eyes were naive to prior treatment. The average number of faricimab injections was 3.8 (range 1 -8) and 13 / 16 eyes received >1 injection. The number of visits in the 52 weeks prior to the first faricimab injection varied from 1 to 10, with 3 eyes receiving a faricimab injection on the first visit and the other 13 having at least 2 visits prior to the first faricimab injection (FIGS. 27 and 28).

[0284] When comparing the macular thickness measurements prior to the first faricimab injection to the thickness measurements after the first injection across all eyes, 14 / 16 eyes showed improved median macular thickness (aggregate median improvement was 40 microns [range -3 to 88.5], p=0.0007, FIGS. 28 and 29). Individual patient trajectories (FIG. 28) showed that macular thickness improved after the first or second injection in 14 / 16 eyes, 13 / 16 eyes had sustained improvement (with or without continued injections), 1 eye worsened after the second injection (patient 10) and 1 eye only received one injection and initially improved but experienced subsequent reaccumulation of SRF without subsequent injections (patient 6). 10 eyes experienced complete resolution of subretinal fluid following the start of the first series of injections at a median of 4 weeks (range 2-15), despite SRF having been present for a median of 30 weeks prior.

[0285] While eyes with clear CNVM on FA and / or hemorrhage on color imaging were excluded, some eyes still have OCT findings that could be interpreted as a CNVM due to a broad hyperreflective PED. As a sensitivity analysis, these eyes (patient / eyes 3 / OD, 11 / OD, 12 / OS, 13 / OD) were further excluded. In the remaining 12 eyes, 10 / 12 eyes showed improved median macular thickness (aggregate median improvement was 29.3 microns [range -3 to 89.5], p=0.004).

[0286] 6 / 16 eyes experienced 2 or more Snellen lines of improvement in visual acuity (FIG. 27) and 6 / 16 eyes experienced only 1 line of visual improvement. The remaining 4 eyes were stable or 1 line worse. Of the 9 eyes that did not experience >2 lines of improvement, 5 / 9 had marked ellipsoid zone (EZ) attenuation at the time of injection and 1 had no significant change in SRF with injection (FIG. 27).

[0287] Summary

[0288] We observed a variable but consistent improvement in macular thickness was observed in 14 / 16 patients with longstanding SRF after receiving faricimab, though the effect on visual acuity was modest (6 / 16 patients experienced >2 lines improvement). Prior to treatment with faricimab, CSC had been diagnosed a median of 4.1 years (range 0.9-8) earlier and SRF (and intraretinal fluid [IRF] in a subset) had been continuously present for a median of 30 weeks (range 9-257). Decreases in macular thickness were observed in 14 / 16 eyes after the first faricimab injection and in 14 / 16 eyes in the full follow-up period compared with prior, 10 of which experienced complete resolution of SRF following the start of the first series of injections at a median of 4 weeks (range 2-25). One eye worsened after the second injection. The median improvement in macular thickness was 40pm [range -3 to 89.5] (P=0.0007). Upon review of OCT images, reductions in macular thickness were consistent with reductions in SRF and / or IRF. Visual acuity improved by 2 lines or more in 6 / 16 eyes.

[0289] Of note, 4 patients with longstanding SRF experienced resolution of SRF 2-3 weeks after the first injection, suggesting that the intervention played a role. The results of this retrospective case series therefore provide motivation to study the effect of modulating the Tie-2 pathway (potentially with more targeted drugs) using additional patients with longer follow-up or a large randomized controlled trial in CSC.

[0290] The standard of care of CSC involves observation, PDT and intravitreal injection of anti-VEGF agents (in cases of CNVM), as well as some other less commonly used treatments. FIG. 28 shows patients who received one or several of these treatments prior to faricimab. In the patients studied here, responses to bevacizumab and aflibercept (in the absence of CNVM) were variable with patients 12 and 13 showing steady worsening despite bevacizumab and aflibercept injections. It is notable here that the effect of faricimab, if any, seems to be short-lived; patients 1 , 6, 8 and 9 had visits ~2 weeks after a faricimab injection (without repeat injection at that visit) and often there was an immediate improvement followed by return of some degree of SRF at the subsequent 4-6 week visit. Of note, patient 3 had variable follow-up times and worsened when these became too long (faricimab #8 was at a visit 77 days after faricimab #7 and showed worsening of macular thickness, but upon return 38 days later for #9, SRF had improved). In general, across several patients studied here, with consistent injections, there was a trend of decreasing macular thickness (with the important previously stated caveat that the natural history of CSC is highly variable).

[0291] Careful inspection of OCTs in this case series indicates that all observed improvements in macular thickening were due to improvement in SRF or IRF and were not driven by PED improvement (FIG. 30).

[0292] For this intervention, these results describe patients with a median of 30 weeks of unrelenting subretinal fluid, followed by a relatively swift improvement on OCT for a subset of patients, which typically would not be expected if they had otherwise gone unchanged for so long.

[0293] Conclusion

[0294] In a retrospective case series, improvement in macular thickness was observed in 14 / 16 eyes with chronic CSC and longstanding SRF immediately following treatment with intravitreal faricimab.

[0295] The above-results were obtained using the following materials and methods.

[0296] Methods

[0297] Study design

[0298] A multi-center retrospective chart review was conducted for patients with chronic CSC (active or recurrent SRF for ~6 months) who received a least one faricimab 6mg injection between January 1 2022 and April 11 2024. The sites involved were Massachusetts Eye and Ear and University of California San Francisco. Institutional review board approval was obtained for the multicenter retrospective chart review from the Mass General Brigham Institutional Review Board (protocol #2024P000543) with an associated data use agreement from the University of San Francisco. Institutional review board approval was determined not to be required by the University of California Institutional Review Board due to only one patient being included. As this was a retrospective study, informed consent was not indicated for inclusion in the study; however standard clinical practice was observed with each patient encounter which included a detailed informed consent discussing the potential risks and benefits of off-label use of intravitreal faricimab injection. The study complied with the Health Insurance Portability and Accountability Act of 1996 and adhered to the tenets of the Declaration of Helsinki.

[0299] Patients with CSC and chronic SRF on optical coherence tomography (OCT) were included (minimum of 8 weeks in duration of SRF as captured on consecutive images), and fluorescein angiography (FA) was used in the majority of cases to confirm the diagnosis (with ICGA additionally being used in some) and to rule out angiographic evidence of CNVM. Patients with evidence of a choroidal neovascular membrane on color photos, optical coherence tomography (OCT) and / or fluorescein angiography were excluded. An additional sensitivity analysis was conducted to further exclude patients with hyperreflective broad-based PEDs that do not leak on FA but are nonetheless concerning for CNVM.

[0300] OCT scanning was performed with the Spectralis device (Heidelberg Engineering, Heidelberg, Germany) in all patients. Images from scans occurring within 52 weeks prior to and 52 weeks following the first faricimab injection were extracted. Automated macular thickness was defined as the distance from Bruch's membrane to the intern al limiting membrane (ILM) (the default setting), and median thickness was calculated as the median of 5 ETDRS regions (the 1 central subfield and the 4 regions in the pericentral ring) (FIG. 26). One patient had active CSC exclusively outside of the central subfield ETRDS region, and in this case the superior and nasal outer ETDRS regions were added and the central subfield was not used. The default segmentation from Bruch's membrane to the ILM was used to calculate thickness unless the segmentation was noted be incorrect, in which case it was manually adjusted. Thickness values were found to be non-normally distributed (Shapiro-Wilk p < 2.2E-16) and thus nonparametric statistics were planned for all calculations. Thickness trajectories were plotted for each patient, and median pre- faricimab and post-faricimab macular thicknesses were compared in aggregate across all patients using the Wilcoxon signed-rank test. When descriptive statistics were used to summarize patients who improved, improvement was defined as a change >10 microns which can correspond to resolution of shallow subretinal fluid but below which cannot be distinguished from normal technical fluctuation in the instrumentation.

[0301] Example 7. A novel genetic variant in VE-PTP is associated with central serous chorioretinopathy and varicose veins

[0302] Further to Examples 1 -6, above, this Example provides additional results showing that a missense variant in PTPRB is associated with disease and that administration of a Tie-2 activator (e.g., faricimab) is useful in treating disease.

[0303] Central serous chorioretinopathy has hallmarks of subretinal fluid, thickened choroid and angiographic leaks. Studies of the genetics of disease points to root-cause biology. CSCR clusters in families; indeed, -50% of asymptomatic family members of CSCR patients have objective findings of CSCR.

[0304] Progress in genome-wide association studies

[0305] A recent meta-analysis identified 5 loci associated to CSCR in 1 ,176 cases and 500,000 controls. Ramo et al. JAMA Ophthalmology 2023. Single cell expression data highlights vascular tissue as an important site.

[0306] Further investigation

[0307] An updated meta-analysis identified 10 loci in 2,319 patients and 750,000 controls (FIG. 31). A rare coding variant in the gene PTPRB caught our attention. Rs113791087 is a missense change (ILE LEU) in PTPRB. PTPRB expression is specific to vascular endothelial cells, such as the capillaries, arteries, and veins of integrated retina, RPE, and choroid, rs 113791087 replicates in 3 cohorts and has a strong effect size in all: Odds Ratio (OR) 2.21 - 8.28 (FIG. 9).

[0308] Other phenotypes associated to the missense variant

[0309] Among 2,469 tested phenotypes in FinnGen and MVP, rs113791087 is associated to risk for varicose veins (FIG. 11).

[0310] Clinical context, mechanism and translation

[0311] PTPRB interacts with VE-cadherin and Tie-2, both of which are involved in endothelial cell tight junctions (Gal et al. Sec. Evo. and Pop. Gen. 2020). A quantitative retrospective review of 16 eyes (15 patients) with chronic CSCR who received faricimab at Mass Eye and Ear or UCSF was performed (FIG. 26). Inclusion criteria were OCT, FA, ICGA with evidence of chronic CSCR; exclusion criteria were patients with CNVM (FA, OCT, exam).

[0312] 16 eyes (15 patients) were included. The age range was 52 - 74, 75% male. SRF was present for a median of 30 weeks [9 - 257], 44% of patients had prior treatments (anti-VEGF, PDT). There was an average of 3.8 faricimab injections, range 1 - 10. Macular thickness decreased in the majority of CSCR patients with Faricimab when comparing measurements during 1 year prior to after injection (FIGS. 30 and 32).

[0313] In summary, high impact genetic variation in PTPRB is associated with CSCR and varicose veins. Overall, the genetics points to vascular tissue as pathologic. Targeting the Tie-2 pathway is helpful in CSCR.

[0314] Example 8. Treatment of ocular diseases or vascular diseases by administration of a Tie-2 activator

[0315] According to the methods disclosed herein, a physician of skill in the art can treat a subject, such as a human patient, having an ocular disease (e.g., central serous chorioretinopathy, venous overload choroidopathy, or separation of retinal layers) or a vascular disease (e.g., a lymphatic disease (e.g., lymphangioma), a venous disease (e.g., varicose veins, venous thromboembolism, chronic peripheral venous insufficiency, or deep vein thrombosis), pleural effusion, hemangioma, capillary nevus, hemorrhoids, or pulmonary embolism). To treat the subject, a physician of skill in the art can administer to the subject a Tie-2 activator, such as an anti-Ang-2 antibody or antigen-binding fragment thereof (e.g., faricimab or nesvacumab), an agent that oligomerizes Tie-2 (e.g., Tie2.1 -hexamer), or an anti-Tie-2 antibody or antigen-binding fragment thereof (e.g., ASP4021 ).

[0316] Activating the Tie-2 pathway improves endothelial cell adherens junctions, which results in decreased leakiness of these vessels and increase in stability of blood vessels. In CSC, this would result in a decrease in subretinal and / or intraretinal fluid. In other vascular diseases, this would result in increased stability of otherwise dilated and leaking blood vessels. Because faricimab resolves subretinal fluid in CSC via blockade of Ang-2 and thus activation of Tie-2, any activation of Tie-2 will have a similar effect. Though faricimab is a bi-specific antibody with both anti-Ang-2 function and anti-VEGF function, anti-VEGF has never been shown to work in CSC (see examples in Figure 28 showing failure of anti- VEGF alone followed by success of faricimab), thus faricimab must exert its effect via the Ang-2 blockade leading to Tie-2 activation.

[0317] To test additional Ang-2 blockers and Tie-2 activators, in vitro experiments are conducted using cultured endothelial cells (HMRECs, human microvascular endothelial cells). Endothelial cell permeability is assessed using trans-endothelial electrical resistance (TEER) measurements and permeability assays with FITC-dextran or Evans Blue dye. Tie-2 activation is assessed via analysis of Tie-2 phosphorylation via Western blot using phospho-Tie-2 antibodies. Downstream signaling pathways of Tie-2 activation are measured by determining the expression of anti-inflammatory (ANGPT1 ) and permeability-related markers (VE-Cadherin, Occludin, Claudin-5) by Western blot, qPCR and immunostaining. Tie-2 activators lead to decreased cell permeability in TEER testing and increased expression of downstream proteins (ANGPT1 , VE-Cadherin, Occludin, Claudin-5) as evaluated according to standard methodologies. Ang-2 blockade has similar effects.

[0318] Ex vivo experiments are conducted using human choroid explants as known in the art. After treatment, structural integrity and angiogenesis are assessed by immunostaining and confocal microscopy.

[0319] OTHER EMBODIMENTS

[0320] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

[0321] All publications, patents, and patent applications (for example, U.S. Patent Application Serial No. 63 / 608,526, filed on December 11 , 2023, and U.S. Patent Application Serial No. 63 / 561 ,276, filed on March 4, 2024) are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Some embodiments of the technology described herein can be defined according to any of the following numbered embodiments:

[0322] E1 . A method of treating a subject having an ocular disease, the method comprising administering to the subject a therapeutically effective amount of a Tie-2 activator.

[0323] E2. The method of E1 , wherein the ocular disease is central serous chorioretinopathy, venous overload choroidopathy, or separation of retinal layers.

[0324] E3. The method of E1 or E2, wherein the Tie-2 activator is an Ang-2 inhibitor.

[0325] E4. The method of E3, wherein the Ang-2 inhibitor binds to both Ang-2 and VEGF.

[0326] E5. The method of E3 or E4, wherein the Ang-2 inhibitor is an anti-Ang-2 antibody or antigen-binding fragment thereof.

[0327] E6. The method of E5, wherein the anti-Ang-2 antibody has a heavy chain sequence having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 1 , 2, or 5.

[0328] E7. The method of E5 or E6, wherein the anti-Ang-2 antibody has a light chain sequence having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 3, 4, or 6.

[0329] E8. The method of any one of E4-E7, wherein the anti-Ang-2 antibody is faricimab.

[0330] E9. The method of any one of E5-E7, wherein the anti-Ang-2 antibody is nesvacumab.

[0331] E10. The method of E1 or E2, wherein the Tie-2 activator is an agent that oligomerizes Tie-2.

[0332] E11 . The method of E10, wherein the agent that oligomerizes Tie-2 is Tie2.1 -hexamer.

[0333] E12. The method of E1 or E2, wherein the Tie-2 activator is an anti-Tie-2 antibody or antigen-binding fragment thereof.

[0334] E13. The method of E12, wherein the anti-Tie-2 antibody is ASP4021.

[0335] E14. The method of any one of E1 -E13, wherein the Tie-2 activator is administered at a dose of about 1 mg to about 10 mg.

[0336] E15. The method of E14, wherein the Tie-2 activator is administered at a dose of about 6 mg.

[0337] E16. The method of any one of E1 -E15, wherein the Tie-2 activator is administered in 1 or 2 doses.

[0338] E17. The method of any one of E1 -E16, wherein the Tie-2 activator is administered intravitreally or intravenously.

[0339] E18. A method of treating a subject having a vascular disease, the method comprising administering to the subject a therapeutically effective amount of a Tie-2 activator.

[0340] E19. The method of E18, wherein the vascular disease is a lymphatic disease, a venous disease, pleural effusion, hemangioma, capillary nevus, hemorrhoids, or pulmonary embolism.

[0341] E20. The method of E19, wherein the lymphatic disease is lymphangioma.

[0342] E21 . The method of E19, wherein the venous disease is varicose veins, venous thromboembolism, chronic peripheral venous insufficiency, or deep vein thrombosis.

[0343] E22. The method of any one of E18-E20, wherein the Tie-2 activator is an Ang-2 inhibitor.

[0344] E23. The method of E22, wherein the Ang-2 inhibitor binds to both Ang-2 and VEGF. E24. The method of E22 or E23, wherein the Ang-2 inhibitor is an anti-Ang-2 antibody or antigenbinding fragment thereof.

[0345] E25. The method of E24, wherein the anti-Ang-2 antibody has a heavy chain sequence having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 1 , 2, or 5.

[0346] E26. The method of E24 or E25, wherein the anti-Ang-2 antibody has a light chain sequence having at least 70% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 3, 4, or 6.

[0347] E27. The method of any one of E23-E26, wherein the anti-Ang-2 antibody is faricimab.

[0348] E28. The method of any one of E24-E26, wherein the anti-Ang-2 antibody is nesvacumab.

[0349] E29. The method of any one of E18-E21 , wherein the Tie-2 activator is an agent that oligomerizes Tie-

[0350] 2.

[0351] E30. The method of E29, wherein the agent that oligomerizes Tie-2 is Tie2.1 -hexamer.

[0352] E31 . The method of any one of E18-E21 , wherein the Tie-2 activator is an anti-Tie-2 antibody or antigen-binding fragment thereof.

[0353] E32. The method of E31 , wherein the anti-Tie-2 antibody is ASP4021 .

[0354] E33. The method of any one of E18-E32, wherein the Tie-2 activator is administered at a dose of about

[0355] 5 mg / kg to about 15 mg / kg.

[0356] E34. The method of any one of E18-E33, wherein the Tie-2 activator is administered every two weeks or every three weeks.

[0357] E35. The method of any one of E18-E34, wherein the Tie-2 activator is administered intravenously.

[0358] Other embodiments are within the following claims.

Claims

CLAIMS1 . A method of treating a subject having an ocular disease, the method comprising administering to the subject a therapeutically effective amount of a Tie-2 activator.

2. The method of claim 1 , wherein the ocular disease is central serous chorioretinopathy, venous overload choroidopathy, or separation of retinal layers.

3. The method of claim 1 , wherein the Tie-2 activator is an Ang-2 inhibitor.

4. The method of claim 3, wherein the Ang-2 inhibitor binds to both Ang-2 and VEGF.

5. The method of claim 3, wherein the Ang-2 inhibitor is an anti-Ang-2 antibody or antigen-binding fragment thereof.

6. The method of claim 5, wherein the anti-Ang-2 antibody is faricimab.

7. The method of claim 5, wherein the anti-Ang-2 antibody is nesvacumab.

8. The method of claim 1 , wherein the Tie-2 activator is an agent that oligomerizes Tie-2.

9. The method of claim 8, wherein the agent that oligomerizes Tie-2 is Tie2.1 -hexamer.

10. The method of claim 1 , wherein the Tie-2 activator is an anti-Tie-2 antibody or antigen-binding fragment thereof.11 . The method of claim 10, wherein the anti-Tie-2 antibody is ASP4021 .

12. The method of claim 1 , wherein the Tie-2 activator is administered at a dose of about 1 mg to about 10 mg.

13. The method of claim 12, wherein the Tie-2 activator is administered at a dose of about 6 mg.

14. The method of claim 1 , wherein the Tie-2 activator is administered in 1 or 2 doses.

15. The method of claim 1 , wherein the Tie-2 activator is administered intravitreally or intravenously.

16. A method of treating a subject having a vascular disease, the method comprising administering to the subject a therapeutically effective amount of a Tie-2 activator.

17. The method of claim 16, wherein the vascular disease is a lymphatic disease, a venous disease, pleural effusion, hemangioma, capillary nevus, hemorrhoids, or pulmonary embolism.

18. The method of claim 17, wherein the lymphatic disease is lymphangioma.

19. The method of claim 17, wherein the venous disease is varicose veins, venous thromboembolism, chronic peripheral venous insufficiency, or deep vein thrombosis.

20. The method of claim 16, wherein the Tie-2 activator is an Ang-2 inhibitor.21 . The method of claim 20, wherein the Ang-2 inhibitor binds to both Ang-2 and VEGF.

22. The method of claim 20, wherein the Ang-2 inhibitor is an anti-Ang-2 antibody or antigen-binding fragment thereof.

23. The method of claim 22, wherein the anti-Ang-2 antibody is faricimab.

24. The method of claim 22, wherein the anti-Ang-2 antibody is nesvacumab.

25. The method of claim 16, wherein the Tie-2 activator is an agent that oligomerizes Tie-2.

26. The method of claim 25, wherein the agent that oligomerizes Tie-2 is Tie2.1 -hexamer.

27. The method of claim 16, wherein the Tie-2 activator is an anti-Tie-2 antibody or antigen-binding fragment thereof.

28. The method of claim 27, wherein the anti-Tie-2 antibody is ASP4021 .

29. The method of claim 16, wherein the Tie-2 activator is administered at a dose of about 5 mg / kg to about 15 mg / kg.

30. The method of claim 16, wherein the Tie-2 activator is administered every two weeks or every three weeks.31 . The method of claim 16, wherein the Tie-2 activator is administered intravenously.

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