EA043390B1 - METHODS FOR TREATING EYE DISEASES WITH APLNR ANTAGONISTS AND VEGF INHIBITORS - Google Patents

Description

List of sequences

This application incorporates by reference the sequence listing provided in machine-readable form as file 10354WO01-Sequence.txt, created on May 4, 2018, containing

16843 bytes.

Field of technology

This invention relates to methods of treating vascular disease of the eye, in particular by administering an apelin receptor antagonist and a vascular endothelial growth factor (VEGF) antagonist to a subject in need thereof.

State of the art

Vascular eye diseases are the leading cause of vision loss in today's aging population. These diseases may be characterized by abnormal leaky blood vessels growing into the retina. The two largest culprits in this population of subjects are diabetic retinopathy and exudative age-related macular degeneration.

Diabetic retinopathy (DR) is a leading cause of visual impairment in the United States (Klein et al., 1984, Ophthalmology, 91: 1464-1474; Moss et al., 1998, Ophthalmology, 105: 998-1003). Diabetic retinopathy results from microvascular decompensation, starting with thickening of the basement membrane (Ruggiero et al., 1997, Diabetes Metab., 23: 30-42), which ultimately leads to vascular occlusion and neovascularization (Porta et al., 2002, Diabetologia, 45: 1617-1634). It is estimated that about 28% of patients aged 40 years and older suffer from DR, and 4.4% have vision-threatening DR (Zhang et al., 2010, JAMA, 304: 649-656). Diabetic macular edema (DME) is a manifestation of DR and is the most common cause of blindness in young and middle-aged people (Klein et al., 1984, Ophthalmology, 91: 1464-1474; Moss et al., 1998, Ophthalmology, 105: 998- 1003).

Age-related macular degeneration (AMD) is the leading cause of severe vision loss in people aged 50 years and older in developed countries. In recent years, significant advances have been made in the treatment of AMD with the introduction of anti-angiogenic agents, offering hope for significant visual restoration in patients with neovascular AMD (Keane et al., 2012, Surv. Ophthalmol., 57: 389-414).

Preproapelin is a 77 amino acid protein expressed in the human central nervous system and peripheral tissues such as lung, heart and mammary gland. Peptides containing C-terminal apelin peptide fragments of varying sizes have been shown to activate the G protein-coupled receptor, APJ receptor (now known as APLNR) (Habata, et al., 1999, Biochem. Biophys. Acta., 1452 : 25-35; Hosoya et al., 2000, JBC, 275 (28): 21061-67; Lee et al., 2000, J. Neurochem., 74: 34-41; Medhurst et al., 2003, J. Neurochem., 84: 1162-1172). Many studies indicate that apelin peptides and analogues mediate cardiovascular and angiogenic effects through their interaction with the APJ receptor (APLNR), such as endothelium-dependent vasodilation (Tatemoto et al., 2001, Regul. Pept., 99: 87-92 ).

The apelin system appears to play a role in pathophysiological angiogenesis. Studies have shown that apelin may be involved in hypoxia-induced retinal angiogenesis (Kasai et al., 2010, Arterioscler. Thromb. Vasc. Biol., 30: 2182-2187). In some reports, certain compositions can inhibit angiogenesis by inhibiting the apelin/APJ pathway (see, for example, US patent No. 7736646), such as APLNR inhibitors, capable of blocking pathological angiogenesis and therefore useful for inhibiting vascularization in the retina (Kojima, Y. , Quertermous, T., 2008, Arterioscler. Thromb. Vasc. Biol., 28: 1687-1688). Thus, interfering with apelin-mediated signaling may also be beneficial for the early prevention of proliferative diabetic retinopathy (Tao et al., 2010, Invest. Opthamol. Visual. Science, 51: 4237-4242; Du, J.H. et al., Int. J. Ophthalmol., 2014 Dec 18, 7 (6): 968-73; Lu, Q. et al., 2013, PLoS One, 8 (7): e69703). Recently, apelin has been implicated in the mechanism of retinopathy of prematurity (AN YF et al., Clin Ophthalmol., 2017 Feb 21, 11: 387-392).

Antivascular endothelial growth factor (VEGF) therapy (eg, aflibercept) is the standard of care for neovascular AMD and DME. The efficacy and safety of aflibercept in these subject populations has been well studied (Dixon et al., 2009, Expert Opin. Investig. Drugs, 18: 1573-80). However, in AMD, although approximately 95% of subjects maintained their vision, only approximately 30% of subjects achieved an improvement of 15 or more best-corrected visual acuity (BCVA) letters at 1 year. In DME, there is also the potential for improved treatment outcomes. As seen, with aflibercept and ranibizumab, less than 50% of patients with vision loss due to DME achieve 15 or more letters of improvement at 1 and 2 years. Additionally, in studies with ranibizumab, clinical signs of proliferative retinopathy developed in 7.2% of subjects receiving 3 years of monthly ranibizumab treatment, with up to 3.2% of patients requiring panretinal photocoagulation, a potentially visually disabling treatment (Brown et al. , 2013, Ophthalmology, 10: 2013-22).

Although both apelin/APLNR and VEGF are known to contribute to angiogenesis and vascular development, the mechanism by which the two signaling pathways interact to promote angiogenesis remains unclear. In particular, these pathways are known to be involved in the formation of retinal vessels, and various

- 1 043390 studies report that apelin and VEGF have positive and negative feedback effects, in which increased expression of one can promote expression of the other, or antagonism of one suppresses expression of the other (Lu et al., 2014, Molecular Vision, 20: 1122-1131 ).

Intravitreal (IVT) delivery of anti-VEGF therapies such as ranibizumab and aflibercept have demonstrated efficacy and safety in chorioretinal diseases. However, there are many additional factors that contribute to vascular permeability, neovascularization, and other vascular dysfunctions.

Brief description of the invention

In one aspect, the present invention provides methods for treating, preventing, or alleviating at least one symptom or sign of an ocular vascular disease or disorder in a subject. The methods include administering a therapeutically effective amount of a pharmaceutical composition containing an APLNR antagonist to a subject in need thereof. In some embodiments, the APLNR antagonist is administered in combination with a vascular endothelial growth factor (VEGF) antagonist, for example, by administering a therapeutically effective amount of a pharmaceutical composition containing a VEGF antagonist.

In some embodiments, the eye disease or disorder is selected from the group consisting of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polypoidal choroidal vasculopathy, choroidal neovascularization (CNV), degenerative myopia (myopic CNV), neovascular glaucoma and retinopathy of prematurity.

In another aspect, the present invention provides methods for inhibiting retinal angiogenesis in a subject. The methods include administering a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist to a subject in need thereof. In some embodiments, retinal angiogenesis is associated with an ocular vascular disease or disorder.

In another aspect, the present invention provides methods for inhibiting retinal neovascularization (eg, in a subject with an ocular disease or angiogenesis-related disorder). The methods include administering a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist to a subject in need thereof.

In another aspect, the present invention provides methods for inhibiting choroidal neovascularization in a subject. The methods include administering a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist to a subject in need thereof.

In another aspect, the present invention provides methods for improving vascular regrowth and reducing pathological neovascularization in a subject. The methods include administering a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist to a subject in need thereof.

In another aspect, the present invention provides methods for promoting retinal revascularization in a subject in need thereof. The methods include administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof; and administering to the subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist.

In another aspect, the present invention provides methods for promoting uniform retinal vascular regrowth in a subject in need thereof. The methods include administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof; and administering to the subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist.

In another aspect, the present invention provides methods for improving vascular regrowth in the retina of a subject in need thereof. The methods include administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof; and administering to the subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist.

In another aspect, the present invention provides methods for promoting uniform growth of blood vessels in the retina of a subject in need thereof. The methods include administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof; and administering to the subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist.

In another aspect, the present invention provides methods for improving the organization of retinal blood vessels in a subject in need thereof. The methods include administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof; and administering to the subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist.

In any of the methods discussed above or herein, the subject may be an individual who has been diagnosed with an eye disease or disorder. In some cases, the eye disease or disorder is selected from the group consisting of diabetic retinopathy, proliferative

- 2 043390 diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polypoidal choroidal vasculopathy, choroidal neovascularization (CNV), degenerative myopia (myopic CNV), neovascular glaucoma and retinopathy of prematurity. In some cases, the eye disease or disorder is age-related macular degeneration. In some cases, the eye disease or disorder is diabetic macular edema. In some cases, the eye disease or disorder is retinopathy of prematurity. In some cases, the eye disease or disorder is proliferative diabetic retinopathy.

Exemplary APLNR antagonists that may be used in the context of the methods or compositions of this invention include small molecule chemical APLNR inhibitors, or biological agents that target APLNR, such as peptides, peptide mimetics, and antibodies. In some embodiments, an APLNR antagonist blocks the interaction of APLNR and apelin.

In accordance with some embodiments of the invention, the APLNR antagonist is an antibody or antigen binding protein that binds the APLNR antagonist and inhibits APLNR signaling. In some embodiments, the anti-APLNR antibody or antigen binding protein comprises heavy chain variable region (HCVR) heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequence of SEQ ID NO: 2 or 13, and a light chain variable region light chain CDR ( LCVR) containing the amino acid sequence of SEQ ID NO: 7 or 18. In some embodiments, the anti-APLNR antibody or antigen binding protein thereof comprises complementarity determining regions (CDRs) of a heavy chain variable region (HCVR) of the anti-APLNR antibody selected from group consisting of H2aM9232N (or H4H9232N) and H1M9207N.

In any of the methods or compositions discussed above or herein, the APLNR antagonist may comprise an anti-APLNR antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof specifically binds to human APLNR. In some cases, an antibody or antigen binding fragment thereof competes for binding to the human apelin receptor (APLNR) with a reference antibody comprising an HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2/7 and 13/18, wherein the antibody or its antigen-binding fragment specifically binds to human APLNR. In some cases, an antibody or antigen binding fragment thereof binds to the same epitope on an APLNR as a reference antibody comprising an HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2/7 and 13/18, wherein the antibody or its the antigen-binding fragment specifically binds to human APLNR. In some cases, the antibody or antigen binding fragment thereof comprises (a) heavy chain variable region (HCVR) complementarity determining regions (CDRs) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 13; and (b) a light chain variable region (LCVR) CDR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 18.

In some cases, the antibody or antigen binding fragment thereof comprises HCDR1HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, selected from the group consisting of: SEQ ID NO: 3-4-5-8-9-10 and 14-15-16 -19-20-21. In some cases, the antibody or antigen-binding fragment contains:

(a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 13; and (b) a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 18.

In some cases, the antibody or antigen binding fragment thereof comprises a heavy and light chain CDR of the amino acid sequence pair HCVR/LCVR selected from the group consisting of SEQ ID NOs: 2/7 and 13/18. In some cases, the antibody or antigen binding fragment thereof comprises a pair of amino acid sequences HCVR/LCVR selected from the group consisting of SEQ ID NOs: 2/7 and 13/18.

In one embodiment of the invention, any of the methods or compositions discussed above or herein, the antibody or antigen binding fragment thereof comprises the amino acid sequence pair HCVR/LCVR SEQ ID NO: 2/7.

In one embodiment of the invention, any of the methods or compositions discussed above or herein, the antibody or antigen binding fragment thereof comprises the amino acid sequence pair HCVR/LCVR SEQ ID NO: 13/18.

In any of the methods or compositions discussed above or herein, the antibody or antigen binding fragment thereof may be a human IgG1 or IgG4 heavy chain constant region antibody. In one embodiment of the invention, the heavy chain constant region is human IgG1. In one embodiment of the invention

- 3 043390 the heavy chain constant region is human IgG4.

In various embodiments of the invention, any of the methods or compositions discussed above or herein, the antibody or antigen binding fragment thereof blocks the interaction of APLNR and apelin. In some cases, an antibody or antigen binding fragment thereof blocks the interaction of APLNR and apelin, exhibiting at least 50% inhibition of binding in a competitive binding assay. In other embodiments, the antibody or antigen binding fragment thereof does not block or only partially blocks the interaction of APLNR and apelin.

VEGF antagonists that can be used in combination with an APLNR antagonist in the compositions and methods of this invention include anti-VEGF antibodies (eg, ranibizumab), small molecule VEGF inhibitors (eg, sunitinib) and VEGF inhibitory fusion proteins (VEGF Traps). An example of a VEGF antagonist that can be used in combination with an APLNR antagonist in the treatment methods of this invention is aflibercept, a VEGF inhibitory fusion protein (see, for example, US Pat. No. 7,087,411).

In any one of the methods or compositions described above or herein, the VEGF antagonist contains a chimeric VEGF receptor-based molecule (VEGF decoys). In some cases, the VEGF trap contains one or more Ig-like domains of VEGFR1, one or more Ig-like domains of VEGFR2, and a multimerizing domain. In some cases, the VEGF trap contains Ig-like domain 2 of VEGFR1, Ig-like domain 3 of VEGFR2, and a multimerizing domain. domain. In some cases, the VEGF decoy is aflibercept or a biosimilar molecule thereof. In some cases, the VEGF antagonist consists of a dimer of two polypeptides consisting of amino acids 27-457 SEQ ID NO: 23.

In another aspect, this disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of an APLNR antagonist and a pharmaceutically acceptable carrier or diluent. In certain embodiments of the invention, the pharmaceutical composition further comprises a VEGF antagonist.

In various embodiments of the invention, this disclosure relates to the use of an APLNR antagonist in combination with a VEGF antagonist in the manufacture of a medicament for treating an eye disease or disorder in a subject, including humans, or to achieve other purposes of any of the methods discussed above or herein. All of the methods discussed above or herein can be implemented as the use or uses of APLNR antagonists and VEGF antagonists for the treatment of eye diseases or disorders, or other purposes of the listed methods. Other embodiments of the invention include APLNR antagonists and VEGF antagonists for use in the methods discussed above or herein.

In another aspect, the present invention provides a composition for treating a vascular eye disease or disorder, wherein the composition contains a therapeutically effective amount of an APLNR antagonist, a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist, and a suitable carrier, excipient or diluent. In various embodiments, the APLNR antagonist or VEGF antagonist compositions may be those discussed above or herein.

Other embodiments of the invention will be apparent from a review of the following detailed description.

Brief description of graphic materials

Fig. 1A and 1B are representative photomicrographs of mouse retinas treated systemically (i.p.) at P2 to P5 and a graph of the calculated vascular area. Residual vascular area was significantly less in retinas treated with aApelinR (anti-APLNR antibody H2aM9232N) (23% at 25 mg/kg, p < 0.05) compared with untreated retinas. Retinal endothelial cells were stained with GS Lectin I. Images were acquired at 20x (for quantification) and 40x. Statistical analysis was performed using Student's t test. Selective inhibition of ApelinR through systemic injection slowed normal vascular sprouting in the developing retina of P5 pups. At a dose of 25 mg/kg, blocking ApelinR slightly inhibits vascular sprouting.

Fig. 2A and 2B are representative photomicrographs of mouse retinas treated systemically (i.p.) at P2 to P5 and a graph of the calculated vascular area. The residual vascular area was significantly smaller in aApelinR-treated (35% at 50 mg/kg, p < 0.005) retinas compared with untreated retinas. Retinal endothelial cells were stained with GS Lectin I. Images were acquired at 20x (for quantification) and 40x. Statistical analysis was performed using Student's t test. Selective inhibition of ApelinR through systemic injection slowed normal vascular sprouting in the developing retina of P5 pups. By increasing the dose to 50 mg/kg, targeting ApelinR further delayed retinal vascular sprouting.

- 4 043390

Fig. 3A and 3B are representative micrographs of mouse retina treated systemically (IP) at P2 to P5 and a plot of the calculated vascular area. In this single-blind study, 50 mg/kg aApelinR reduced vascular growth by 29.8% (p<0.0001) compared to Fc-treated controls. Retinal endothelial cells were stained with GS Lectin I. Images were acquired at 20x (for quantification) and 40x. Statistical analysis was performed using Student's t test. Selective inhibition of ApelinR through systemic injection slows normal vascular sprouting in the developing retina of P5 pups.

Fig. 4A and 4B are representative photomicrographs of mouse retinas treated systemically (i.p.) at P2 to P5 and a graph of the calculated vascular area. Residual vessel area was significantly less with the combination (aApelinR+aflibercept) (62% at 50 mg/kg, p<0.0001) compared to the individual reagents, aApelinR alone (31% at 50 mg/kg, p<0.001) or aflibercept (43% at 50 mg/kg, p<0.005). Retinal endothelial cells were stained with GS Lectin I. Note the effect on dose and relative area of vascularization. Images were taken at 20x (for quantification) and 40x. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. With both systemic and intravitreal administration (see Figures 5A and 5B), administration of aApelinR and aflibercept resulted in regression of the normally developing retinal vasculature. Blocking both ApelinR and VEGFA by systemic injection is more effective in preventing vascular growth compared to blocking ApelinR or VEGFA alone. In this model, combination treatment further reduced vascular area by 46% (p < 0.0005) compared with aApelinR and by 32% (p < 0.001) compared with aflibercept.

Fig. 5A and 5B are representative photomicrographs of a mouse retina treated by intravitreal (IVT) injection at P4-P6 and a graph of the calculated vascular area. Residual vessel area was significantly less in the combination (aApelinR + aflibercept) (68% at 5 μg/kg, p < 0.0001) compared to the individual reagents, aApelinR alone (43% at 5 μg/kg, p < 0.0001 ) or aflibercept (65% at 5 μg/kg, p<0.0001). Retinal endothelial cells were stained with GS Lectin I. Note the effect on dose and relative area of vascularization. Images were taken at 20x (for quantification) and 40x. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. Both systemic and intravitreal administration of aApelinR and aflibercept leads to regression of the normally developing retinal vasculature. Blocking both ApelinR and VEGFA by intravitreal injection is even more effective in preventing vascular growth compared to blocking ApelinR and VEGFA alone. IVT combination treatment further reduced vascular area by 50% (p<0.0005) compared with aApelinR and by 34% (p<0.001) compared with aflibercept.

Fig. 6A and 6B are representative photomicrographs of OIR mouse retinas treated systemically (IP) at P12 to P16 and plots of calculated avascular area. Residual avascular area was significantly smaller in aApelinR (29%, p<0.05) and aflibercept (27.5%, p<0.01) retinas compared with Fc (control) retinas. Note the significant reduction in neovascular bundles in samples treated with aApelinR (67%, p<0.0001) and aflibercept (94%, p<0.0005) compared to Fc. Retinal endothelial cells were stained with GS Lectin I. Images were acquired at 20x (quantification of avascular area) and 40x (quantification of abnormal neovascular area). Statistical analysis was performed using Student's t test. Inhibition of ApelinR and VEGFA by systemic injection promotes retinal vascular sprouting and reduces pathological neovascularization.

In fig. 7A and 7B are representative photomicrographs of a mouse retina with induced retinopathy (OIR) treated intravitreally at P12 to P16 and plots of the calculated avascular area. Residual avascular area was significantly reduced in the aApelinR condition (27.5% p<0.05) and increased in the aflibercept condition (32%, p<0.0001) compared with Fc controls. Note the significant reduction in neovascular tufts in aApelinR (60%, p < 0.0001) and complete disappearance of tufts in aflibercept samples. Retinal endothelial cells were stained with GS Lectin I. Note the effect on dose and relative area of vascularization. Images were taken at 20x (quantification of avascular area) and 40x (quantification of abnormal neovascular area). Statistical analysis was performed using one-way ANOVA followed by Tukey's test. Both systemically (see Figures 6A and 6B) and intravitreal administration, selective inhibition of ApelinR promotes retinal vascular sprouting and reduces neovascularization in OIR mice. Inhibition of ApelinR by intravitreal injection improves vascular regrowth and reduces abnormal neovascularization, whereas inhibition of VEGFA stops vascular regrowth and completely stops any neovascularization.

Fig. 8A and 8B are representative micrographs of OIR mouse retinas,

- 5 043390 treated systemically (20x) in P12 to P16, and graphs of the calculated avascular area. The rate of regrowth in the combination treatment was similar for each agent alone, vascular regrowth was improved compared with control hFc-treated retinas, and there was a significant change in avascular area between treatment groups (p<0.0005).

Fig. 9A and 9B are representative photomicrographs of OIR mouse retinas treated systemically (40x) at P12 to P16 and plots of calculated abnormal vascular area. Combination treatment showed fewer pruned vessels compared with aflibercept and fewer abnormal neovascularizations compared with anti-APLNR and Fc control (Fig. 9A). Fig. 9B demonstrates that there were significant changes in abnormal vascular area between treatment groups (p<0.0005). Abnormal vascular area was significantly reduced with anti-APLNR (***, p<0.0005), with aflibercept (*** p<0.005) and with the combination (***, p<0.0005) compared with control Fc, and abnormal vascular area were also significantly reduced in the combination treatment group (*, p<0.05) compared with the anti-APLNR treatment group

In fig. 10 shows examples of image processing and measurement used to calculate the vessel area and vessel length in Example 7. The source images are the 40x micrographs shown in FIG. 9A.

Fig. 11A and 11B are representative processed images showing vascular organization and uniformity from micrographs (40x) of OIR mouse retinas processed systemically at P12 to P16, and plots of calculated vessel area. As shown in FIG. 11A, the combination of anti-APLNR antibody and aflibercept resulted in retinal vessels that are more organized and uniform compared with anti-APLNR alone, and less sparse (with fewer pruned vessels) compared with aflibercept alone. Fig. 11B demonstrates that there were significant changes in vessel area between treatment groups (p<0.0005). Vessel area was significantly increased with anti-APLNR (***, p<0.0005) and decreased with aflibercept (***, p<0.005) and combination therapy (*, p<0.05) compared to Fc control . Vessel area was significantly reduced with aflibercept (####, p<0.0005) and with the combination (####, p<0.0005) compared with anti-APLNR. In contrast, vessel area was significantly increased with combination therapy (&&&, p<0.005) compared with aflibercept.

Fig. 12A and 12B are representative processed images showing vessel density from photomicrographs (40x) of OIR mouse retinas processed systemically at P12 to P16, and plots of calculated vessel length. As shown in FIG. 12A, the combination of anti-APLNR antibody and aflibercept produced retinal vessels of intermediate density compared to anti-APLNR (higher density) or aflibercept alone (lower density). Fig. 12B demonstrates that there were significant changes in vessel length between treatment groups (p<0.0005). Vessel length was significantly reduced with aflibercept (####, p<0.0005) and with the combination (####, p<0.0005) compared with anti-APLNR. In contrast, vessel length was significantly increased with combination therapy (&&&, p<0.005) compared with aflibercept.

Detailed Description of the Invention

Before describing this specification, it should be understood that this disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is intended solely to describe certain embodiments and should not be construed as limiting as the scope of this description will be limited only by the appended claims.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this description belongs. As used herein, the term about, when used in relation to a specific numerical value given, means that the value may differ from the given value by no more than 1%. For example, as used herein, the expression about 100 includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein may be practiced or tested herein, preferred methods and materials are described herein. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

This invention and its disclosure are based in part on the unexpected discovery that a combination of an APLNR antagonist and a VEGF antagonist (e.g., an anti-APLNR antibody and a VEGF decoy) can produce more organized and orderly intermediate-density revascularization (with fewer pruned vessels) in the retina of a subject. than is observed with an APLNR antagonist or a VEGF antagonist alone.

Definitions.

The expressions apelin receptor, APLNR, ApelinR, APJ receptor, etc., as used in

- 6 043390 herein refer to the human APLNR protein having the amino acid sequence represented as

MEEGGDFDNYYGADNQSECEYTDWKSSGALIPAIYMLVFLLGTTGNGLVLWTVFRSSR EKRRSADIFIASLAVADLTFVVTLPLWATYTYRDYDWPFGTFFCKLSSYLIFVNMYASVF CLTGLSFDRYLAIVRPVANLRLRVSGAVATAVLWVLAALLAMPVMVLRTTGDLENT TKVQCYMDYSMVATVSSEWAWEVGLG VSSTTVGFVVPFTIMLTCYFFIAQTIAGHFRK ERIEGLRKRRRLLSIIVVLVVTFALCWMPYHLVKTLYMLGSLLHWPCDFDLFLMNIFPY CTCISYVNSCLNPFLYAFFDPRFRQACTSMLCCGQSRCAGTSHSSSGEKSASYSSGHSQG PGPNMGKGGEQMHEKSIPYSQETLVVD (SEQ ID NO: 22), or essentially, similar to the amino acid sequence of SEQ ID NO: 22. All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the corresponding protein, polypeptide or a protein fragment unless explicitly stated to be from a non-human species (eg, mouse APLNR, simian APLNR, etc.).

As used herein, the term antibody or antigen binding fragment that binds APLNR or anti-APLNR antibody includes immunoglobulin molecules, antibodies and antigen binding fragments thereof that bind the APLNR protein fragment. APLNR molecules include natural APLNR proteins as well as variants of recombinant APLNR proteins, such as, for example, monomeric and dimeric APLNR constructs. In embodiments of the invention, the antibody that binds APLNR or an antigen binding fragment thereof is an APLNR antagonist.

As used herein, the term antagonist refers, in part, to a moiety that binds to a receptor at or near the same site as an agonist (e.g., an endogenous ligand), but that does not activate the intracellular response normally initiated by the active form of the receptor and thereby inhibits or neutralizes the intracellular response with an agonist or partial agonist. The term antagonist may also refer in part to a moiety that binds to a receptor agonist, thereby sequestering the agonist from interaction with its cognate receptor. An example of an antagonist that binds an agonist is a ligand decoy, such as a VEGF decoy. In some cases, antagonists do not reduce the basal intracellular response in the absence of an agonist or partial agonist. The antagonist need not act as a competitive inhibitor, but may act by sequestering the agonist or indirectly modulating the downstream effect.

As used herein, the term APLNR antagonist may refer to a moiety that binds to an APLNR receptor at or near the same site as an agonist (eg, apelin) but does not activate the intracellular response normally initiated by the active form of the receptor, and thereby inhibiting or neutralizing the intracellular APLNR response. In embodiments of the invention, the APLNR antagonist is an antibody or antigen-binding fragment thereof that binds APLNR. Examples of APLNR antagonists can be found in International Patent Publication No. WO2015077491, published May 28, 2015, which is specifically incorporated herein by reference in its entirety. Other APLNR antagonists may include small molecules and other biological entities such as peptides.

The term immunoglobulin (Ig) refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) chains and one pair of heavy (H) chains, all four of which may be linked together by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology, ch. 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989)).

The term antibody, as used herein, means any antigen-binding molecule or molecular complex containing at least one complementarity determining region (CDR) that specifically binds or interacts with a particular antigen (eg, APLNR). The term antibody includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds, as well as their multimers (for example, IgM), as well as immunoglobulin molecules, including a fragment of one or heavier chains or a fragment of one or more light chains (eg, Fab, F(ab') ₂ or scFv fragments), as described herein. Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or _VH ) and a heavy chain constant region. The heavy chain constant region includes three domains, _CH1 , CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The V _H and V _L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FR). Each V _H and V _L consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present invention, the FR of the anti-APLNR antibody (or antigen binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. A consensus amino acid sequence can be determined based on parallel analysis of two or more CDRs.

As used herein, the term antibody also includes antigen-binding portions of complete antibody molecules. The terms antigen binding region of an antibody, antigen binding fragment of an antibody, and the like as used herein include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding antibody fragments can be derived, for example, from full-length antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving processing and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA may be sequenced and processed chemically or using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration or to introduce codons, create cysteine residues, modify, add or remove amino acids, etc. Such methods can also be used to synthesize any antibody fusion molecule containing an antigen binding fragment derived from a complete antibody molecule.

Non-limiting examples of antigen binding fragments include (i) Fab fragments;

(ii) F(ab')2 fragments;

(iii) Fd fragments;

(iv) Fv fragments;

(v) single chain Fv molecules (scFv);

(vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic a hypervariable region of an antibody (eg, a dedicated complementarity determining region (CDR) such as a CDR3 peptide) or a conformationally restricted peptide FR3-CDR3-FR4.

Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, mini-bodies, nanobodies (e.g. monovalent nanobodies, divalent nanobodies, etc.) , small modular protein (SMIP) immunopharmaceuticals and shark IgNAR variable domains are also included in the expression of the antigen binding fragment used herein.

An antigen binding fragment of an antibody typically contains at least one variable domain. The variable domain can be of any size or amino acid composition and typically comprises at least one CDR that is adjacent or in frame with one or more framework sequences. In antigen binding fragments having a VH domain associated with a VL domain, the _VH and VL domains may be arranged relative to each other in any suitable order. For example, the variable region may be dimeric and contain dimers VH-VH, VH-V _L or V _L -V _L . Alternatively, the antigen binding antibody fragment may comprise a monomeric V _H or VL domain.

In certain embodiments of the invention, the antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen binding fragment of an antibody of the present invention include (i) Vh-Ch1;

(ii) Vh-Ch2;

(iii) Vh-Ch3;

(iv) Vh-Ch1-Ch2;

(v) Vh-Ch1-Ch2-Ch3;

(vi) Vh-Ch2-Ch3;

(vii) Vh-Cl;

(viii) Vl-Ch1;

(ix) Vl-Ch2;

(x) Vl-Ch3;

(xi) Vl-Ch1-Ch2;

(xii) Vl-Ch1-Ch2-Ch3;

- 8 043390 (xiii) Vl-Ch2-Ch3; and (xiv) Vl-Cl.

In any configuration of variable and constant domains, including any of the exemplary configurations above, the variable and constant domains may be directly linked to each other or may be linked by a full-length or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen binding fragment of an antibody herein may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more monomeric _VH- or VL- domains (eg via disulfide bond(s)).

As with full-length antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen binding antibody fragment will typically comprise at least two different variable domains, with each variable domain being capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats described herein, can be adapted for use in the context of an antigen binding fragment of an antibody herein using standard techniques available in the art.

The phrase antibody fusion proteins includes recombinant polypeptides and proteins derived from antibodies of the invention that have been engineered to contain an antibody or antigen binding fragment as described herein. The peptide component can be fused to an anti-APLNR antibody or antigen binding fragment at either the N-terminus or C-terminus of the light or heavy chain of the antibody, with or without peptide linkers. The phrase fused to, as used herein, means (but is not limited to) a polypeptide formed by expressing a chimeric gene obtained by combining more than one sequence, typically by cloning one gene into an expression vector in frame with a second gene so that the two genes encode one continuous polypeptide. Recombinant cloning techniques such as polymerase chain reaction (PCR) and restriction endonuclease cloning are well known in the art. In addition to being produced by recombinant technology, portions of a polypeptide can be fused to each other by chemical reaction or other methods known in the art to produce non-standard polypeptides.

In some embodiments, the components or amino acids of the antibody fusion protein are separated by a linker (or spacer) peptide. Such peptide linkers are well known in the art (eg, polyglycine or Gly-Ser linkers) and typically allow one or both components of the antibody fusion protein to be correctly folded. The linker provides a flexible attachment site for the fusion protein component, allowing the two ends of the molecule to move independently, and may play an important role in maintaining the respective functions of each of the two groups. Therefore, the junction site acts in some cases both as a linker, which joins two parts together, and as a spacer, which allows each of the two parts to form its own biological structure and not interfere with the other part. In addition, the junction site must create an epitope that will not be recognized by the subject's immune system as foreign, in other words, will not be considered immunogenic. The choice of linker can also influence the binding activity and therefore the biological activity of the fusion protein (see Huston et al., 1988, PNAS, 85: 16: 5879-83; Robinson & Bates, 1998, PNAS, 95 (11): 5929-34; and Arai et al., 2001, PEDS, 14 (8): 529-32; Chen, X. et al., 2013, Advanced Drug Delivery Reviews, 65: 1357-1369.). In one embodiment of the invention, the apelin peptide is linked to the C-terminus or N-terminus of the light chain or heavy chain of an antibody or antigen binding fragment thereof through one or more peptide linkers.

Antibodies of the present invention may function through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Complement-dependent cytotoxicity (CDC) refers to the lysis of antigen-expressing cells by an antibody of the invention in the presence of complement. Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcR) (eg, natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on the target cell and thereby leads to lysis of the target cell. CDC and ADCC can be measured using assays that are well known and available in the art (see, for example, US Pat. Nos. 5,500,362; and 5,821,337; and Clynes et al., 1998, Proc. Natl. Acad. Sci. (USA ), 95: 652-656). The antibody constant region is important for the antibody's ability to fix complement and mediate cell-dependent cytotoxicity. Thus, the antibody isotype can be selected based on whether

- 9 043390 is it possible for an antibody to mediate cytotoxicity.

In another aspect, an antibody can be engineered in its Fc domain to activate all, some, or none of the normal Fc effector functions without affecting the desired pharmacokinetic properties of the antibody. Therefore, antibodies with engineered Fc domains that alter Fc receptor binding may have reduced side effects. Thus, in one embodiment of the invention, the protein comprises a chimeric or otherwise modified Fc domain. As an example of a chimeric Fc domain, see WO 2014/121087 A1 published August 7, 2014, which is incorporated herein by reference in its entirety.

The term EC ₅₀ or EC ₅₀ as used herein refers to half the maximum effective concentration, which includes the concentration of the ligand that induces a response, eg, a cellular response, halfway between the baseline value and the maximum after a specified exposure time. The EC50 is essentially the concentration of a ligand at which 50% of its maximum effect is observed. Thus, with regard to cellular signaling, increased receptor activity is observed with a reduced EC ₅₀ value, i.e. half the maximum effective concentration (less ligand needed to produce a greater response).

As used herein, the term IC ₅₀ or IC ₅₀ refers to the half maximum inhibitory concentration of a cellular response. In other words, a measurement of the effectiveness of a particular fragment (eg, protein, compound or molecule) in inhibiting the biological or biochemical function of a receptor, where the assay quantifies the amount of such fragment required to inhibit a given biological process. Thus, with regard to cell signaling, higher inhibitory activity is observed with a reduced IC50 value.

Methods for treating or improving vascular eye diseases or disorders

This description includes methods of treating, preventing, or alleviating at least one symptom or sign of an ocular vascular disease or disorder in a subject. The methods of this aspect of the disclosure include administering a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist to a subject in need thereof. In some embodiments, the APLNR antagonist is administered subcutaneously, intravenously, or intravitreally. In embodiments of the invention, the APLNR antagonist is administered in combination with a VEGF antagonist. In some embodiments, the APLNR antagonist is administered intravitreally in combination with a VEGF antagonist. In some embodiments, the APLNR antagonist is administered as a single combination dosage formulation with the VEGF antagonist. In some embodiments, the APLNR antagonist is administered in combination with a VEGF antagonist, wherein the APLNR antagonist is administered intravenously and the VEGF antagonist is administered intravitreally. The VEGF antagonist can be administered before, after, or simultaneously with the APLNR antagonist.

This invention is based in part on Applicant's discovery that a combination of antagonism directed against the VEGF and apelin/APLNR pathways can advantageously influence unwanted pathological vascularization of the eye. In particular, the applicant has discovered that such combinations can be used to treat or prevent conditions such as diabetic retinopathy, including proliferative diabetic retinopathy, retinopathy of prematurity and age-related macular degeneration. The addition of an APLNR antagonist could improve the anti-angiogenic effects of VEGF antagonism when VEGF pathway antagonism was saturated.

As used herein, the terms treat, treat, or the like. means the relief of symptoms, the elimination of the causes of symptoms either temporarily or permanently, or to prevent or delay the onset of symptoms of a neovascular eye disease or disorder. In certain embodiments of the invention, these methods are useful for treating or improving at least one symptom or indication, including, but not limited to, retinal angiogenesis, neovascularization, vascular leakage, retinal thickening within 500 μm of the center of the fovea, hard yellow exudates within 500 µm of the center of the fovea with adjacent retinal thickening and at least 1 disc area of retinal thickening, any part of which is within 1 disc diameter of the center of the fovea, blurred vision, floaters, loss of contrast, double vision and possible vision loss. In the context of methods of treating a vascular ocular disease such as AMD or DME, the term means that from the start of treatment the subject exhibits an improvement of one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) letters on the Early Treatment Diabetic Retinopathy (EDTRS) visual acuity chart. In certain embodiments of the invention, this term means that, from the start of treatment, the subject is prevented from losing vision greater than or equal to 15 letters.

As used herein, the terms prevent, prevents, or the like. means preventing the development of a symptom, indication or complication of vascular eye disease. In the context of methods for treating a vascular ocular disease such as AMD or DME, the term means that moderate to severe vision loss is prevented in a subject upon initiation of treatment.

As used herein, the term vascular disease or eye disorder refers to

- 10 043390 refers to eye disease or disorders that affect the blood vessels of the eye. The diseases may be caused by abnormal angiogenesis (the formation of new blood vessels) or occlusion or blockage of blood vessels. As used herein, the term includes eye diseases or disorders associated with angiogenesis. The term includes, but is not limited to, an eye disease or disorder selected from the group consisting of diabetic retinopathy (including proliferative diabetic retinopathy), diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polypoidal choroidal vasculopathy, choroidal neovascularization (CNV), degenerative myopia (myopic CNV), neovascular glaucoma and retinopathy of prematurity. In some embodiments, the term neovascular ocular disease or disorder may be used interchangeably with angiogenesis-related ocular disease or disorder.

In certain embodiments of the invention, this disclosure includes methods for treating, preventing, or ameliorating at least one symptom or sign of an eye disease or disorder associated with angiogenesis in a subject, wherein the disease or disorder is selected from the group consisting of pathological neovascularization, diabetic retinopathy (including proliferative diabetic retinopathy), diabetic macular edema, age-related macular degeneration, retinal neovascularization, polypoidal choroidal vasculopathy, choroidal neovascularization (CNV), degenerative myopia (myopic CNV), neovascular retinal glaucoma, predisposition to glaucoma, and retinopathy of prematurity. In some embodiments, administration of an APLNR antagonist also promotes normal retinal revascularization, such as in oxygen-induced retinopathy (OIR).

Diabetic macular edema (DME) as used herein refers to a serious eye condition that affects people with diabetes (type 1 or type 2). Macular edema occurs when fluid from the blood vessels in the retina leaks into the macula, and deposits of fluid and protein accumulate on or under the eye's macula (the yellow central area of the retina), causing it to thicken and swell (edema). Swelling can distort a person's central vision because the macula is located near the center of the retina at the back of the eyeball. Primary symptoms of DME include, but are not limited to, blurred vision, spots before the eyes, loss of contrast, double vision, and possible vision loss. The pathology of DME is characterized by a breakdown of the blood-brain barrier, which normally prevents the movement of water in the retina, allowing fluid to accumulate in the retinal tissue, and the presence of retinal thickening. Currently, DME is diagnosed during an eye examination consisting of a visual acuity test that determines the smallest letters a person can read on a standardized chart, a dilated eye examination to check for signs of the disease, imaging tests such as optical coherence tomography (OCT) or fluorescence angiography (FA) and tonometry, a tool that measures the pressure inside the eye. The following tests are also performed to determine treatment: optical coherence tomography (OCT), fluorescein angiography, and stereo color fundus photography. DME can be broadly divided into two main categories: focal and diffuse. Focal DME is characterized by specific areas of isolated and distinct leaks in the macula with sufficient macular blood flow. Diffuse DME occurs as a result of leakage of the entire capillary bed surrounding the macula due to the breakdown of the internal blood-retina barrier. In addition to focal and diffuse, DME is also classified based on clinical examination findings for clinically significant macular edema (CSME), non-CSME, and CSME with central involvement (CSME-CI), which includes the fovea. This description includes methods of treating the above categories of DME.

Age-related macular degeneration (AMD), as used herein, refers to a serious eye condition in which the small central part of the retina, known as the macula, deteriorates. Wet AMD is characterized by the growth of abnormal blood vessels from the choroid under the macula. This is called choroidal neovascularization (CNV). These blood vessels allow blood and fluid to flow into the retina, causing distorted vision in which straight lines appear wavy, as well as blind spots and loss of central vision. These abnormal blood vessels eventually become scarred, leading to permanent loss of central vision. Symptoms of AMD include dark, blurry areas in the center of vision; and decreased or altered color perception. AMD can be detected during a routine eye examination. One of the most common early signs of macular degeneration is the presence of drusen - tiny yellow deposits under the retina - or clumping of pigment.

Retinopathy of prematurity (ROP), as used herein, also known as retrolental fibroplasia and Terry syndrome, refers to a condition affecting preterm, low birth weight, early gestational age infants. Retinopathy of prematurity occurs when the development of normal retinal blood vessels is interrupted by birth before full gestation, resulting in abnormal retinal blood vessel development. If the condition

- 11 043390 progresses, the growth of scar tissue can lead to retinal detachment and deterioration of vision or loss of vision. The pathogenesis of ROP involves two distinct phases: the vasoobliterative phase and the vasoproliferative phase. Normal retinal vascular growth is delayed in the first phase as a consequence of exposure to the hyperoxic environment, whereas the second phase involves a rapid increase in neovascularization followed by retinal detachment. Ablation of the avascular retina using cyrotherapy or laser photocoagulation is considered as the primary treatment for ROP, and anti-VEGF therapies (eg, anti-VEGF antibodies) are being studied. Despite these treatment options, the condition remains a leading cause of vision impairment throughout life, with incidence rates remaining relatively constant for over twenty years.

Diabetic retinopathy (DR), as used herein, is a chronic, progressive disease of the retinal microvasculature associated with prolonged hyperglycemia. DR is the most common microvascular complication of diabetes and is a growing global problem as the prevalence of diabetes continues to rise worldwide. Proliferative DR (PDR), as used herein, is a sight-threatening complication of DR and is characterized by the development of abnormal new vessels in the retina, optic nerve head, or anterior segment of the eye. PDR is a late stage of DR that is caused by hypoxia and the expression of proangiogenic growth factors that stimulate the aberrant formation of new blood vessels in the retina that protrude into the preretinal space. Retinal neovascularization can cause severe vision loss when it results in vitreous hemorrhage or tractional retinal detachment. Laser photocoagulation has been the standard of care for the treatment of PDR for many years, with newer treatments including intraocular treatment with anti-VEGF (eg, anti-VEGF antibodies) and steroid agents, as well as vitreoretinal surgery. Despite these therapeutic advances, there remain unmet needs for treatment, particularly noninvasive, nondestructive, and longer lasting treatment options.

As used herein, the expression subject in need means a human or non-human mammal that exhibits one or more symptoms or signs, and/or has been diagnosed with an eye disease or angiogenesis-related disorder. The term subject in need thereof may also include, for example, subjects who, prior to treatment, exhibit (or have demonstrated) one or more signs of neovascular ocular disease, such as, for example, retinal angiogenesis, neovascularization, vascular leakage, retinal thickening within 500 µm from the center of the fovea, hard yellow exudates within 500 µm of the center of the fovea with adjacent retinal thickening and at least 1 disc area of retinal thickening, any part of which is within 1 disc diameter of the center of the fovea, blurred vision, front floaters eyes, loss of contrast, double vision and possible vision loss.

As used herein, a subject in need thereof also includes a human or non-human mammal that has a vascular disease or ocular disorder selected from the group consisting of diabetic retinopathy (including proliferative diabetic retinopathy), diabetic macular edema, age-related macular degeneration, neovascularization retinal vein occlusion, central retinal vein occlusion, branched retinal vein occlusion, polypoidal choroidal vasculopathy, choroidal neovascularization (CNV), degenerative myopia (myopic CNV), neovascular glaucoma and retinopathy of prematurity.

As used herein, the subject in need thereof may include a subset of the population that is more susceptible to DME or AMD, or may exhibit elevated levels of a DME-related or AMD-related biomarker, or a ROP- or PDR-related biomarker. For example, a subject in need thereof may include a subject who has had diabetes for more than 10 years, or a subject who frequently has high blood sugar levels or high fasting blood glucose levels. In some embodiments, the term subject in need thereof includes a subject who has been diagnosed or diagnosed with diabetes prior to or during administration of the APLNR antagonist and/or VEGF antagonist. In some embodiments, the term subject in need thereof includes a subject who was over 50 years of age before or at the time of administration of the APLNR antagonist and/or VEGF antagonist. In some embodiments, the term subject in need includes subjects who are smokers, or subjects with high blood pressure or high cholesterol.

This description includes methods of treating, preventing, or reducing the severity of vascular disease of the eye, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist to a subject in need thereof, wherein the pharmaceutical composition is administered to the subject in multiple doses, for example, as part of a specific therapeutic dosing regimen. For example, a therapeutic dosing regimen may include administering to a subject multiple doses of the pharmaceutical composition at a frequency of about once per day, once every two days, once every three days, once every four days, once every five days, once every six days , once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every four months or less. In certain embodiments of the invention, a therapeutic dosing regimen may include administering to a subject multiple doses of the pharmaceutical composition at a frequency of once daily or twice daily or more.

This disclosure also includes methods for inhibiting, reducing, or suppressing vascular leakage in a subject. In some embodiments, the methods in accordance with this aspect of the disclosure include administering to a subject one or more doses of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist to reduce or inhibit vascular fluid leakage in the eye of the subject. In some embodiments, vascular fluid leak is inhibited for more than 3 weeks, more than 4 weeks, more than 8 weeks, or more than 10 weeks compared to a subject administered the VEGF antagonist alone.

The methods herein, in accordance with some embodiments of the invention, comprise administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist. In some embodiments, an APLNR antagonist can be administered in combination with therapy, including laser treatment, to stop leakage into the macula. As used herein, in combination with means that the pharmaceutical composition containing an APLNR antagonist is administered to a subject simultaneously with, immediately before, or immediately after administration of the VEGF antagonist. As used herein, in combination with means that the pharmaceutical composition containing an APLNR antagonist is administered to a subject simultaneously with, immediately before, or immediately after administration of the VEGF antagonist. In certain embodiments of the invention, the VEGF antagonist is co-administered with an APLNR antagonist. In a related embodiment, this disclosure includes methods including administering a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist to a subject to provide greater therapeutic effect or synergistic effect compared to administering a VEGF antagonist alone. The subject may be on a therapeutic regimen of a VEGF antagonist via intravitreal administration. In some embodiments, an APLNR antagonist is added to this therapeutic regimen, in which one or more intravitreal injections of the VEGF antagonist may be reduced or the duration between successive intravitreal injections may be increased.

The methods herein are useful for the treatment or prevention of vascular ocular disorders in subjects who have been diagnosed with or are at risk of having an ocular vascular disorder. Typically, the methods herein demonstrate effectiveness within 36 weeks of starting the treatment regimen (with the initial dose administered at week 0), for example, by the end of week 6, by the end of week 12, by the end of week 18, by the end of week 24, and etc. In the context of treatments for angiogenic ocular disorders such as AMD and DME, efficacy means that the subject has experienced a loss of 10 or fewer letters on the Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart since the start of treatment. In some embodiments, effectiveness means an increase of one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) letters on the ETDRS chart since the start of treatment.

For example, a therapeutic dosing regimen may include administering to a subject multiple doses of the pharmaceutical composition at a frequency of about once per day, once every two days, once every three days, once every four days, once every five days, once every six days , once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every four months or less. In certain embodiments of the invention, a therapeutic dosing regimen may include administering to a subject multiple doses of the pharmaceutical composition at a frequency of once daily or twice daily or more.

APLNR antagonists.

This description includes methods of treating, preventing, or alleviating at least one symptom or sign of an ocular vascular disease or disorder in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition containing an APLNR antagonist to a subject in need thereof. APLNR antagonists include antibodies or antigen binding fragments thereof, small molecule inhibitors of the apelin receptor signaling pathway and peptide inhibitors of the apelin receptor signaling pathway. Illustrative small molecule inhibitors or antagonists of APLNR can be found in US Patent Publications No. US 2014000518 and US 20150125459 and International Patent Publications Nos. WO 2004081198 and WO 2015140296. Illustrative peptide inhibitors or antagonists of APLNR can be found in US Patent Nos. 9593153 and 7736646 and international patent publication WO 2004081198 Other APLNR antagonists include MM54, MM07, N-alpha-acetyl-nona-D-arginine amide (ALX40-4C), ML221, apelin-13 mutant (F13A), 4-oxo-6-((pyrimidin-2-ylthio )methyl)-4H-pyran-3-yl-4-nitrobenzoate (M1221) and E339-3D6.

APLNR antagonists include antibodies or antigen-binding fragments thereof selected from

- 13 043390 group of antibodies described in international patent publication No. WO 2015077491, published on May 5, 2015, which is specifically included in this document in its entirety and in the patent

US No. 9493554. In some embodiments, the antibodies are

H2aM9232N, H4H9232N or H1M9207N. In one embodiment, the anti-APLNR antibody is H4H9232N.

APLNR antagonists include antibodies or antigen binding fragments thereof selected from the group consisting of a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 13, or a substantially similar sequence thereof having at least at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

APLNR antagonists include antibodies or antigen binding fragments thereof containing a light chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 18, or a substantially similar sequence thereof having at least 90% similarity, at least 95%, at least 98%, or at least 99% sequence identity.

In certain embodiments, the antibody or antigen binding fragment thereof comprises heavy and light chain CDR sequences encoded by nucleic acid sequences SEQ ID NOs: 1 and 6 (e.g., H1M9207N) and SEQ ID NOs: 12 and 17 (e.g., H2aM9232N or H4H9232N) .

Certain non-limiting, illustrative antibodies and antigen binding fragments are described to contain HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having amino acid sequences selected from the group consisting of SEQ ID NO: 3-4-5-8-9-10 (for example, H1M9207N); and SEQ ID NO: 14-15-16-19-20-21 (for example, H2aM9232N or H4H9232N). Antibodies designated H2aM9232N and H4H9232N share the same human variable regions (HCVR and LCVR). H2aM9232N contains the mouse IgG2a heavy chain constant region, while H4H9232N contains the human IgG4 heavy chain constant region.

APLNR antagonists include antibodies or antigen binding fragments thereof containing a pair of HCVR and LCVR sequences (HCVR/LCVR) selected from the group consisting of SEQ ID NOs: 2/7 and 13/18.

APLNR antagonists include antibodies or antigen binding fragments thereof containing a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 16, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 21, or a substantially similar sequence thereof, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

In some embodiments, APLNR antagonists include antibodies or antigen binding fragments thereof comprising an HCDR3/LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NOs: 5/10 and 16/21.

APLNR antagonists include antibodies or antigen binding fragments thereof containing a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 14, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; heavy chain CDR2 domain (HCDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 15, or a substantially similar sequence thereof, having at least 90%, at least 95%, at least 98 % or at least 99% sequence identity; light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 19, or a substantially similar sequence thereof, having at least 90%, at least 95%, at least 98 % or at least 99% sequence identity; and a light chain CDR2 (LCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 20, or a substantially similar sequence thereof, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

Some non-limiting exemplary anti-APLNR antibodies include an antibody or antigen binding fragment of an antibody that specifically binds APLNR, wherein the antibody or antigen binding fragment comprises heavy and light chain CDR domains contained in a heavy and light chain variable region (HCVR/LCVR) selected from the group , consisting of SEQ ID NO: 2/7 and 13/18.

Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in said HCVR and/or LCVR amino acid sequences described herein. Exemplary conventions that may be used to define CDR boundaries include, for example, the Kabat definition, the Hotia definition, and the AtM definition. In general, the Kabat definition is based on sequence variability, the Hotia definition is based on the position of structural loop regions, and the AtM definition is a compromise between the Kabat and Hotia approaches. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948; Martin et al., 1989, Proc. Natl. Acad. Sci., USA, 86: 9268-9272. Public databases are also available to identify CDR sequences in an antibody.

This description relates to antibodies or antigen binding fragments thereof containing a heavy chain (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 13, or a substantially similar sequence thereof having at least 90%, according to at least 95%, at least 98%, or at least 99% sequence identity.

This description also relates to an antibody or antigen binding fragment of an antibody containing a light chain (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 18, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

This invention also relates to an antibody or antigen-binding fragment thereof containing a pair of amino acid sequences HC and LC (HC/LC).

In certain embodiments, the antibody or antigen binding fragment thereof comprises heavy and light chain CDR sequences encoded by antibody nucleic acid sequences H1M9207N or H2aM9232N (or H4H9232N).

This description includes anti-APLNR antibodies having a modified glycosylation pattern. In some applications, a modification to remove unwanted glycosylation sites, or an antibody that lacks the fucose moiety present in the oligosaccharide chain, may be useful, for example, to enhance the function of antibody-dependent cellular cytotoxicity (ADCC) (see Shield et al., 2002, JBC, 277:26733). In other applications, galactosylation can be modified to modify complement dependent cytotoxicity (CDC).

For example, this disclosure includes APLNR antagonists that block or inhibit apelin-mediated signaling in cells expressing human APLNR with an IC ₅₀ of less than about 20 nM, less than about 10 nM, less than about 2 nM, less than about 1 nM , less than about

900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about

500 pM, less than about 400 pM, less than about 350 pM, less than about 300 pM, less than about

250 pM, less than about 200 pM, less than about 150 pM, less than about 100 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, or less than about 10 pM, as measured in a blocking or inhibition bioassay on cells, for example, using the assay format as defined in Examples 5, 8 , 9 or 11 WO 2015/077491 or a substantially similar analysis.

This disclosure includes APLNR antagonists that inhibit the APLNR-mediated pERK ratio in the presence of apelin with an IC ₅₀ of less than about 50 nM, less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less less than about 5 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, or less than about 300 pM as measured in the APLNR-induced pERK assay.

In other embodiments, however, certain APLNR antagonists of the present invention, while capable of inhibiting or attenuating APLNR-mediated signaling, do not or only partially block the interaction of APLNR and apelin. Such antibodies and antigen binding fragments thereof may be referred to herein as indirect blockers. Without being bound by theory, indirect characterization blockers are believed to function by binding to APLNR at an epitope that does not overlap or partially overlaps with the N-terminal ligand binding domain of APLNR but still interferes with APLNR-mediated signaling without directly blocking the APLNR/apelin interaction .

This disclosure includes APLNR antagonists that bind soluble APLNR molecules with high affinity and/or specificity. For example, this disclosure includes antibodies and antigen-binding antibody fragments that bind APLNR with a binding coefficient greater than about 20, as measured by fluorescence-activated cell sorting (FACS) analysis, for example, using the assay format as defined in WO Example 4 2015/077491. In some embodiments, the antibodies or antigen-binding fragments of the invention bind APLNR with a binding coefficient of greater than about 15, greater than about

- 15 043390

20, more than about 100, more than about 200, more than about 300, more than about 400, less than about 500, more than about 1000, more than about 1500, or more than about 2000, as measured, for example, by FACS or a substantially similar analysis.

This disclosure also includes anti-APLNR antibodies and antigen-binding fragments thereof that specifically bind to APLNR with a dissociative half-life (?/ ₂ ) greater than about 10 minutes, as measured by surface plasmon resonance at 25 or 37°C, for example, using the well-known BIAcore™ analysis format or a substantially similar analysis.

Antibodies of this invention may have one or more of the above biological characteristics or any combinations thereof. Other biological characteristics of the antibodies herein will become apparent to one skilled in the art from a review of this disclosure, including the worked examples provided herein.

Anti-APLNR antibodies may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily identified by comparing the amino acid sequences described herein with germline sequences available, for example, from public antibody databases. This description includes antibodies and antigen binding fragments thereof that are derived from any of the amino acid sequences described herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated to the corresponding residue(s) ) the germline sequence from which the antibody originated, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as germline mutations). One of skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can readily produce numerous antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In certain embodiments, all framework residues and/or CDR residues in the VH and/or _VL domains are mutated back to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework residues and/or CDR residues are mutated to the corresponding residue(s) of a different germline sequence (ie, a germline sequence that is different from the germline sequence from which the antibody was originally derived). In addition, antibodies herein may contain any combination of two or more germline mutations in framework regions and/or CDR regions, for example, wherein certain individual residues are mutated to the corresponding residue of a certain germline sequence, while certain other residues that differ from the original germline sequence are retained or mutated to the corresponding residue of another germline sequence. Once produced, antibodies and antigen binding fragments that contain one or more germline mutations can be readily screened for one or more desired properties, such as increased binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (depending on circumstances), weakened immunogenicity, etc. Antibodies and antigen binding fragments obtained using this general approach are included herein.

This disclosure also includes anti-APLNR antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences described herein having one or more conservative substitutions. For example, this description includes anti-APLNR antibodies having amino acid sequences HCVR, LCVR and/or CDR, for example, with 10 or less, 8 or less, 6 or less, 4 or less, and other conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences described herein.

The term epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in a variable region of an antibody molecule known as a paratope. One antigen can have more than one epitope. Thus, different antibodies can bind to different regions on the antigen and can have different biological effects. Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, the epitope may include saccharide moieties, phosphoryl groups, or sulfonyl groups on the antigen.

The term significant identity or substantially identical, when referring to a nucleic acid or fragment thereof, indicates that when optimally aligned with corresponding nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is at least about 95% nucleotide sequence identity and more preferably, at least about 96, 97, 98, or 99% of the nucleotide bases, as measured by any well-known sequence identity algorithm such as FASTA, BLAST, or GAP, as described below. A nucleic acid molecule substantially identical to a reference nucleic acid molecule in certain cases encodes a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

When applied to polypeptides, the term significant similarity or substantially similar means that two peptide sequences, when optimally aligned, for example using the GAP or BESTFIT programs using default gap opening penalties, have a sequence identity of at least 95%, even more preferably at least 98 or 99%. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). Typically, a conservative amino acid substitution will not essentially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson, 1994, Methods Mol. Biol., 24: 307-331, incorporated herein by reference. Examples of amino acid groups that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine;

(2) aliphatic hydroxyl side chains: serine and threonine;

(3) amide-containing side chains: asparagine and glutamine;

(4) aromatic side chains: phenylalanine, tyrosine and tryptophan;

(5) main side chains: lysine, arginine and histidine;

(6) acidic side chains: aspartate and glutamate; and (7) sulfur-containing side chains - cysteine and methionine.

Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagyne glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix described in Gonnet et al., 1992, Science, 256: 1443-1445, incorporated herein by reference. A moderately conservative substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software finds similar sequences using similarity measurements assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit that can be used with default parameters to determine sequence homology or sequence identity between related polypeptides, such as homologous polypeptides from different species or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, programs in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignment identity and percentage sequence identity of the regions of best overlap between the target sequence and the search sequence (Pearson (1994), supra). Another preferred algorithm when comparing a sequence as described herein to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al., 1990, J. Mol. Biol., 215: 403-410; and Altschul et al., 1997, Nucleic. Acids. Res., 25: 3389-402, each of which is incorporated herein by reference.

This disclosure further includes anti-APLNR antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (eg, H1M9207N and H2aM9232N and H4H9232N). Likewise, this description also includes

- 17043390 anti-APLNR antibodies that compete for binding to APLNR with any of the specific exemplary antibodies described herein (eg, H1M9207N and H2aM9232N and H4H9232N).

Whether an antibody binds to or competes for binding to the same epitope as a reference anti-APLNR antibody can be readily determined using standard methods known in the art and exemplified herein. For example, to determine whether a test antibody binds to the same epitope as a reference anti-APLNR antibody of the invention, the reference antibody is allowed to bind to the APLNR protein. The ability of the test antibody to bind to the APLNR molecule is then determined. If the test antibody is able to bind to APLNR after saturating binding to the reference anti-APLNR antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-APLNR antibody. On the other hand, if the test antibody is unable to bind to the APLNR molecule after saturating binding to the reference anti-APLNR antibody, then the test antibody may bind to the same epitope as the epitope associated with the reference anti-APLNR antibody of the description. An additional standard experiment (e.g., peptide mutation and binding assay) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody or whether steric blocking (or other phenomenon) is responsible. for lack of observed binding. Experiments of this type can be performed using ELISA, RIA (radioimmunoassay), BIAcore™, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. In accordance with some embodiments of the present invention, two antibodies bind to the same (or overlapping) epitope if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other by at least at least 50%, but preferably 75, 90% or even 99%, as measured in a competitive binding assay (see, for example, Junghans et al., 1990, Cancer Res., 50: 1495-1502). Alternatively, two antibodies bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies are considered to have overlapping epitopes if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other.

To determine whether an antibody competes for binding (or cross-competes for binding) with a reference anti-APLNR antibody, the binding procedure described above is performed in two directions: in the first direction, the reference antibody is bound to the APLNR protein under saturation conditions, followed by a binding assay of the test antibody. antibodies with the APLNR molecule. In the second direction, the antibody of interest is bound to the APLNR molecule under saturation conditions, followed by determination of the binding of the reference antibody to the APLNR molecule. If in both directions only the first (saturating) antibody is able to bind to the APLNR molecule, then it is concluded that the test antibody and the reference antibody are competing for binding to APLNR. As one skilled in the art will appreciate, an antibody that competes for binding to a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

In certain embodiments, the anti-APLNR antibodies herein are human antibodies. The term human antibody as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDR and, in particular , CDR3. However, the term human antibody as used herein is not intended to include antibodies in which CDR sequences derived from the germ line of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In various embodiments, the antibodies discussed herein are human IgG heavy chain constant region antibodies. In some cases, the antibodies have a heavy chain constant region of the human IgG1 or IgG4 isotype.

Antibodies of the invention may, in some embodiments, be recombinant human antibodies. The term recombinant human antibody, as used herein, is intended to include all human antibodies thereof that are produced, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below ), antibodies isolated from a recombinant, combinatorial library of human antibodies (described further below), antibodies isolated from an animal (for example, a mouse) that is transgenic for human immunoglobulin genes (see, for example, Taylor et al., 1992, Nucl. Acids Res., 20: 6287-6295), or antibodies produced, expressed, created or isolated by any other method that involves splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using an animal transgenic for human Ig sequences, somatic mutagenesis in vivo) and thus the amino acid sequences of the VH and _VL regions of the recombinant antibodies are sequences that , although derived from and related to human germline VH and _VL sequences, cannot naturally exist in the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms, which are related to the heterogeneity of the hinge. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by a heavy chain disulfide bond. In the second form, the dimers are not linked through interchain disulfide bonds, and a molecule of about 75-80 kDa is formed, consisting of a covalently linked light and heavy chain (half-antibody). These forms are extremely difficult to separate, even after affinity purification.

The frequency of occurrence of the second form in different intact IgG isotypes is due to, but not limited to, structural differences associated with the isotype of the antibody hinge region. A single amino acid substitution in the hinge region of human IgG4 can significantly reduce the occurrence of the second form (Angal et al., 1993, Molecular Immunology, 30: 105) to levels typically observed using the human IgG1 hinge. This description covers antibodies having one or more mutations in the hinge, CH2 or CH3 region, which may be desirable, for example, in production, to improve the yield of the desired form of the antibody.

Antibodies may be described as isolated antibodies. An isolated antibody, as used herein, means an antibody that has been identified and separated and/or extracted from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of the body, or from a tissue or cell in which the antibody naturally exists or is produced is an isolated antibody for purposes of this description. The isolated antibody also includes the antibody in situ in the recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In certain embodiments of the invention, the isolated antibody may be substantially free of other cellular material and/or chemicals.

This description includes neutralizing and/or blocking anti-APLNR antibodies. A neutralizing or blocking antibody, as used herein, is intended to mean an antibody whose binding to APLNR (i) interferes with the interaction between APLNR or a fragment of APLNR and a component of the APLNR receptor (eg, apelin peptide, etc.); and/or (ii) results in inhibition of at least one biological function of APLNR.

Inhibition caused by an APLNR neutralizing or blocking antibody does not need to be complete if it can be detected by an appropriate assay.

VEGF antagonists.

As used herein, the term VEGF antagonist is any agent that binds or interacts with VEGF, inhibits the binding of VEGF to its receptors (VEGFR1 and VEGFR2), and/or inhibits the biological signaling and activity of VEGF. VEGF antagonists include molecules that interfere with the interaction between VEGF and the natural VEGF receptor, for example, molecules that bind to VEGF or the VEGF receptor and prevent or otherwise interfere with the interaction between VEGF and the VEGF receptor. Specific exemplary VEGF antagonists include anti-VEGF antibodies (eg, ranibizumab [LUCENTIS®]), anti-VEGF receptor antibodies (eg, anti-VEGFR1 antibodies, anti-VEGFR2 antibodies, etc.), small molecule VEGF inhibitors (eg, sunitinib ) and VEGF receptor chimeric molecules or VEGF inhibitory fusion proteins (also referred to herein as VEGF decoys), such as aflibercept and ziv-aflibercept. Other examples of VEGF traps are ALT-L9, M710, FYB203 and CHS-2020. Additional examples of VEGF traps can be found in US Pat. Nos. 7,070,959, 7,306,799, 7,374,757, 7,374,758, 7,531,173, 7,608,261, 5,952,199, 6,100,071, 6,383,486, 6,897,294, and 7,771,721, which are expressly incorporated herein by reference. . Additional VEGF inhibitors and/or antagonists include the small molecule inhibitors pazopanib, sorafenib, axitinib, ponatinib, regorafenib, cabozantinib, vandetanib, cabozantinib, and lenvatinib; and VEGF inhibitory antibodies: bevacizumab and ramucirumab or their biosimilar molecules.

VEGF receptor chimeric molecules include chimeric polypeptides that

- 19 043390 contain two or more immunoglobulin (Ig)-like domains of the VEGF receptor, such as VEGFR1 (also called Flt1) and/or VEGFR2 (also called Flk1 or KDR), and may also contain a multimerizing domain (for example, an Fc domain, which facilitates multimerization [eg, dimerization] of two or more chimeric polypeptides). An exemplary VEGF receptor chimeric molecule is the molecule designated VEGFR1R2-FcΔC1(a) (also known as aflibercept; marketed under the product name EYLEA®). In some embodiments, aflibircept contains an amino acid sequence represented as

MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSP

NITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHR QTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLK TQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCP APELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQIDNO: 23).

The amount of VEGF antagonist contained in the pharmaceutical compositions of this invention may vary depending on the specific properties desired for the compositions, as well as the specific circumstances and purposes for which the compositions are intended for use. In certain embodiments of the invention, the pharmaceutical compositions are liquid formulations that may contain from 5±0.75 to 150±22.5 mg/ml of VEGF antagonist; from 10±1.5 to 100±15.0 mg/ml VEGF antagonist; from 20±3 to 80±12 mg/ml VEGF antagonist; from 30±4.5 to 70±10.5 mg/ml VEGF antagonist or 40±6.0 mg/ml VEGF antagonist. For example, the compositions of this invention may contain about 20 mg/ml; about 30 mg/ml; about 40 mg/ml; about 50 mg/ml; or about 60 mg/ml VEGF antagonist.

The methods of this invention include administering to a subject in need thereof a therapeutic composition containing a VEGF antagonist.

Therapeutic composition and administration.

This disclosure provides pharmaceutical compositions containing at least one APLNR antagonist, such as an anti-APLNR antibody or antigen-binding fragment thereof, that specifically binds to human APLNR. In accordance with some other embodiments of the invention, this invention relates to pharmaceutical compositions containing additional therapeutic agents. This disclosure further provides pharmaceutical preparations containing at least one VEGF antagonist, for example, for use in combination with a pharmaceutical composition containing at least one APLNR antagonist.

The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients and other agents that provide improved transport, delivery, tolerability and the like. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, emulsions such as oil in water or water in oil, emulsions based on carbowax (polyethylene glycols with different molecular weights), semi-solid gels and semi-solid mixtures containing carbowax. See also Powell et al., Compendium of excipients for parenteral formulations, PDA (1998), J. Pharm. Sci. Technol., 52:238-311.

The dose of an APLNR antagonist, such as an anti-APLNR antibody or an antigen-binding fragment thereof that specifically binds to human APLNR, administered to a subject may vary depending on the age and size of the subject, the target disease, conditions, route of administration, and the like. The preferred dose is usually based on body weight or body surface area. When an APLNR antagonist is used to treat a condition or disease, it may be advantageous to administer intravenously the antibody of the present invention, typically in a single dose of about 0.01 mg/kg to about 50 mg/kg body weight. In other cases, it may be advantageous to administer the APLNR antagonist intravenously, for example, at a concentration of about 0.01 mg/kg to about 50 mg/kg body weight. Depending on the severity of the pathological condition, the frequency and duration of treatment can be adjusted. Effective dosages and schedules of administration of an APLNR antagonist, such as an anti-APLNR antibody, can be determined experimentally; for example, the subject's progress can be monitored through periodic assessment and the dose adjusted accordingly. In addition, interspecies dose scaling can be accomplished using methods well known in the art (eg, Mordenti et al., 1991, Pharmaceut. Res., 8: 1351).

The dose of the VEGF antagonist may vary depending on the age and size of the subject, the target disease, conditions, route of administration, and the like. The preferred dose is usually based on body weight or body surface area. When a VEGF antagonist is used for le

- 20 043390 treatment of a condition or disease, it may be advantageous to administer intravenously an antibody of this invention, typically in a single dose of about 0.01 mg/kg to about 50 mg/kg body weight. In other cases, it may be advantageous to administer the VEGF antagonist intravenously, for example, at a concentration of about 0.01 mg/kg to about 50 mg/kg body weight. Depending on the severity of the pathological condition, the frequency and duration of treatment can be adjusted. Effective doses and schedules of administration of a VEGF antagonist can be determined empirically; for example, the subject's progress can be monitored through periodic assessment and the dose adjusted accordingly. Various delivery systems are known and can be used to administer the pharmaceutical composition herein, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J Biol Chem 262: 4429-4432). Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, intravitreal, subcutaneous, intranasal, epidural, and oral routes of administration. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous membranes (for example, oral mucosa, rectal and small intestinal mucosa, etc.) , and can also be administered together with other biologically active agents. Administration may be systemic or local.

This description includes methods that include administering an APLNR antagonist to a subject, wherein the APLNR antagonist is contained in a pharmaceutical composition. In certain embodiments of the invention, the pharmaceutical composition further comprises a VEGF antagonist. In alternative embodiments, the APLNR antagonist and the VEGF antagonist may each be in their own separate pharmaceutical formulation. The pharmaceutical compositions of the invention may be formulated with suitable carriers, excipients and other agents that provide suitable transport, delivery, tolerability and the like. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water or water emulsions. in oil, emulsions based on carbowax (polyethylene glycols with different molecular weights), semi-solid gels and semi-solid mixtures containing carbowax. See also Powell et al., Compendium of excipients for parenteral formulations, PDA (1998), J. Pharm. Sci. Technol., 52: 238-311.

As used herein, the expression pharmaceutical composition means a combination of at least one active ingredient (e.g., small molecule, macromolecule, compound, etc., which is capable of producing a biological effect in a human or non-human animal), and at least one an inactive ingredient that, in combination with an active ingredient or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal. The term formulation as used herein means a pharmaceutical formulation unless specifically stated otherwise.

The amount of APLNR antagonist, such as an anti-APLNR antibody or antigen-binding fragment thereof, contained in the pharmaceutical compositions of this invention may vary depending on the specific properties desired for the compositions, as well as the specific circumstances and purposes for which the compositions are intended for use. In certain embodiments of the invention, the pharmaceutical preparations are liquid preparations that may contain from 5±0.75 to 150±22.5 mg/ml of antibody; from 7.5±1.125 to 140±21 mg/ml antibody; from 10±1.5 to 130±19.5 mg/ml antibody; 10±1.5 mg/ml antibody; 20±3 mg/ml antibody; 60±9 mg/ml antibody; or 120±18 mg/ml antibody. For example, the compositions of this invention may contain about 10 mg/ml; about 20 mg/ml; about 40 mg/ml; about 60 mg/ml; about 80 mg/ml; about 100 mg/ml; about 120 mg/ml; or about 140 mg/ml of an antibody or antigen-binding fragment thereof that specifically binds to human APLNR.

In some embodiments, the pharmaceutical compositions are liquid preparations that may contain from 5±0.75 to 100±15 mg/ml of VEGF antagonist. For example, the compositions of this invention may contain about 5 mg/ml; about 10 mg/ml; about 15 mg/ml; about 20 mg/ml; about 25 mg/ml; about 30 mg/ml; about 35 mg/ml; about 40 mg/ml; about 50 mg/ml; about 60 mg/ml; about 70 mg/ml; about 80 mg/ml; about 90 mg/ml; or about 100 mg/ml of a VEGF antagonist such as aflibercept.

In certain embodiments of the invention, the pharmaceutical compositions are stable liquid co-formulations containing from about 5 mg/ml to about 150 mg/ml of an APLNR antagonist and from about 5 to 100 mg/ml of a VEGF antagonist.

Pharmaceutical preparations herein contain one or more excipients. Ter- 21 043390 min excipient as used herein means any non-therapeutic agent added to a formulation to provide the desired consistency, viscosity or stabilizing effect.

Exemplary compositions containing a VEGF antagonist that can be used in the context of this description are described, for example, in US patent No. 7531173 and 7608261. Exemplary pharmaceutical compositions containing an APLNR antagonist that can be used in the context of this description are described, for example, in the publication of the application US Patent No. 20130186797. Exemplary pharmaceutical compositions containing an APLNR antagonist that may be used in the context of this disclosure are described, for example, in International Patent Publication No. WO 2016085750 and US Patent Application Publication No. US 20110027286.

Combined types of therapy.

The methods herein, in accordance with some embodiments of the invention, comprise administering to a subject an APLNR antagonist in combination with a VEGF antagonist. As used herein, the expression in combination with means that the VEGF antagonist is administered before, after or simultaneously with a pharmaceutical composition containing an APLNR antagonist. The term in combination with also includes sequential or concomitant administration of an APLNR antagonist and a VEGF antagonist. For example, when administered before, a pharmaceutical composition containing an APLNR antagonist may be administered more than 72 hours, about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes before administration of the pharmaceutical composition containing the VEGF antagonist. When administered after, the pharmaceutical composition containing the APLNR antagonist can be administered over about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, or more than 72 hours after administration of a pharmaceutical composition containing a VEGF antagonist. Coadministration with a pharmaceutical composition containing an APLNR antagonist means that the VEGF antagonist is administered to a subject in a separate dosage form within less than 5 minutes (before, after, or at the same time) of administration of a pharmaceutical composition containing an APLNR antagonist, or is administered to a subject at in the form of a single combined dosage form containing an APLNR antagonist and a VEGF antagonist.

Combination therapy may include an APLNR antagonist and a VEGF antagonist (eg, aflibercept, a VEGF decoy, see, for example, US Pat. No. 7,087,411 (also referred to herein as a VEGF inhibitory fusion protein), an anti-VEGF antibody (eg, ranibizumab, etc.) ), small molecule VEGF receptor kinase inhibitor (for example, sunitinib, sorafenib or pazopanib), etc.

The methods of this invention include administering an APLNR antagonist in combination with a VEGF antagonist for additive or synergistic activity to treat or ameliorate at least one symptom or sign of an eye disease or disorder selected from the group consisting of diabetic retinopathy (including proliferative diabetic retinopathy), diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polypoidal choroidal vasculopathy, choroidal neovascularization (CNV), degenerative myopia (myopic CNV), neovascular glaucoma and retinopathy of prematurity.

Containers and methods of administration.

Various delivery systems are known and can be used to administer the pharmaceutical composition herein, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J Biol Chem 262: 4429-4432). Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, intravitreal, subcutaneous, intranasal, epidural, and oral routes of administration. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous membranes (for example, oral mucosa, rectal and small intestinal mucosa, etc.) , and can also be administered together with other biologically active agents.

For the treatment of eye diseases, the pharmaceutical compositions of the invention can be administered, for example, as eye drops, subconjunctival injection, subconjunctival implant, intravitreal injection, intravitreal implant, sub-Tenon injection or sub-Tenon implant.

The pharmaceutical composition herein can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, in the case of subcutaneous delivery, the pen syringe easily finds applications in the delivery of the pharmaceutical composition according to this description. This syringe pen can be reusable or disposable. A reusable pen typically uses a replaceable cartridge that contains a pharmaceutical composition. Immediately after all of the pharmaceutical composition in the cartridge is administered and the cartridge becomes empty, the empty cartridge can be immediately discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen can then be reused. The disposable syringe pen does not have a replaceable cartridge. Preferably, the disposable pen comes filled with a pharmaceutical composition held in a reservoir within the product. Immediately after the reservoir is emptied of the pharmaceutical composition, the entire product is discarded.

In certain situations, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment of the invention, a pump may be used. In another embodiment of the invention, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment of the invention, the controlled release system can be placed in close proximity to the target of the composition, requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in Langer, 1990, Science, 249: 1527-1533.

Injectable forms may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable forms can be prepared by known methods. For example, injectable forms can be prepared, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof described above in a sterile aqueous or oily medium commonly used for injection. As the aqueous injection medium, there are, for example, saline solution, isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with a corresponding solubilizing agent such as alcohol (for example, ethanol), polyalcohol ( for example, propylene glycol, polyethylene glycol), nonionic surfactant [for example, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil], etc. The oily medium used is, for example, sesame oil, soybean oil etc., which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus obtained is preferably filled into a suitable ampoule.

Preferably, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms in a single dose suitable to match the dose of the active substances. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

The pharmaceutical compositions herein may be contained in any container suitable for storing or administering drugs and other therapeutic compositions. For example, pharmaceutical compositions may be contained in a sealed and sterilized plastic or glass container having a defined volume, such as a vial, ampoule, syringe, cartridge, bottle or intravenous bag. Various types of bottles can be used to contain the compositions herein, including, for example, clear and opaque (eg, amber) glass or plastic bottles. Likewise, any type of syringe can be used to contain or administer the pharmaceutical compositions herein.

The pharmaceutical compositions herein may be contained in normal tungsten syringes or low tungsten syringes. As will be appreciated by those skilled in the art, the process of making glass syringes typically involves the use of a hot tungsten rod which serves to pierce the glass, thereby creating an opening from which liquids can be drawn and expelled from the syringe. This process results in the deposition of trace amounts of tungsten on the inside surface of the syringe. Post-rinsing and other processing steps can be used to reduce the amount of tungsten in the syringe. As used herein, the term normal tungsten means that the syringe contains greater than or equal to 500 parts per billion (ppm) tungsten. The term low tungsten means that the syringe contains less than 500 ppb tungsten. For example, a low tungsten syringe herein may contain less than about 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90 , 80, 70, 60, 50, 40, 30, 20, 10 or less parts per billion tungsten.

Rubber pistons used in syringes and rubber stoppers used to seal the openings of vials may be coated to prevent contamination of the medicinal contents of the syringe or vial or to maintain their stability. Thus, the pharmaceutical compositions herein, in accordance with some embodiments of the invention, may be contained in a syringe that includes a coated plunger, or in a vial that is sealed with a coated rubber stopper. For example, the piston or plug may be coated with a fluorocarbon film. Examples of coated stoppers or plungers suitable for use with vials and syringes containing pharmaceutical compositions herein are mentioned, for example, in US Pat. No. 4,997,423; 5908686; 6286699; 6645635; and 7226554, the contents of which are incorporated herein by reference in their entirety. Specific illustrative coated rubber plugs and pistons that may be used in the context of this invention are commercially available under the trade name FluroTec®, available from West Pharmaceutical Services, Inc. (Lyonville, Pennsylvania). FluroTec® is an example of a fluorocarbon coating used to minimize or prevent drug product adhesion to a rubber surface.

In accordance with some embodiments of the present invention, pharmaceuticals may be contained in a low tungsten syringe that includes a fluorocarbon coated plunger.

The pharmaceutical compositions may be administered to a subject by parenteral routes, such as injection (eg, subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or transdermal, mucosal, nasal, pulmonary, or oral administration. Numerous reusable pens or auto-injection delivery devices can be used for subcutaneous delivery of the pharmaceutical compositions described herein. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75/25™ pen, syringe -HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Indiana), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lake, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pens or autoinjector delivery devices that are used for subcutaneous delivery of the pharmaceutical composition herein include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ ( Eli Lilly), SURECLICK™ auto-injector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ pen (Abbott Labs, Abbott Park, IL).

The use of a microinfuser for the delivery of pharmaceutical compositions as described herein is also discussed herein. As used herein, the term microinfuser means a subcutaneous delivery device designed to slowly administer large volumes (e.g., up to about 2.5 mL or more) of a therapeutic formulation over an extended period of time (e.g., about 10, 15, 20, 25, 30 or more minutes). See, for example, US Pat. No. 6,629,949; US Patent No. 6659982; and Meehan et al., J. Controlled Release, 46: 107-116 (1996). Microinfusers are particularly useful for delivering large doses of therapeutic proteins contained in high concentrations (eg, about 100, 125, 150, 175, 200 or more mg/ml) or viscous solutions.

In one embodiment of the invention, the pharmaceutical composition is administered through an IV drip such that the composition is reconstituted in an intravenous bag containing a physiologically acceptable solution. In one embodiment of the invention, the pharmaceutical composition is a compounded sterile preparation in an intravenous infusion bag such that a single dose of the drug product is diluted in 100, 250 ml (or other similar amount suitable for intravenous drip delivery) of physiological buffer (for example, 0. 9% saline). In some embodiments, the infusion bag is made of polyvinyl chloride (eg, VIAFLEX, Baxter, Deerfield, IL). In some embodiments, the infusion bag is made of polyolefin (EXCEL IV Bags, Braun Medical Inc., Bethlehem, PA).

Administration schemes.

This disclosure includes methods comprising administering to a subject a pharmaceutical composition containing an APLNR antagonist, such as an anti-APLNR antibody or antigen binding fragment thereof, at a dosing frequency of about four times per week, twice per week, once per week, once every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every eight weeks, every twelve weeks, or less frequently until a therapeutic response is achieved. In some embodiments, the methods include administering a pharmaceutical composition comprising an APLNR antagonist, such as an anti-APLNR antibody or an antigen binding fragment thereof, and a VEGF antagonist, at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks , every three weeks, every four weeks, every five weeks, every six weeks, every eight weeks, every nine weeks, every twelve weeks, or less frequently until a therapeutic response is achieved.

In accordance with some embodiments of this disclosure, multiple doses of an APLNR antagonist, such as an anti-APLNR antibody or an antigen-binding fragment thereof, and a VEGF antagonist may be administered to a subject over a period of time. Methods in accordance with this aspect of the disclosure include sequentially administering to a subject multiple doses of an APLNR antagonist, such as an anti-APLNR antibody or antigen binding fragment thereof, and a VEGF antagonist. As used herein, the term sequential administration means that each dose of an APLNR antagonist, such as an anti-APLNR antibody or antigen-binding fragment thereof, and a VEGF antagonist is administered to a subject at a different point in time, for example, on different days separated by a predetermined interval (for example, hours , days, weeks or months). This description includes methods

- 24 043390 which involve sequentially administering to a subject one initial dose of an APLNR antagonist, such as an anti-APLNR antibody or an antigen-binding fragment thereof, and a VEGF antagonist, followed by one or more secondary doses of an APLNR antagonist, such as an anti-APLNR antibody or an antigen-binding fragment thereof , and a VEGF antagonist, and optionally accompanied by one or more tertiary doses of an APLNR antagonist, such as an anti-APLNR antibody or antigen-binding fragment thereof, and a VEGF antagonist.

In accordance with some embodiments of this disclosure, multiple doses of a co-formulation comprising an APLNR antagonist, such as an anti-APLNR antibody or an antigen binding fragment thereof, and a VEGF antagonist may be administered to a subject over a period of time. Methods in accordance with this aspect of the disclosure include sequentially administering to a subject multiple doses of a co-formulation comprising an APLNR antagonist, such as an anti-APLNR antibody or antigen binding fragment thereof, and a VEGF antagonist. As used herein, the term sequential administration means that each dose of an APLNR antagonist in combination with a VEGF antagonist is administered to a subject at a different point in time, eg, on different days separated by a predetermined interval (eg, hours, days, weeks, or months). This disclosure includes methods that include sequentially administering to a subject one initial dose of a co-formulation comprising an APLNR antagonist and a VEGF antagonist, followed by one or more secondary doses of the co-formulation of APLNR and a VEGF antagonist, and optionally followed by one or more tertiary doses of the co-formulation an APLNR antagonist and a VEGF antagonist.

The terms initial dose, secondary doses and tertiary doses refer to the time sequence of administration. Thus, the starting dose is the dose that is administered at the beginning of the treatment regimen (also called the base dose); secondary doses are doses that are administered after the initial dose; and tertiary doses are doses that are administered after secondary doses. The initial, secondary and tertiary doses may contain the same amount of APLNR antagonist (or a combined formulation containing an APLNR antagonist and a VEGF antagonist), but generally may differ from each other in terms of frequency of administration. In some embodiments of the invention, however, the amount contained in the initial, secondary and/or tertiary doses differs from each other (eg, adjusted up or down as needed) during the course of treatment. In certain embodiments of the invention, one or more (eg, 1, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses followed by subsequent doses that are administered less frequently (eg, maintenance doses). For example, an APLNR antagonist (or a co-formulation containing an APLNR antagonist and a VEGF antagonist) can be administered to a subject with an ocular disease or disorder at a loading dose of about 6 mg followed by one or more maintenance doses.

In one illustrative embodiment of this disclosure, each secondary and/or tertiary dose is administered from 1 to 14 (e.g., 1, ₁ ^1/2 , 2, 21/2, 3, 31/2 _{, 4} , 41/2, 5, 51/2, 6, 61/2, 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/ 2, 14, 141/2 or more) weeks after the immediately preceding dose. The phrase immediately preceding dose, as used herein, means, in a sequence of multiple administrations, the dose of an APLNR antagonist (or a combination formulation containing an APLNR antagonist and a VEGF antagonist) that is administered to a subject before the administration of the very next dose in the sequence, with no intervening doses.

The methods of this aspect of the disclosure may include administering to the subject any number of secondary and/or tertiary doses of an anti-APLNR antagonist (or a combination formulation comprising an APLNR antagonist and a VEGF antagonist). For example, in certain embodiments of the invention, only one secondary dose is administered to the subject. In other embodiments, the subject is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses. Likewise, in certain embodiments of the invention, only one tertiary dose is administered to the subject. In other embodiments, the subject is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses.

In embodiments of the invention that include multiple secondary doses, each secondary dose can be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the subject 1-2 weeks after the immediately preceding dose. Likewise, in embodiments of the invention involving multiple tertiary doses, each tertiary dose can be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the subject 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which secondary and/or tertiary doses are administered to a subject may vary over the course of treatment. The frequency of administration may also be adjusted by the physician during the course of treatment depending on the needs of the individual subject after clinical evaluation.

This disclosure includes methods comprising sequentially administering an APLNR antagonist and a VEGF antagonist to a subject for the treatment of DME, AMD, ROP or PDR. In some embodiments, these methods include administering one or more doses of an APLNR antagonist followed by one or more doses of a VEGF antagonist. In certain embodiments of the invention,

- 25 043390 these methods involve administering one dose of a VEGF antagonist followed by one or more doses of an APLNR antagonist.

Examples

The following examples are provided to provide those skilled in the art with complete disclosure and description of how to make and use the methods and compositions herein, and are not intended to limit the scope of what the inventors consider their description to be. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example 1.

To evaluate the in vivo performance of selected anti-APLNR antibodies of the invention, their ability to block APLNR-mediated angiogenesis in the ocular vasculature was measured.

The retinal vascular development (RVD) model was used to evaluate the effect of an antagonistic anti-APLNR antibody on blood vessel sprouting in the normally developing retina in pups that had a mixed strain background (75% C57BL6 and 25% Sv129), and were homozygous for expressing human APLNR instead mouse APLNR (humanized APLNR mice).

Humanized (Hu) ApelinR mice were systemically (IP) administered 25 and 50 mg/kg anti-Apelin receptor antibody (aAR; H2aM9232N) on postnatal day (P)2. Reagents were masked and labeled as solution A and solution B to prevent experimenter bias. On postnatal day 5, tissue samples were collected and then fixed in PBS containing 4% paraformaldehyde. Fixed tissue samples (retinal endothelial cells) were washed with PBS and then stained with GS Lectin I (Vector Laboratories, #FL-1101) diluted 1:200 in 1x PBS containing 1% BSA in 0.25% TritonX 100 overnight at 25 °C to visualize the retinal vasculature. The next day, stained samples were washed several times with PBS, mounted flat on glass slides, and then coverslipped using Prolong Gold (Invitrogen, #P36930). Images were acquired at 20× magnification using an epifluorescence microscope (Nikon Eclipse 80). Retinal vascular areas were measured from the resulting images obtained in this analysis using an advanced version of Adobe Photoshop CS6. Only after retinal vasculature area measurements and statistical analyzes were completed were sample identifiers unmasked.

In fig. 1A and 1B show representative photomicrographs of mouse retinas treated systemically at P2 to P5 and a plot of calculated vascular area (images at 20x, quantification, and 40x; statistical analysis was performed using Student's T test).

Residual vascular area was significantly less in aApelinR retinas (23% at 25 mg/kg, p < 0.05) compared with untreated retinas. Selective inhibition of ApelinR through systemic injection slowed normal vascular sprouting in the developing retina of P5 pups. At a dose of 25 mg/kg, blocking ApelinR slightly inhibits vascular sprouting.

Example 2.

The subsequent experiment in the RVD model was carried out similarly to example 1 at the highest dose and analyzed using unmasked identifiers. Briefly, pups were administered i.p. 50 mg/kg Fc (control) or aAR. Fig. 2A and 2B are representative photomicrographs of mouse retinas treated systemically at P2 to P5 and a graph of the calculated vascular area. Residual vascular area was significantly less in aApelinR retinas (35% at 50 mg/kg, p < 0.005) compared with untreated retinas. Retinal endothelial cells were stained with GS Lectin I. Images were acquired at 20x (for quantification) and 40x. Statistical analysis was performed using Student's t test. Selective inhibition of ApelinR through systemic injection slowed normal vascular sprouting in the developing retina of P5 pups. By increasing the dose to 50 mg/kg, targeting ApelinR further delayed retinal vascular sprouting.

Example 3.

In a subsequent experiment in the RVD model (performed similarly to Example 1), Hu pups at P2 were dosed IP with 50 mg/kg Fc, aAR, or aflibercept or a combination (aAR and aflibercept) and collected at P5. Fig. 3A and 3B are representative photomicrographs of mouse retinas treated systemically at P2 to P5 and a graph of the calculated vascular area. In this single-blind study, 50 mg/kg aApelinR reduced vascular growth by 29.8% (p<0.0001) compared to Fc-treated controls. Retinal endothelial cells were stained with GS Lectin I. Images were acquired at 20x (for quantification) and 40x. Statistical analysis was performed using Student's t test. Selective inhibition of ApelinR through systemic injection slows normal vascular sprouting in the developing retina of P5 pups.

- 26 043390

Example 4.

In another RVD model experiment (performed similarly to Example 1), Hu pups at P4 were intravitreally (IVT) administered with 5 μg of Fc, αAR, or aflibercept or a combination (aAR and aflibercept) and collected at P6. Fig. 4A and 4B are representative photomicrographs of mouse retinas treated systemically at P2 to P5 and a graph of calculated vascular area. Residual vessel area was significantly less with the combination (aApelinR+aflibercept) (62% at 50 mg/kg, p<0.0001) compared to the individual reagents, aApelinR alone (31% at 50 mg/kg, p<0.001) or aflibercept (43% at 50 mg/kg, p<0.005). Retinal endothelial cells were stained with GS Lectin I. Note the effect on dose and relative area of vascularization. Images were taken at 20x (for quantification) and 40x. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. With both systemic and intravitreal administration (see Example 5 below), combination therapy with aApelinR and aflibercept resulted in regression of normally developing retinal vasculature. Blocking both ApelinR and VEGFA by systemic injection is more effective in preventing vascular sprouting compared to blocking ApelinR alone or VEGFA alone.

When aAR was administered in P2, vascular proliferation was reduced by 23% (n=6 eyes/group, p<0.05) at 25 mg/kg and by 35% (n=6 eyes/group, p<0.005) at 50 mg /kg in P5. Our masking study was able to confirm our observations with a 29% (n=5 eyes per group, p<0.0001) reduction in vascular sprouting compared to the Fc control.

Combined inhibition of ApelinR and VEGFA following IP and IVT injections significantly limited vascular proliferation by 62% at 50 mg/kg (n=4 eyes per group, p<0.0001) and 68% at 5 μg (n=4 eyes) /group, p<0.0001). Aflibercept alone caused a 43% reduction in blood vessel proliferation at 50 mg/kg (n=4 eyes/group, p<0.001) and a 65% reduction at 5 μg (n=3 eyes/group, p<0.005). Additionally, aAR alone caused a 31% reduction in blood vessel proliferation at 50 mg/kg (n=4 eyes/group p<0.001) and a 43% reduction at 5 μg (n=4 eyes/group p<0.005) .

Example 5.

In another RVD experiment, IVT mice were administered aApelinR (H2aM9232N) (5 μg), aflibercept (5 μg), or a combination (admixture) of aApelinR and aflibercept. Fig. 5A and 5B are representative photomicrographs of mouse retina treated with intravitreal injection at P4 to P6 and a graph of the calculated vascular area. Residual vessel area was significantly less in the combination (aApelinR + aflibercept) (68% at 5 μg/kg, p < 0.0001) compared to the individual reagents, aApelinR alone (43% at 5 μg/kg, p < 0.0001 ) or aflibercept (65% at 5 μg/kg, p<0.0001). Note the effect on dose and relative vascularized area. Images were taken at 20x (for quantification) and 40x. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. Both systemic and intravitreal administration of aApelinR and aflibercept leads to regression of the normally developing retinal vasculature. Blocking both ApelinR and VEGFA by intravitreal injection is even more effective in preventing vascular growth compared to blocking ApelinR and VEGFA alone.

Example 6.

The mouse model of oxygen-induced ischemic retinopathy (OIR) is a well-characterized model of pathological neovascularization associated with increased expression of the important proangiogenic factor, VEGF (Smith et al., 1994, Invest. Ophthalmol. Vis. Sci., 35: 101-111; Neely et al., 1998, Am. J. Pathol., 153: 665-670; Saint-Geniez et al., 2004, Int. J. Dev. Biol., 48: 1045-1058) and are thus related to pathological angiogenesis associated with various diseases (Ferrarra et al., 2005, Nature, 438: 967-974).

A study was conducted to determine the effect of anti-APLNR antibody on retinal vessel growth, vascular abnormality formation, and retinal perfusion in oxygen-induced ischemic retinopathy (OIR).

To determine whether apelin/APLNR signaling plays a role in pathological angiogenesis as well as during normal development, the OIR model was used. In the OIR model. exposure of pups to hyperoxia at P6 results in rapid obliteration of capillaries in the central retina. After returning to room air at P11, the avascular zone becomes severely hypoxic, which in turn causes extensive abnormal neovascularization, characterized by ectopic growth of vessels into the vitreous (epiretinal vascular bundles) and the formation of abnormal arteriovenous shunts; the central parts of the retina remain largely avascular for a long period.

C57/B16 mice (Taconic) were used to study the effect of VEGF decoy or neutralizing antibody to APLNR on retinal neovascularization in OIR. OIR was produced using the method developed by Smith et al., 1994 (supra). Briefly, humanized mouse pups were placed in a hyperoxic environment (75% O ₂ ) at P6 and returned to room air at P11. Exposure to hyperoxia causes rapid vasoobliteration in the central retina. When mice are returned to room air (P11), vascular loss from the central retina leads to severe hypoxia/ischemia, which in turn stimulates pathological vascular changes. Pups were systemically administered 50 mg/kg

Fc (control), a-ApelinR or aflibercept at P12 and collected at P16. Retinal endothelial cells were stained with GS Lectin I.

Fig. 6A and 6B are representative photomicrographs of OIR mouse retinas treated systemically at P12 to P16 and plots of the calculated avascular area. Residual avascular area was significantly smaller in aApelinR (29%, p<0.05) and aflibercept (27.5%, p<0.01) retinas compared with Fc (control) retinas. Note the significant reduction in neovascular bundles in samples treated with aApelinR (67%, p<0.0001) and aflibercept (94%, p<0.0005) compared to Fc. Retinal endothelial cells were stained with GS Lectin I. Images were acquired at 20x (quantification of avascular area) and 40x (quantification of abnormal neovascular area). Statistical analysis was performed using one-way ANOVA followed by Tukey's test. When administered either systemically or intravitreally, selective inhibition of ApelinR promotes retinal vascular sprouting and reduces neovascularization in OIR mice. Inhibition of ApelinR and VEGFA by systemic injection promotes retinal vascular sprouting and reduces pathological neovascularization.

OIR mice. were treated similarly to the above study, except that OIR IVT mice were injected with 5 μg of Fc, aAR, or aflibercept at P12 and collected at P16. In fig. 7A and 7B are representative photomicrographs of an OIR mouse retina treated intravitreally at P12 to P16 and plots of the calculated avascular area. Avascular area was significantly reduced in the aApelinR condition (27.5%, p<0.05) and increased in the aflibercept condition (32%, p<0.0001) compared with Fc controls. Note the significant reduction in neovascular tufts in aApelinR (60%, p < 0.0001) and complete disappearance of tufts in aflibercept samples. Retinal endothelial cells were stained with GS Lectin I. Images were acquired at 20x (quantification of avascular area) and 40x (quantification of abnormal neovascular area). Statistical analysis was performed using one-way ANOVA followed by Tukey's test. When administered either systemically or intravitreally, selective inhibition of ApelinR promotes retinal vascular sprouting and reduces neovascularization in OIR mice. Inhibition of ApelinR by intravitreal injection improves vascular regrowth and reduces abnormal neovascularization, whereas inhibition of VEGFA stops vascular regrowth and completely stops any neovascularization.

In the OIR model, aAR and aflibercept were able to promote vascular regrowth. When pups were treated IP with OIR at P12, aAR and aflibercept significantly reduced avascular areas by 29 and 27% (n=6 eyes/group, p<0.05) and neovascularization by 69 and 94% (n=6 eyes/group , p<0.0001) at P16, respectively. When administered IVT, aflibercept reduced vascular regrowth by 30% (n=4 eyes/group, p<0.0001) and eliminated all neovascularization, whereas aAR was able to stimulate vascular regrowth by 30% (n=4 eyes/group, p< 0.05) and reduce neovascularization by 60% (n=4 eyes/group, p<0.0001).

A combination of an APLNR antagonist and a VEGF scavenger, such as aflibercept, also reversed pathological angiogenesis and promoted normal vascular sprouting in the OIR model, as discussed in Example 7. Therefore, this combination is expected to treat pathological angiogenesis observed in various ocular diseases, including retinopathy of prematurity .

Example 7.

In the following in vivo experiment, the OIR model, as discussed above in Example 6, was used to evaluate the organization and density of revascularization. Briefly, humanized mouse pups were placed in a hyperoxic environment (75% O ₂ ) at P6 and returned to room air at P11. Exposure to hyperoxia causes rapid vasoobliteration in the central retina. When mice are returned to room air (P11), vascular loss from the central retina leads to severe hypoxia/ischemia, which in turn stimulates pathological vascular changes. Pups were systemically administered 50 mg/kg Fc (control), aAPLNR (H4H9232N), or aflibercept (VEGF trap) or 25 mg/kg combination of aAPLNR and aflibercept at P12 and collected at P16. Retinal endothelial cells were stained with GS Lectin I.

Fig. 8A and 8B are representative photomicrographs of OIR mouse retinas treated systemically (20x) at P12 to P16 and plots of calculated avascular area. The rate of regrowth with the combination treatment is similar to each individual agent, and to this end, vascular regrowth is improved in all treatment conditions compared to control hFc-treated retinas, with the combination treatment demonstrating a slightly lower mean avascular area. Fig. 8B demonstrates that there were significant changes in avascular area between treatment groups (p<0.0005). Vessel area was significantly reduced with anti-APLNR antibody (**, p<0.05), with VEgF trap (***, p<0.005) and with the combination (***, p<0.05) compared with

-

Claims (10)

Fc control. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. Fig. 9A and 9B are representative photomicrographs of OIR mouse retinas treated systemically (40x) at P12 to P16 and plots of calculated abnormal vascular area. The combination treatment showed fewer pruned vessels compared to VEGF trap and fewer abnormal neovascularizations compared to anti-APLNR and Fc control (Fig. 9A). Fig. 9B demonstrates that there were significant changes in abnormal vascular area between treatment groups (p<0.0005). Abnormal vascular area was significantly reduced with anti-APLNR (***, p<0.0005), with VEGF trap (***, p<0.005) and with the combination (***, p<0.0005) compared with Fc control. Abnormal vascular area was also significantly reduced in the combination treatment group (*, p<0.05) compared with the anti-APLNR treatment group. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. In fig. Figure 10 shows examples of image processing and measurement used to calculate the vessel area and vessel length. The original images are the 40x micrographs shown in FIG. 9A. Threshold images and binary images are discussed more specifically with reference to FIGS. 11A, 11B, 12A and 12B below. Fig. 11A and 11B are representative processed images showing vascular organization and uniformity from micrographs (40x) of OIR mouse retinas processed systematically at P12 to P16, and plots of calculated vessel area. As shown in FIG. 11A, the combination of anti-APLNR antibody and VEGF trap resulted in retinal vessels that are more organized and uniform compared with anti-APLNR alone, and less sparse (with fewer pruned vessels) compared with VEGF trap alone. Fig. 11B demonstrates that there were significant changes in vessel area between treatment groups (p<0.0005). Vessel area was significantly increased with anti-APLNR (***, p<0.0005) and decreased with VEGF trap (***, p<0.005) and combination therapy (*, p<0.05) compared with Fc control. Vessel area was significantly reduced with VEGF trap (####, p<0.0005) and with the combination (####, p<0.0005) compared with anti-APLNR. In contrast, vessel area was significantly increased with combination therapy (&&&, p<0.005) compared with VEGF trap. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. Fig. 12A and 12B are representative processed images showing vessel density from photomicrographs (40x) of OIR mouse retinas processed systemically at P12 to P16, and plots of calculated vessel length. As shown in FIG. 12A, the combination of anti-APLNR antibody and VEGF trap produced retinal vessels of intermediate density compared to anti-APLNR (higher density) or VEGF trap alone (lower density). Fig. 12B demonstrates that there were significant changes in vessel length between treatment groups (p<0.0005). Vessel length was significantly reduced with VEGF trap (####, p<0.0005) and with the combination (####, p<0.0005) compared with anti-APLNR. In contrast, vessel length was significantly increased with combination therapy (&&&, p<0.005) compared with VEGF trap. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. As shown in FIG. 8A-12B and discussed above, the combination of anti-APLNR antibody and VEGF trap promotes revascularization, resulting in a decrease in avascular area compared to Fc controls. In addition, combination treatment inhibited pathological neovascularization and had an intermediate effect on vessel area and vessel length compared with treatment with anti-APLNR antibody or VEGF trap alone. Specifically, the combination treatment promoted microvascular growth between major blood vessels compared with VEGF decoy treatment, and improved blood vessel organization and homogeneity compared with anti-APLNR antibody treatment. This description is not intended to limit the scope to the specific embodiments described herein. Indeed, various modifications of this specification in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. CLAIM 1. A method of treating a vascular eye disease or an ocular vascular disorder, comprising administering a therapeutically effective amount of an APLNR antagonist to a patient, wherein the APLNR antagonist is an anti-APLNR antibody or antigen-binding fragment thereof that specifically binds to human APLNR and contains six complementarity determining regions, HCDR1 , HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, containing the amino acid sequences SEQ ID NO: 14, 15, 16, 19, 20 and 21, respectively; and administering a therapeutically effective amount of a vascular endothelial growth factor antagonist - 29 043390 (VEGF) to a patient where the VEGF antagonist is aflibercept. 2. The method of claim 1, wherein the patient has been diagnosed with an eye disease or disorder that is selected from the group consisting of diabetic retinopathy, proliferative diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion retinal veins, polypoidal choroidal vasculopathy, choroidal neovascularization (CNV), degenerative myopia (myopic CNV), neovascular glaucoma and retinopathy of prematurity. 3. The method of claim 2, wherein the eye disease or disorder is age-related macular degeneration. 4. The method of claim 2, wherein the eye disease or disorder is diabetic macular edema. 5. The method according to any one of claims 1-4, where the antibody or antigen-binding fragment contains a heavy chain variable region containing an amino acid sequence SEQ ID NO: 13; and a light chain variable region containing the amino acid sequence SEQ ID NO: 18. 6. The method according to any one of claims 1 to 5, wherein the antibody or antigen binding fragment thereof is a human antibody with an IgG1 or IgG4 heavy chain constant region. 7. A composition for the treatment of a vascular disease or vascular disorder of the eye, containing a therapeutically effective amount of an APLNR antagonist, where the APLNR antagonist is an anti-APLNR antibody or antigen-binding fragment thereof that specifically binds to human APLNR and contains six complementarity determining regions, HCDR1, HCDR2 , HCDR3, LCDR1, LCDR2 and LCDR3, containing the amino acid sequences SEQ ID NO: 14, 15, 16, 19, 20 and 21, respectively; a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist, wherein the VEGF antagonist is aflibercept; and a suitable carrier, excipient or diluent. 8. The composition according to claim 7, where the antibody or antigen-binding fragment thereof contains a heavy chain variable region containing an amino acid sequence SEQ ID NO: 13; and a light chain variable region containing the amino acid sequence SEQ ID NO: 18. 9. The composition according to claim 7 or 8, wherein the antibody or antigen-binding fragment thereof is a human antibody with an IgG1 or IgG4 heavy chain constant region. 10. The composition according to any one of claims 7 to 9, wherein the eye disease or disorder is selected from the group consisting of diabetic retinopathy, proliferative diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion , polypoidal choroidal vasculopathy, choroidal neovascularization (CNV), degenerative myopia (myopic CNV), neovascular glaucoma and retinopathy of prematurity. -