WO2018208625A1 - Methods of treating eye disorders with aplnr antagonists and vegf inhibitors - Google Patents

Methods of treating eye disorders with aplnr antagonists and vegf inhibitors Download PDF

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Publication number
WO2018208625A1
WO2018208625A1 PCT/US2018/031255 US2018031255W WO2018208625A1 WO 2018208625 A1 WO2018208625 A1 WO 2018208625A1 US 2018031255 W US2018031255 W US 2018031255W WO 2018208625 A1 WO2018208625 A1 WO 2018208625A1
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Prior art keywords
antibody
aplnr
antagonist
antigen
vegf
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PCT/US2018/031255
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English (en)
French (fr)
Inventor
Jingtai Cao
Eunice CHEUNG
Ivan B. Lobov
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Regeneron Pharmaceuticals, Inc.
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Priority to EA201992630A priority Critical patent/EA201992630A1/ru
Priority to IL270267A priority patent/IL270267B1/en
Priority to EP18729829.4A priority patent/EP3618876A1/en
Priority to JP2019560665A priority patent/JP7161494B2/ja
Priority to CN201880037918.XA priority patent/CN110709104A/zh
Priority to KR1020197035953A priority patent/KR102667021B1/ko
Priority to AU2018266324A priority patent/AU2018266324B2/en
Priority to MX2019012985A priority patent/MX2019012985A/es
Priority to CA3062418A priority patent/CA3062418A1/en
Publication of WO2018208625A1 publication Critical patent/WO2018208625A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • the present disclosure relates to methods of treating vascular eye disease, in particular, by administering an apelin receptor antagonist and a vascular endothelial growth factor (VEGF) antagonist to a subject in need thereof.
  • VEGF vascular endothelial growth factor
  • 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. Two of the largest contributors to this subject population are diabetic retinopathy and exudative age-related macular degeneration.
  • Diabetic retinopathy is a major cause of visual impairment in the United States (Klein et al., 1984, Ophthalmology 91 : 1464-1474; Moss et al., 1998,
  • Diabetic retinopathy results from microvascular decompensation beginning with basement membrane thickening (Ruggiero et al., 1997, Diabetes Metab. 23:30-42), and eventually leading to vascular occlusion and neovascularization (Porta et al., 2002, Diabetologia 45: 1617-1634). It is estimated that about 28% of subjects 40 years and older with diabetes have 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 frequent cause of blindness in young and mid-aged adults (Klein et al. , 1984, Ophthalmology 91 : 1464- 1474; Moss et al., 1998, Ophthalmology 105:998-1003).
  • Age-related macular degeneration is the leading cause of severe visual loss in people aged 50 years or older in the developed world. In recent years, major advances have been made in the treatment of AMD, with the introduction of anti-angiogenic agents, offering hope of significant visual recovery for subjects with neovascular AMD (Keane et al., 2012, Surv Ophthalmol. 57: 389-414).
  • Preproapelin is a 77 amino acid protein expressed in the human CNS and peripheral tissues, e.g., lung, heart, and mammary gland.
  • APJ receptor now known as APLNR
  • APLNR G protein-coupled receptor
  • apelin peptides and analogues convey cardiovascular and angiogenic actions through their interaction with the APJ receptor (APLNR), such as endothelium-dependent vasodilation (Tatemoto et al., 2001 , Regul Pept 99:87-92).
  • APLNR APJ receptor
  • compositions may inhibit angiogenesis by inhibiting the apelin/APJ pathway (see, e.g., US Patent No. 7,736,646), such as APLNR inhibitors capable of blocking pathological angiogenesis and therefore useful in inhibiting vascularization in the retina (Kojima, Y. and Quertermous, T., 2008, Arterioscler Thromb Vase Biol 28: 1687-1688).
  • VEGF vascular endothelial growth factor
  • IVT Intravitreal
  • the present invention provides methods for treating, preventing or ameliorating at least one symptom or indication of a vascular eye disease or disorder in a subject.
  • the methods include administering a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist to a subject in need thereof.
  • the APLNR antagonist is administered in combination with a vascular endothelial growth factor (VEGF) antagonist, for example by administration of a therapeutically effective amount of a pharmaceutical composition comprising a VEGF antagonist.
  • VEGF vascular endothelial growth factor
  • 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.
  • CNV choroidal neovascularization
  • myopic CNV degenerative myopia
  • neovascular glaucoma retinopathy of prematurity
  • the present invention provides methods for inhibiting retinal angiogenesis in a subject.
  • the methods comprise administering a
  • the retinal angiogenesis is associated with a vascular eye disease or disorder.
  • the present invention provides methods for inhibiting retinal neovascularization (e.g., in a subject with an eye disease or disorder associated with angiogenesis).
  • the methods comprise administering a
  • a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist to the subject in need thereof.
  • the present invention provides methods for inhibiting choroidal neovascularization in a subject.
  • the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist to the subject in need thereof.
  • the present invention provides methods for improving vascular regrowth and decreasing abnormal neovascularizations in a subject.
  • the methods comprise administering a therapeutically effective amount of a
  • composition comprising an APLNR antagonist in combination with a VEGF antagonist to the subject in need thereof.
  • the present invention provides methods for promoting revascularization of a retina in a subject in need thereof.
  • the methods comprise administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof; and administering a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist, to the subject.
  • VEGF vascular endothelial growth factor
  • the present invention provides methods for promoting uniform regrowth of retinal vessels in a subject in need thereof.
  • the methods comprise administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof; and administering a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist, to the subject.
  • VEGF vascular endothelial growth factor
  • the present invention provides methods of improving vessel outgrowth in a retina of a subject in need thereof.
  • the methods comprise administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof; and administering a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist, to the subject.
  • VEGF vascular endothelial growth factor
  • the present invention provides methods for promoting uniform blood vessel growth in a retina in a subject in need thereof.
  • the methods comprise administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof; and administering a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist, to the subject.
  • VEGF vascular endothelial growth factor
  • the present invention provides methods for improving blood vessel organization of a retina in a subject in need thereof.
  • the methods comprise administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof; and administering a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist, to the subject.
  • VEGF vascular endothelial growth factor
  • the subject may be an individual that has been diagnosed with an eye disease or disorder.
  • 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.
  • the eye disease or disorder is age-related macular degeneration.
  • the eye disease or disorder is diabetic macular edema.
  • the eye disease or disorder is retinopathy of prematurity.
  • the eye disease or disorder is
  • Exemplary APLNR antagonists that can be used in the context of the methods or compositions of the present disclosure include small molecule chemical inhibitors of APLNR, or biological agents that target APLNR, such as peptides, peptide mimetics and antibodies.
  • the APLNR antagonist blocks the interaction of APLNR and apelin.
  • the APLNR antagonist is an antibody or antigen-binding protein that binds the APLNR antagonist and inhibits APLNR signaling.
  • the anti-APLNR antibody or antigen-binding protein comprises the heavy chain complementarity determining regions (HCDRs) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ I D NO: 2 or 13 and the light chain CDRs of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 7 or 18.
  • the anti-APLNR antibody or antigen-binding protein comprises the complementarity determining regions (CDRs) of a heavy chain variable region (HCVR) of an anti-APLNR antibody selected from the group consisting of
  • H2aM9232N (or H4H9232N) and H1 M9207N.
  • the APLNR antagonist may comprise an anti-APLNR antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds human APLNR.
  • the antibody or antigen-binding fragment thereof competes for binding to 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 antigen- binding fragment thereof specifically binds human APLNR.
  • the antibody or antigen-binding fragment thereof binds to the same epitope on 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 antigen- binding fragment thereof specifically binds human APLNR.
  • the antibody or antigen-binding fragment comprises: (a) the complementarity
  • the antibody or antigen-binding fragment comprises HCDR1 -HCDR2-HCDR3- LCDR1 -LCDR2-LCDR3 domains, respectively, selected from the group consisting of: SEQ ID NOs: 3-4-5-8-9-10 and 14-15-16-19-20-21 .
  • the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ I D 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.
  • the antibody or antigen-binding fragment comprises the heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 2/7 and 13/18.
  • the antibody or antigen- binding fragment comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 2/7 and 13/18.
  • the antibody or antigen-binding fragment thereof comprises the HCVR/LCVR amino acid sequence pair of SEQ I D NOs: 13/18.
  • the antibody or antigen-binding fragment can be a human antibody with an lgG1 or lgG4 heavy chain constant region.
  • the heavy chain constant region is human lgG1 .
  • the heavy chain constant region is human lgG4.
  • the antibody or antigen-binding fragment thereof blocks the interaction of APLNR and apelin. In some cases, the antibody or antigen-binding fragment thereof blocks the interaction of APLNR and apelin exhibiting at least 50% binding inhibition 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 may be used in combination with an APLNR antagonist in the compositions and methods of the present disclosure include anti- VEGF antibodies (e.g., ranibizumab), small molecule VEGF inhibitors (e.g., dueinib), and VEGF-inhibiting fusion proteins ("VEGF Traps").
  • An example of a VEGF antagonist that may be used in combination with the APLNR antagonist in the methods of treatment of the present disclosure is aflibercept, a VEGF-inhibiting fusion protein (see e.g. US 7,087,41 1 ).
  • the VEGF antagonist comprises a VEGF receptor-based chimeric molecule (VEGF Trap).
  • VEGF Trap comprises one or more immunoglobulin (lg)- like domains of VEGFR1 , one or more Ig-like domains of VEGFR2, and a
  • the VEGF Trap comprises Ig-like domain 2 of VEGFR1 , Ig-like domain 3 of VEGFR2, and a multimerizing domain.
  • the VEGF Trap is aflibercept, or a biosimilar molecule thereof.
  • the VEGF antagonist consists of a dimer of two polypeptides consisting of amino acids 27-457 of SEQ ID NO: 23.
  • the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of an APLNR antagonist and a pharmaceutically acceptable carrier or diluent, in certain embodiments, the pharmaceutical composition further comprises a VEGF antagonist.
  • the present disclosure provides for the use of an APLNR antagonist in conjunction with a VEGF antagonist in the manufacture of a medicament to treat an eye disease or disorder in a subject, including humans, or to perform the other objectives of any of the methods discussed above or herein.
  • All of the methods discussed above or herein can be embodied as a use or uses of the APLNR antagonists and VEGF antagonists for the treatment of eye diseases or disorders or other objectives of the recited methods.
  • Other embodiments include the APLNR antagonists and VEGF antagonists for use in the methods discussed above or herein.
  • the present invention provides a composition for treating a vascular eye disease or disorder, in which the composition comprises a
  • an APLNR antagonist 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.
  • VEGF vascular endothelial growth factor
  • suitable carrier excipient or diluent.
  • the APLNR antagonist or the VEGF antagonist can be as discussed above or herein.
  • Figures 1 A and 1 B are representative photomicrographs of mouse retinas treated systemically (IP) at P2 to P5 and a graph of the calculated vascular area. Residual vascular area was significantly smaller in aApelinR (anti-APLNR antibody H2aM9232N) treated (23% at 25 mg/kg, p ⁇ 0.05) retinas compared to untreated retinas. Retinal endothelial cells were stained with GS Lectin I . Images were taken at 20x (for quantification) and 40x. Statistical analysis was done with Student T-test. Selective inhibition of ApelinR via systemic injection delayed normal vascular outgrowth in the developing retina in P5 pups. At 25mg/kg Dose, blocking ApelinR slightly inhibits vascular outgrowth.
  • FIGS 2A and 2B are representative photomicrographs of mouse retinas treated systemically (IP) at P2 to P5 and a graph of the calculated vascular area. Residual vascular area was significantly smaller in aApelinR treated (35% at 50 mg/kg, p ⁇ 0.005) retinas compared to untreated retinas. Retinal endothelial cells were stained with GS Lectin I. Images were taken at 20x (for quantification) and 40x. Statistical analysis was done with Student T-test. Selective inhibition of ApelinR via systemic injection delayed normal vascular outgrowth in the developing retina in P5 pups. By increasing the dose to 50mg/kg Dose, targeting ApelinR further delays retinal vascular outgrowth.
  • FIGS 3A and 3B are representative photomicrographs of mouse retinas treated systemically (IP) at P2 to P5 and a graph of the calculated vascular area.
  • IP systemically
  • 50 mg/kg aApelinR decreased vascular growth by 29.8% (p ⁇ 0.0001 ) compared to Fc treated controls.
  • Retinal endothelial cells were stained with GS Lectin I. Images were taken at 20x (for quantification) and 40x.
  • Statistical analysis was done with Student T-test. Selective inhibition of ApelinR via systemic injection delays normal vascular outgrowth in the developing retina in P5 pups.
  • Figures 4A and 4B are representative photomicrographs of mouse retinas treated systemically (IP) at P2 to P5 and a graph of the calculated vascular area. Residual vascular area was significantly smaller in combination (aApelinR + aflibercept) (62% at 50 mg/kg, p ⁇ 0.0001 ) compared to single reagents, aApelinR (31 % at 50mg/kg, p ⁇ 0.001 ) or aflibercept (43% at 50mg/kg, p ⁇ 0.005) alone.
  • Retinal endothelial cells were stained with GS Lectin I. Note the effects on dose and relational vascularized area. Images were taken at 20x (for quantification) and 40x. Statistical analysis was done with one-way ANOVA with post-hoc Tukey test. In both systemic and intravitreal (see Figs. 5A and 5B) administration, aApelinR, and aflibercept combination therapy resulted in regression of normal developing retinal vasculature. Blocking both ApelinR and VEGFA via systemic injection is more effective in hindering vascular outgrowth compared to blocking ApelinR or VEGFA alone. In this model, combination treatment further decreased vascular area by 46% (p ⁇ 0.0005) compared to aApelinR and 32% (p ⁇ 0.001 ) compared to aflibercept.
  • FIGS 5A and 5B are representative photomicrographs of mouse retinas treated via intravitreal (IVT) injection at P4 to P6 and a graph of the calculated vascular area. Residual vascular area was significantly smaller in combination (aApelinR + aflibercept) (68% at 5 ⁇ g, p ⁇ 0.0001 ) compared to single reagents, aApelinR (43% at 5 ⁇ g, p ⁇ 0.0001 ) or aflibercept (65% at 5 ⁇ g, p ⁇ 0.0001 ) alone. Retinal endothelial cells were stained with GS Lectin I. Note the effects on dose and relational vascularized area. Images were taken at 20x (for quantification) and 40x.
  • combination treatment further decreased vascular area by 50% (p ⁇ 0.0005) compared to aApelinR and 34% (p ⁇ 0.001 ) compared to aflibercept.
  • Figures 6A and 6B are representative photomicrographs of OIR mouse retinas treated systemically (I P) at P12 to P16 and graphs 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 to Fc (control) retinas. Notice the significant decrease in neovascular tufts in aApelinR (67%, p ⁇ 0.0001 ) and aflibercept (94%, p ⁇ 0.0005) treated samples compared to Fc. Retinal endothelial cells were stained with GS Lectin I.
  • FIGS 7A and 7B are representative photomicrographs of oxygen induced retinopathy (OIR) mouse retinas treated intravitreally at P12 to P16, and graphs of calculated avascular area. Residual avascular area was significantly decreased in aApelinR (27.5% p ⁇ 0.05) and increased in aflibercept (32%, p ⁇ 0.0001 ) conditions compared to Fc controls. Notice the significant decrease 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 effects on dose and relational vascularized area.
  • OIR oxygen induced retinopathy
  • Figures 8A and 8B are representative photomicrographs (20x) of OIR mouse retinas treated systemically at P12 to P16 and graphs of the calculated avascular area.
  • the rate of regrowth with the combination treatment is similar to each agent alone, vascular regrowth is improved compared to hFc-treated control retinas, and there were significant changes in avascular area between treatment groups
  • Figures 9A and 9B are representative photomicrographs (40x) of OIR mouse retinas treated systemically at P12 to P16 and graphs of the calculated abnormal vascular area.
  • the combination treatment shows fewer pruned vessels compared to aflibercept, and fewer abnormal neovascularizations compared to anti-APLNR and Fc control (Figure 9A).
  • Figure 9B shows that there were significant changes in abnormal vascular area between treatment groups (p ⁇ 0.0005).
  • Abnormal vascular area was significantly decreased with anti-APLNR (***, p ⁇ 0.0005), with aflibercept (*** p ⁇ 0.005), and with the combination (***, p ⁇ 0.0005) compared to Fc control, and the abnormal vascular area was also significantly decreased in the combination treatment group (*, p ⁇ 0.05) compared to the anti-APLNR treatment group
  • Figure 10 is representative of the image processing and measurement techniques used to calculate vessel area and vessel length in Example 7.
  • the "original images” are the 40x photomicrographs shown in Figure 9A.
  • Figures 1 1 A and 1 1 B are representative processed images showing the organization and uniformity of vessels from the photomicrographs (40x) of OIR mouse retinas treated systemically at P12 to P16 and graphs of the calculated vessel area.
  • the combination of anti-APLNR antibody and aflibercept produced retinal vessels that are more organized and uniform compared to anti-APLNR alone, and less sparse (with fewer pruned vessels) compared to aflibercept alone.
  • Figure 1 1 B shows that there were significant changes in vessel area between the treatment groups (p ⁇ 0.0005). Vessel area was significantly increased with anti-APLNR (***, p ⁇ 0.0005) and decreased with aflibercept (***, p ⁇ 0.005) and the combination treatment (*, p ⁇ 0.05) compared to Fc control.
  • FIGS 12A and 12B are representative processed images showing the density of vessels from the photomicrographs (40x) of OI R mouse retinas treated systemically at P12 to P16 and graphs of the calculated vessel length. As shown in Figure 12A, the combination of anti-APLNR antibody and aflibercept produced retinal vessels of intermediate density compared to anti-APLNR (greater density) or aflibercept (lesser density) alone.
  • FIG 12B shows that there were significant changes in vessel length between the treatment groups (p ⁇ 0.0005). Vessel length was significantly decreased with aflibercept (####, p ⁇ 0.0005) and with the combination (####, p ⁇ 0.0005) compared to anti-APLNR. In contrast, vessel length was significantly increased with the combination treatment (&&&, p ⁇ 0.005) compared to aflibercept.
  • the present invention and disclosure is based, in part, on the surprising discovery that the combination of an APLNR antagonist and a VEGF antagonist (e.g. , an anti-APLNR antibody and a VEGF Trap) can produce more organized and ordered revascularization of intermediate density (with fewer pruned vessels) in the retina of a subject than is observed with either an APLNR antagonist or a VEGF antagonist, alone.
  • a VEGF antagonist e.g. , an anti-APLNR antibody and a VEGF Trap
  • APLNR Apelin receptor
  • APLNR ApelinR
  • APJ receptor a human APLNR protein having the amino acid sequence set forth as:
  • proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species (e.g., “mouse APLNR,” “monkey APLNR,” etc.).
  • an antibody or antigen-binding fragment that binds APLNR or an “anti-APLNR antibody” includes immunoglobulin molecules, antibodies and antigen-binding fragments thereof that bind a fragment of an APLNR protein.
  • APLNR molecules include natural APLNR proteins as well as recombinant APLNR protein variants such as, e.g., monomeric and dimeric APLNR constructs.
  • an antibody that binds APLNR or antigen binding fragment thereof is an APLNR antagonist.
  • antagonist refers, in part, to a moiety that binds to the receptor at the same site or near the same site as an agonist (for example, the endogenous ligand), but which does not activate the intracellular response typically initiated by the active form of the receptor, and thereby inhibits or neutralizes the intracellular response by an agonist or partial agonist.
  • 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 trap, such as a VEGF trap. In some cases, antagonists do not diminish the baseline intracellular response in the absence of an agonist or partial agonist.
  • An antagonist does not necessarily have to function as a competitive binding inhibitor, but may work by sequestering an agonist, or indirectly modulating a downstream effect.
  • APLNR antagonist may refer to a moiety that binds to the APLNR receptor at the same site or near the same site as an agonist (for example, apeiin), but which does not activate the intracellular response typically initiated by the active form of the receptor, and thereby inhibits or neutralizes the intracellular response of APLNR.
  • an 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 can include small molecules and other biological entities, such as peptides.
  • immunoglobulin 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, which may all four be inter-connected by disulfide bonds.
  • L light
  • H heavy
  • the structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W. , ed. , 2nd ed. Raven Press, N. Y. (1989)).
  • antibody means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., APLN R).
  • CDR complementarity determining region
  • the term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g. , IgM), as well as immunoglobulin molecules including a fragment of one or more heavy chains or a fragment of one or more light chains, (e.g. Fab, F(ab')2 or scFv fragments), as described herein.
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR ⁇ ⁇ / ⁇ ) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CH1 , CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain (Ci_1 ).
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • the FRs of the anti-APLNR antibody may be identical to the human germline sequences, or may be naturally or artificially modified.
  • An amino acid consensus sequence may be defined based on a side-by- side analysis of two or more CDRs.
  • antibody also includes antigen-binding fragments of full antibody molecules.
  • antigen-binding portion of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by 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 delete amino acids, etc.
  • Such techniques may also be employed to synthesize any antibody-fusion molecule containing an antigen-binding fragment derived from a full 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 (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMI Ps), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.
  • SMI Ps small modular immunopharmaceuticals
  • shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment," as used herein.
  • An antigen-binding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers.
  • the antigen- binding fragment of an antibody may contain a monomeric VH or VL domain.
  • an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) V H -C H 1 ; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1 -C H 2; (v) V H -C H 1 - CH2-C h 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1 ; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L - CH1 -C h 2; (xii) V L -CH1 -CH2-C h 3; (xiii) V L -C H
  • variable and constant domains including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
  • a hinge region may consist of at least 2 (e.g. , 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
  • an antigen- binding fragment of an antibody of the present disclosure may comprise a homo- dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
  • antigen-binding fragments may be monospecific or multispecific (e.g., bispecific).
  • a multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.
  • Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.
  • antibody-fusion proteins includes recombinant polypeptides and proteins derived from antibodies of the disclosure that have been engineered to contain an antibody or antigen-binding fragment as described herein.
  • the peptide component may be fused to the anti-APLNR antibody or antigen-binding fragment either at the N-terminus or the C-terminus of the antibody light chain or heavy chain, with or without peptide linkers.
  • fused to means (but is not limited to) a polypeptide formed by expression of a chimeric gene made by combining more than one sequence, typically by cloning one gene into an
  • the components or amino acids of an antibody-fusion protein are separated by a linker (or "spacer") peptide.
  • linker or "spacer” peptide.
  • Such peptide linkers are well known in the art (e.g., polyglycine or Gly-Ser linkers) and typically allow for proper folding of one or both of the components of the antibody-fusion protein.
  • the linker provides a flexible junction region of the component of the fusion protein, allowing the two ends of the molecule to move independently, and may play an important role in retaining each of the two moieties' appropriate functions. Therefore, the junction region acts in some cases as both a linker, which combines the 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.
  • junction region should create an epitope that will not be recognized by the subject's immune system as foreign, in other words, will not be considered immunogenic.
  • Linker selection may also have an effect on binding activity, and thus the bioactivity, of the fusion protein.
  • the apelin peptide is connected to the C-terminus or to the N-terminus of the light chain or heavy chain of the antibody or antigen-binding fragment thereof, via one or more peptide linkers.
  • the antibodies of the present disclosure may function through complement- dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • NK Natural Killer
  • the constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity.
  • the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity.
  • the antibody may be engineered at its Fc domain to activate all, some, or none of the normal Fc effector functions, without affecting the antibody's desired pharmacokinetic properties. Therefore, antibodies with
  • the protein comprises a chimeric or otherwise modified Fc domain.
  • a chimeric Fc domain see International Publication No. WO 2014/121087 A1 , published August 7, 2014, which is herein incorporated by reference in its entirety.
  • EC50 refers to the half maximal effective concentration, which includes the concentration of a ligand that induces a response, for example a cellular response, halfway between the baseline and maximum after a specified exposure time.
  • the EC50 essentially represents the concentration of a ligand where 50% of its maximal effect is observed.
  • increased receptor activity is observed with a decreased EC50 value, i.e. half maximal effective concentration value (less ligand needed to produce a greater response).
  • IC50 refers to the half maximal inhibitory concentration of a cellular response.
  • a particular moiety e.g. protein, compound, or molecule
  • an assay quantitates the amount of such moiety needed to inhibit a given biological process.
  • the present disclosure includes methods for treating, preventing, or ameliorating at least one symptom or indication of a vascular eye disease or disorder in a subject.
  • the methods according to this aspect of the disclosure comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist to the subject in need thereof.
  • the APLNR antagonist is administered subcutaneously, intravenously or intravitreally. In embodiments, the APLNR antagonist is administered in combination with a VEGF antagonist. In some embodiments, the APLNR antagonist is intravitreally administered in combination with the VEGF antagonist. In some embodiments, the APLNR antagonist is administered as a single combined dosage formulation with the VEGF antagonist. In some embodiments, the APLNR antagonist is administered in combination with the VEGF antagonist, wherein the APLNR antagonist is administered intravenously and the VEGF antagonist is administered intravitreally. The VEGF antagonist may be administered before, after or
  • the present invention is based, in part , on Applicant's discovery that a combination of antagonism directed against both VEGF and the apelin/APLNR pathways can advantageously impact unwanted pathological vascularization of the eye.
  • 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
  • APLNR an antagonist of APLNR can improve the anti- angiogenic effects of VEGF antagonism where antagonism of the VEGF pathway has been saturated.
  • the terms “treat”, “treating”, or the like mean to alleviate symptoms, eliminate the causation of symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of a neovascular eye disease or disorder.
  • the present methods are useful for treating or ameliorating at least one symptom or indication including, but not limited to, retinal angiogenesis, neovascularization, vascular leak, retinal thickening within 500 ⁇ of the center of the fovea, hard, yellow exudates within 500 ⁇ 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, blurry vision, floaters, loss of contrast, double vision, and eventual loss of vision.
  • the term means that, from the initiation of treatment, the subject exhibits gain of one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) letters on the Early Treatment Diabetic Retinopathy Study (EDTRS) visual acuity chart.
  • ETRS Early Treatment Diabetic Retinopathy Study
  • the term means that, from initiation of treatment, vision loss of greater than or equal to 15 letters is prevented in the subject.
  • the terms “prevent”, “preventing”, or the like mean to prevent development of a symptom, indication or a complication of a vascular eye disease.
  • a vascular eye disease such as AMD or DME
  • the term means, from initiation of treatment, moderate or severe vision loss is prevented in a subject.
  • vascular eye disease or disorder refers to eye disease or disorders that affect blood vessels in the eye.
  • the diseases may be caused due to abnormal angiogenesis (formation of new blood vessels) or occlusion or blockage of blood vessels.
  • the term includes eye diseases or disorders associated with angiogenesis.
  • the term includes, but is not limited to 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
  • CNV neovascularization
  • myopic CNV degenerative myopia
  • neovascular glaucoma neovascular glaucoma
  • retinopathy of prematurity neovascularization
  • myopic CNV degenerative myopia
  • neovascular glaucoma neovascular glaucoma
  • retinopathy of prematurity neovascularization glaucoma
  • neovascular eye disease or disorder may be used interchangeably with the term “eye disease or disorder associated with angiogenesis.”
  • the present disclosure includes methods for treating, preventing, or ameliorating at least one symptom or indication 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 glaucoma, and retinopathy of prematurity.
  • administration of an APLNR antagonist also promotes normal revascularization of the retina, for example in oxygen induced retinopathy (OIR).
  • OIR oxygen induced retinopathy
  • DME Diabetic Macular Edema
  • DME Diabetic Macular Edema
  • the swelling may distort a person's central vision, as the macula is near the center of the retina at the back of the eyeball.
  • the primary symptoms of DME include, but are not limited to, blurry vision, floaters, loss of contrast, double vision, and eventual loss of vision.
  • the pathology of DME is characterized by breakdown of the blood-retinal barrier, normally preventing water movement in the retina, thus allowing fluid to accumulate in the retinal tissue, and presence of retinal thickening. DME is presently diagnosed during an eye
  • DME can be broadly characterized into two main categories— Focal and Diffuse. Focal DME is characterized by specific areas of separate and distinct leakage in the macula with sufficient macular blood flow.
  • Diffuse DME results from leakage of the entire capillary bed surrounding the macula, resulting from a breakdown of the inner blood-retina barrier of the eye.
  • DME is also categorized based on clinical exam findings into clinically significant macular edema (CSME), non-CSME and CSME with central involvement (CSME-CI), which involves the fovea.
  • CSME macular edema
  • CSME-CI central involvement
  • the present disclosure includes methods to treat the above-mentioned categories of DME.
  • the wet form of AMD is characterized by the growth of abnormal blood vessels from the choroid underneath the macula. This is called choroidal
  • CNV neovascularization
  • Retinopathy of Prematurity refers to a condition affecting premature infants of low birth weight and young gestational age. Retinopathy of prematurity occurs when the development of normal retinal blood vessels is interrupted by birth prior to full gestational term, resulting in abnormal development of retinal blood vessels. If the condition progresses, the growth of scar tissue can lead to retinal detachment and vision impairment or vision loss.
  • the pathogenesis of ROP involves two discrete phases: a vasoobliterative phase and a vasoproliferative phase.
  • DR Diabetic retinopathy
  • PDR Proliferative DR
  • PDR 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 the advanced stage of DR, which is driven by hypoxia and expression of proangiogenic growth factors, which stimulate the aberrant formation of new blood vessels in the retina that protrude into the preretinal space.
  • Retinal neovascularization can result in sever vision loss when it leads to vitreous hemorrhage or fractional retinal detachment.
  • photocoagulation has been the standard for treatment of PDR for many years, with more recent therapies including intraocular treatment with anti-VEGF (e.g., anti- VEGF antibodies) and steroid agents, and vitreoretinal surgery.
  • anti-VEGF e.g., anti- VEGF antibodies
  • steroid agents e.g., steroid agents
  • vitreoretinal surgery e.g., vitreoretinal surgery.
  • a subject in need thereof means a human or non-human mammal that exhibits one or more symptoms or indications of, and/or who has been diagnosed with an eye disease or disorder associated with
  • a subject in need thereof may also include, e.g., subjects who, prior to treatment, exhibit (or have exhibited) one or more indications of a neovascular eye disease such as, e.g., retinal angiogenesis, neovascularization, vascular leak, retinal thickening within 500 ⁇ of the center of the fovea, hard, yellow exudates within 500 ⁇ 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, blurry vision, floaters, loss of contrast, double vision, and eventual loss of vision.
  • a neovascular eye disease such as, e.g., retinal angiogenesis, neovascularization, vascular leak, retinal thickening within 500 ⁇ of the center of the fovea, hard, yellow exudates within 500 ⁇ of the center of the fovea with adjacent retinal thickening, and at least 1
  • a "subject in need thereof” also includes a human or non-human mammal who has a vascular 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.
  • 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
  • CNV choroidal neovascularization
  • a subject in need thereof may include a subset of population which is more susceptible to DME or AMD or may show an elevated level of a DME-associated or an AMD-associated biomarker, or a biomarker associated with ROP or PDR.
  • a subject in need thereof may include a subject suffering from diabetes for more than 10 years, or a subject who has frequent high blood sugar levels or high fasting blood glucose levels.
  • the term "a subject in need thereof” includes a subject who, prior to or at the time of administration of the APLNR antagonist and/or VEGF antagonist, has been or is diagnosed with diabetes.
  • the term "a subject in need thereof” includes a subject who, prior to or at the time of administration of the APLNR antagonist and/or VEGF antagonist, is more than 50 years old. In some embodiments, the term “a subject in need thereof” includes subjects who are smokers, or subjects with high blood pressure or high cholesterol.
  • the present disclosure includes methods for treating, preventing or reducing the severity of a vascular eye disease 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, e.g., as part of a specific therapeutic dosing regimen.
  • the therapeutic dosing regimen may comprise administering multiple doses of the pharmaceutical composition to the subject at a frequency of about once a 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 frequently.
  • the therapeutic dosing regimen may comprise administering multiple doses of the pharmaceutical composition to the subject at a frequency of once a day or 2 times a day or more.
  • the present disclosure also includes methods for inhibiting or reducing or suppressing vascular leak in a subject.
  • the methods according to this aspect of the disclosure comprise administering to the subject one or more doses of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist to reduce or inhibit vascular leak in the eye of a subject.
  • the vascular 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 who has been administered the VEGF antagonist alone.
  • the methods of the present disclosure comprise administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist.
  • the APLNR antagonist may be administered in combination with therapy including laser treatment to stop leakage into the macula.
  • the phrase 'in combination with means that the pharmaceutical composition comprising an APLNR antagonist is administered to the subject at the same time as, just before, or just after administration of a VEGF antagonist.
  • the phrase 'in combination with means that the pharmaceutical composition comprising an APLNR antagonist is administered to the subject at the same time as, just before, or just after administration of the VEGF antagonist.
  • the VEGF antagonist is administered as a co- formulation with the APLNR antagonist.
  • the present disclosure includes methods comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist to a subject to provide a greater therapeutic effect or synergistic effect as compared to administration of the VEGF antagonist alone.
  • the subject may be on a therapeutic regimen of intravitreally administered VEGF antagonist.
  • the APLNR antagonist is added to this therapeutic regimen, wherein one or more intravitreal injections of the VEGF antagonist may be reduced or the duration between successive intravitreal injections may be increased.
  • the methods of the present disclosure are useful for treating or preventing vascular eye disorders in subjects that have been diagnosed with or are at risk of being afflicted with a vascular eye disorder.
  • the methods of the present disclosure demonstrate efficacy within 36 weeks of the initiation of the treatment regimen (with the initial dose administered at "week 0"), e.g., by the end of week 6, by the end of week 12, by the end of week 18, by the end of week 24, etc.
  • "efficacy” means that, from the initiation of treatment, the subject exhibits a loss of 10 or fewer letters on the Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart.
  • EDRS Early Treatment Diabetic Retinopathy Study
  • "efficacy” means a gain of one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or more) letters on the ETDRS chart from the time of initiation of treatment.
  • the therapeutic dosing regimen may comprise administering multiple doses of the pharmaceutical composition to the subject at a frequency of about once a 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 frequently.
  • the therapeutic dosing regimen may comprise administering multiple doses of the pharmaceutical composition to the subject at a frequency of once a day or 2 times a day or more.
  • the present disclosure includes methods for treating, preventing, or ameliorating at least one symptom or indication of a vascular eye disease or disorder in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist to the subject in need thereof.
  • the 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.
  • Exemplary small molecule inhibitors or antagonists of APLNR can be found in US Patent Publication Nos. US2014000518 and US20150125459 and International Patent Publication Nos. WO2004081 198 and WO2015140296.
  • Exemplary peptide inhibitors or antagonists of APLNR can be found in US Patent Nos. 9,593, 153 and 7,736,646 and International Patent Publication WO2004081 198.
  • Other APLNR antagonists include MM54, MM07, N-alpha-acetyl-nona-D-arginine amide acetate (ALX40-4C), ML221 , the mutant apelin-13(F13A), 4-oxo-6-((pyrimidin- 2-ylthio) methyl)-4H-pyran-3-yl-4- nitrobenzoate (MI221 ) and E339-3D6.
  • the APLNR antagonists include antibodies or antigen-binding fragments thereof selected from the group of antibodies disclosed in International Patent Publication No. WO 2015077491 , published May 5, 2015, which is specifically incorporated herein in its entirety and US Patent No. 9,493,554. In some
  • the antibodies are H2aM9232N, H4H9232N or H1 M9207N.
  • the anti-APLNR antibody is H4H9232N.
  • the APLNR antagonists include antibodies, or antigen-binding fragments thereof, selected from the group comprising a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ I D NO: 2 and 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • HCVR heavy chain variable region
  • the APLNR antagonists include antibodies or antigen-binding fragments thereof comprising a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 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.
  • LCVR light chain variable region
  • the antibody or antigen-binding fragment thereof comprises the heavy and light chain CDR sequences encoded by the nucleic acid sequences of SEQ ID NOs: 1 and 6 (e.g. H1 M9207N), and SEQ ID NOs: 12 and 17 ⁇ e.g. H2aM9232N or H4H9232N).
  • Certain non-limiting, exemplary antibodies and antigen-binding fragments of the disclosure comprise HCDR1 -HCDR2-HCDR3-LCDR1 -LCDR2-LCDR3 domains, respectively, having the amino acid sequences selected from the group consisting of: SEQ ID NOs: 3-4-5-8-9-10 (e.g. H1 M9207N); and SEQ ID NOs: 14-15-16-19-20-21 (e.g. H2aM9232N or H4H9232N).
  • the antibodies designated H2aM9232N and H4H9232N share the same human variable regions (HCVR and LCVR).
  • H2aM9232N comprises a murine lgG2a heavy chain constant region
  • H4H9232N comprises a human lgG4 heavy chain constant region.
  • the APLNR antagonists include antibodies or antigen-binding fragments thereof comprising a HCVR and LCVR (HCVR/LCVR) sequence pair selected from the group consisting of SEQ ID NO: 2/7, and 13/18.
  • the APLNR antagonists include antibodies or antigen-binding fragments thereof comprising a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 16, 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 CDR3 (LCDR3) domain having an amino acid sequence selected from the group consisting of SEQ I D NO: 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.
  • HCDR3 heavy chain CDR3
  • LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ I D NO: 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.
  • the APLNR antagonists include antibodies or antigen-binding fragments thereof comprises a HCDR3/LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NO: 5/10 and 16/21 .
  • the APLNR antagonists include antibodies or antigen-binding fragments thereof comprising a heavy chain CDR1 (HCDR1 ) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 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; a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from the group consisting of SEQ I D NO: 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; a light chain CDR1 (LCDR1 ) domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 9 and 20, or a substantially similar sequence thereof having at least 90%
  • Certain 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 the heavy and light chain CDR domains contained within heavy and light chain variable region (HCVR/LCVR) sequences selected from the group consisting of SEQ ID NO: 2/7 and 13/18.
  • HCVR/LCVR heavy and light chain variable region
  • Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition.
  • the Kabat definition is based on sequence variability
  • the Chothia definition is based on the location of the structural loop regions
  • the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat,
  • the present disclosure provides antibodies or antigen-binding fragments thereof comprising a heavy chain (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • HCVR heavy chain
  • the present disclosure also provides an antibody or antigen-binding fragment of an antibody comprising a light chain (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 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.
  • LCVR light chain
  • the present disclosure also provides an antibody or antigen-binding fragment thereof comprising a HC and LC (HC/LC) amino acid sequence pair.
  • the antibody or antigen-binding fragment thereof comphses the heavy and light chain CDR sequences encoded by the nucleic acid sequences of antibodies H1 M9207N, or H2aM9232N (or H4H9232N).
  • the present disclosure includes anti-APLNR antibodies having a modified glycosylation pattern.
  • modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al., 2002, JBC 277:26733).
  • ADCC antibody dependent cellular cytotoxicity
  • modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).
  • the present disclosure includes APLNR antagonists thereof that block or inhibit apelin-mediated signaling in cells expressing human APLNR, with an IC50 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 90 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 cell-based blocking or inhibition
  • the present disclosure includes APLNR antagonists that inhibit APLNR- mediated ratio of pERK, in the presence of apelin, with an IC50 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 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 an APLNR-induced pERK assay.
  • certain APLNR antagonists of the present disclosure despite having the ability to inhibit or attenuate APLNR-mediated signaling, do not block 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.”
  • the indirect blockers of the disclosure function by binding to APLNR at an epitope that does not overlap, or overlaps only partially, with the N-terminal ligand binding domain of APLNR, but nonetheless interferes with APLNR-mediated signaling without blocking the APLNR/apelin interaction directly.
  • the present disclosure includes APLNR antagonists that bind soluble APLNR molecules with high affinity and/or specificity.
  • the present disclosure includes antibodies and antigen-binding fragments of antibodies that bind APLNR with a binding ratio of greater than about 20 as measured by a fluorescent activated cell sorting (FACS) assay, e.g., using the assay format as defined in Example 4 of WO2015/077491 .
  • FACS fluorescent activated cell sorting
  • the antibodies or antigen- binding fragments of the present disclosure bind APLNR with a binding ratio of greater than about 15, greater than about 20, greater than about 100, greater than about 200, greater than about 300, greater than about 400, greater than about 500, greater than about 1000, greater than about 1500, or greater than about 2000, as measured by e.g., FACS, or a substantially similar assay.
  • the present disclosure also includes anti-APLNR antibodies and antigen- binding fragments thereof that specifically bind to APLNR with a dissociative half-life (t1 ⁇ 2) of greater than about 10 minutes as measured by surface plasmon resonance at 25°C or 37°C, e.g., using the well-known BIAcoreTMassay format, or a substantially similar assay.
  • t1 ⁇ 2 dissociative half-life
  • the antibodies of the present disclosure may possess one or more of the aforementioned biological characteristics, or any combinations thereof. Other biological characteristics of the antibodies of the present disclosure will be evident to a person of ordinary skill in the art from a review of the present disclosure including the working Examples herein.
  • the anti-APLNR antibodies may comprise 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 as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
  • the present disclosure includes antibodies and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, 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").
  • Germline mutations A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof.
  • all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived.
  • only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1 , CDR2 or CDR3.
  • one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).
  • the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
  • the present disclosure also includes anti-APLNR antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions.
  • the present disclosure includes anti-APLNR antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc.
  • epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope.
  • a single antigen may have more than one epitope.
  • different antibodies may bind to different areas on an antigen and may have different biological effects.
  • Epitopes may be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
  • a linear epitope is one produced by adjacent amino acid residues in a polypeptide chain.
  • an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
  • nucleic acid or fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below.
  • a nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.
  • the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • phenylalanine, tyrosine, and tryptophan (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine- arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
  • a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., 1992, Science 256: 1443-1445, herein incorporated by reference.
  • a “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
  • Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1 . Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1 .
  • FASTA e.g., FASTA2 and FASTA3
  • FASTA2 and FASTA3 provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 1994, supra).
  • Another preferred algorithm when comparing a sequence of the disclosure 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, e.g., Altschul et al., 1990, J. Mol. Biol. 215:403-410 and Altschul et al., 1997, Nucleic Acids Res. 25:3389-402, each herein incorporated by reference.
  • the present disclosure further includes anti-APLNR antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g. H1 M9207N and H2aM9232N and H4H9232N). Likewise, the present disclosure also includes anti-APLNR antibodies that compete for binding to APLNR with any of the specific exemplary antibodies described herein (e.g. H1 M9207N and H2aM9232N and H4H9232N).
  • test antibody may bind to the same epitope as the epitope bound by the reference anti-APLNR antibody of the disclosure. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, BIAcoreTM, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.
  • two antibodies bind to the same (or overlapping) epitope if, e.g., a 1 -, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., 1990, Cancer Res. 50: 1495-1502).
  • two antibodies are deemed to bind to the same epitope if essentially 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 deemed to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • an antibody competes for binding (or cross-competes for binding) with a reference anti-APLNR antibody the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to an APLNR protein under saturating conditions followed by assessment of binding of the test antibody to the APLNR molecule. In a second orientation, the test antibody is allowed to bind to an APLNR molecule under saturating conditions followed by assessment of binding of the reference antibody to the APLNR molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the APLNR molecule, then it is concluded that the test antibody and the reference antibody compete for binding to APLNR.
  • an antibody that competes for binding with 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 an overlapping or adjacent epitope.
  • the anti-APLNR antibodies of the disclosure 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.
  • the human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term “human antibody”, as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the antibodies discussed herein are human antibodies with an IgG heavy chain constant region. In some cases, the antibodies have a heavy chain constant region of human lgG1 or lgG4 isotype.
  • the antibodies of the disclosure may, in some embodiments, be any antibodies of the disclosure.
  • recombinant human antibody is intended to include all human antibodies thereof that are prepared, expressed, created 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 human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al., 1992, Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond.
  • the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody).
  • the frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody.
  • a single amino acid substitution in the hinge region of the human lgG4 hinge can significantly reduce the appearance of the second form (Angal et al., 1993, Molecular Immunology 30: 105) to levels typically observed using a human lgG1 hinge.
  • the instant disclosure encompasses 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 antibody form.
  • the antibodies of the disclosure may be isolated antibodies.
  • An "isolated antibody,” as used herein, means an antibody that has been identified and separated and/or recovered 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 an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an “isolated antibody” for purposes of the present disclosure.
  • An isolated antibody also includes an antibody in situ within a
  • Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • a “neutralizing” or “blocking” antibody is intended to refer to an antibody whose binding to APLNR: (i) interferes with the interaction between APLNR or an APLNR fragment and an APLNR receptor component (e.g., apelin peptide, etc.); and/or (ii) results in inhibition of at least one biological function of APLNR.
  • the inhibition caused by an APLNR neutralizing or blocking antibody need not be complete so long as it is detectable using an appropriate assay.
  • VEGF antagonist is any agent that binds to 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 which interfere with the interaction between VEGF and a natural VEGF receptor, e.g., molecules which bind to VEGF or a VEGF receptor and prevent or otherwise hinder the interaction between VEGF and a VEGF receptor.
  • Specific exemplary VEGF antagonists include anti-VEGF antibodies (e.g., ranibizumab
  • anti-VEGF receptor antibodies e.g., anti-VEGFR1 antibodies, anti- VEGFR2 antibodies, etc.
  • small molecule inhibitors of VEGF e.g., sunitinib
  • VEGF receptor-based chimeric molecules or VEGF-inhibiting fusion proteins also referred to herein as "VEGF-Traps”
  • VEGF-Traps VEGF-Traps
  • aflibercept and ziv-aflibercept 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 U.S. Patent Nos.
  • Additional VEGF inhibitors and/or antagonist include the small molecules inhibitors: pazopanib, sorafenib, axitinib, ponatinib, regorafenib, cabozantinib, vandetanib, cabozantinib, and lenvatinib; and VEGF inhibitory antibodies: bevacizumab and ramucirumab, or biosimilar molecules thereof.
  • VEGF receptor-based chimeric molecules include chimeric polypeptides which comprise two or more immunoglobulin (Ig)-like domains of a VEGF receptor such as VEGFR1 (also referred to as Flt1 ) and/or VEGFR2 (also referred to as Flk1 or KDR), and may also contain a multimerizing domain (e.g., an Fc domain which facilitates the multimerization [e.g., dimerization] of two or more chimeric VEGFR1 (also referred to as Flt1 ) and/or VEGFR2 (also referred to as Flk1 or KDR), and may also contain a multimerizing domain (e.g., an Fc domain which facilitates the multimerization [e.g., dimerization] of two or more chimeric VEGFR1 (also referred to as Flt1 ) and/or VEGFR2 (also referred to as Flk1 or KDR), and may also contain a multimerizing domain (e.g., an
  • VEGFR1 R2-FcAC1 (also known as aflibercept; marketed under the product name EYLEA®).
  • aflibercept comprises the amino acid sequence set forth as
  • the amount of the VEGF antagonist contained within the pharmaceutical formulations of the present disclosure may vary depending on the specific properties desired of the formulations, as well as the particular circumstances and purposes for which the formulations are intended to be used.
  • the pharmaceutical formulations are liquid formulations that may contain 5 ⁇ 0.75 mg/mL to 150 ⁇ 22.5 mg/mL of VEGF antagonist; 10 ⁇ 1 .5 mg/mL to 100 ⁇ 15.0 mg/mL of VEGF antagonist; 20 ⁇ 3 mg/mL to 80 ⁇ 12 mg/mL of VEGF antagonist; 30 ⁇ 4.5 mg/mL to 70 ⁇ 10.5 mg/mL of VEGF antagonist or 40 ⁇ 6.0 mg/mL of the VEGF antagonist.
  • the formulations of the present disclosure may comprise about 20 mg/mL; about 30 mg/mL; about 40 mg/mL; about 50 mg/mL; or about 60 mg/mL of a VEGF antagonist.
  • the methods of the present disclosure comprise administering to a subject in need thereof a therapeutic composition comprising an VEGF antagonist.
  • the present disclosure provides pharmaceutical formulations comprising at least one APLNR antagonist, such as an anti-APLNR antibody, or an antigen-binding fragment thereof, which binds specifically to human APLNR. According to certain other embodiments, the present disclosure provides pharmaceutical formulations comprising additional therapeutic agents. The present disclosure further provides pharmaceutical formulations comprising at least one VEGF antagonist, for example, for use in conjunction with a pharmaceutical formulation comprising at least one APLNR antagonist.
  • compositions of the disclosure are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
  • suitable carriers excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
  • a multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTI NTM, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various 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 52:238-31 1 .
  • APLNR antagonist such as an anti-APLNR antibody, or an antigen-binding fragment thereof, which binds specifically to human APLNR
  • administered to a subject may vary depending upon the age and the size of the subject, target disease, conditions, route of administration, and the like.
  • the preferred dose is typically calculated according to body weight or body surface area.
  • an APLNR antagonist when used for treating a condition or disease, it may be advantageous to intravenously administer the antibody of the present disclosure normally at a single dose of about 0.01 to about 50 mg/kg body weight. In other instances it may be advantageous to administer an APLNR antagonist intravitreally, for example at a concentration of about 0.01 to about 50 mg/kg body weight.
  • Effective dosages and schedules for administering APLNR antagonist, such as anti-APLNR antibodies may be determined empirically; for example, subject progress can be monitored by periodic assessment, and the dose adjusted accordingly.
  • interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991 ,
  • the dose of VEGF antagonist may vary depending upon the age and the size of the subject, target disease, conditions, route of administration, and the like.
  • the preferred dose is typically calculated according to body weight or body surface area.
  • a VEGF antagonist when used for treating a condition or disease, it may be advantageous to intravenously administer the antibody of the present disclosure normally at a single dose of about 0.01 to about 50 mg/kg body weight. In other instances it may be advantageous to administer a VEGF antagonist intravitreally, for example at a concentration of about 0.01 to about 50 mg/kg body weight.
  • the frequency and the duration of the treatment can be adjusted.
  • Effective dosages and schedules for administering VEGF antagonist may be determined empirically; for example, subject progress can be monitored by periodic assessment, and the dose adjusted accordingly.
  • Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, intravitreal, subcutaneous, intranasal, epidural, and oral routes.
  • composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • Administration can be systemic or local.
  • the present disclosure includes methods which comprise administering an APLNR antagonist to a subject wherein the APLNR antagonist is contained within a pharmaceutical composition.
  • the pharmaceutical composition in certain embodiments, the pharmaceutical
  • composition further comprises a VEGF antagonist.
  • the APLNR antagonist and the VEGF antagonist may each be in its own separate pharmaceutical dosage formulation.
  • the pharmaceutical compositions of the disclosure may be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various 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 52:238-31 1 .
  • the expression "pharmaceutical formulation” means a combination of at least one active ingredient (e.g., a small molecule, macromolecule, compound, etc. which is capable of exerting a biological effect in a human or non- human animal), and at least one inactive ingredient which, when combined with the active ingredient or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal.
  • active ingredient e.g., a small molecule, macromolecule, compound, etc. which is capable of exerting a biological effect in a human or non- human animal
  • inactive ingredient which, when combined with the active ingredient or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal.
  • formulation means “pharmaceutical formulation” unless specifically indicated otherwise.
  • APLNR antagonist such as anti-APLNR antibody, or antigen- binding fragment thereof, contained within the pharmaceutical formulations of the present disclosure may vary depending on the specific properties desired of the formulations, as well as the particular circumstances and purposes for which the formulations are intended to be used.
  • the pharmaceutical formulations are liquid formulations that may contain 5 ⁇ 0.75 mg/mL to 150 ⁇ 22.5 mg/mL of antibody; 7.5 ⁇ 1 .125 mg/mL to 140 ⁇ 21 mg/mL of antibody; 10 ⁇ .5 mg/mL to 130 ⁇ 19.5 mg/mL of antibody; 10 ⁇ .5 mg/mL of antibody; 20 ⁇ 3 mg/mL of antibody; 60 ⁇ 9 mg/mL of antibody; or 120 ⁇ 18 mg/mL of antibody.
  • the pharmaceutical formulations are liquid formulations that may contain 5 ⁇ 0.75 mg/mL to 150 ⁇ 22.5 mg/mL of antibody; 7.5 ⁇ 1 .125 mg/mL to 140 ⁇ 21 mg/mL of antibody; 10 ⁇ .5 mg/mL to 130 ⁇ 19.5 mg/mL of antibody; 10 ⁇ .5 mg/mL of antibody; 20 ⁇ 3 mg/mL of antibody; 60 ⁇ 9 mg/mL of antibody; or 120 ⁇ 18 mg/mL of antibody.
  • the pharmaceutical formulations are liquid formulations that may contain 5 ⁇ 0.75 mg
  • formulations of the present disclosure may comprise 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 an antigen-binding fragment thereof that binds specifically to human APLNR.
  • the pharmaceutical formulations are liquid formulations that may contain 5 ⁇ 0.75 mg/mL to 100 ⁇ 15 mg/mL of a VEGF antagonist.
  • the formulations of the present disclosure may comprise 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.
  • the pharmaceutical formulations are stable liquid co-formulations comprising about 5 mg/mL to about 150 mg/mL of the APLNR antagonist and about 5 to 100 mg/mL of the VEGF antagonist.
  • the pharmaceutical formulations of the present disclosure comprise one or more excipients.
  • excipient means any non-therapeutic agent added to the formulation to provide a desired consistency, viscosity or stabilizing effect.
  • Exemplary formulations comprising a VEGF antagonist that can be used in the context of the present disclosure are disclosed, e.g., in U.S. Pat. Nos. 7,531 , 173 and 7,608,261 .
  • Exemplary pharmaceutical compositions comprising an APLNR antagonist that can be used in the context of the present disclosure are disclosed, e.g., in US Patent Application Publication No. 20130186797.
  • compositions comprising an APLNR antagonist that can be used in the context of the present disclosure are disclosed, e.g., in International Patent Publication No. WO2016085750 and US Patent Application Publication No.
  • the methods of the present disclosure comprise administering to the subject an APLNR antagonist in combination with a VEGF antagonist.
  • a VEGF antagonist in combination with a VEGF antagonist.
  • the expression "in combination with” means that the VEGF antagonist is administered before, after, or concurrent with the
  • the pharmaceutical composition comprising the 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 prior to the administration of the pharmaceutical composition comprising the VEGF antagonist.
  • the pharmaceutical composition comprising the APLNR antagonist may be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 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 the administration of the pharmaceutical composition comprising the VEGF antagonist.
  • Administration "concurrent" with the pharmaceutical composition comprising the APLNR antagonist means that the VEGF antagonist is administered to the subject in a separate dosage form within less than 5 minutes (before, after, or at the same time) of administration of the pharmaceutical composition comprising the APLNR antagonist, or administered to the subject as a single combined dosage formulation comprising the APLNR antagonist and the VEGF antagonist.
  • Combination therapies may include an APLNR antagonist and a VEGF antagonist (e.g., aflibercept, a VEGF-Trap, see, e.g., U.S. Pat. No. 7,087,41 1 (also referred to herein as a "VEGF-inhibiting fusion protein"), anti-VEGF antibody (e.g., ranibizumab, etc.), a small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib or pazopanib), etc.
  • a VEGF antagonist e.g., aflibercept, a VEGF-Trap, see, e.g., U.S. Pat. No. 7,087,41 1 (also referred to herein as a "VEGF-inhibiting fusion protein"
  • anti-VEGF antibody e.g., ranibizumab, etc.
  • the methods of the disclosure comprise administering an APLNR antagonist in combination with a VEGF antagonist for additive or synergistic activity to treat or ameliorate at least one symptom or indication 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.
  • 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
  • Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, micro-particles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262: 4429-4432).
  • Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, intravitreal, subcutaneous, intranasal, epidural, and oral routes.
  • composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
  • infusion or bolus injection by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • the pharmaceutical formulations of the disclosure may be administered, e.g., by eye drops, subconjunctival injection, subconjunctival implant, intravitreal injection, intravitreal implant, sub-Tenon's injection or sub-Tenon's implant.
  • a pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe.
  • a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure.
  • Such a pen delivery device can be reusable or disposable.
  • a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been
  • the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition.
  • the pen delivery device can then be reused.
  • a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used.
  • polymeric materials can be used; see, Medical Applications of
  • a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 1 15-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527-1533.
  • the injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc.
  • injectable preparations may be prepared by known methods.
  • the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections.
  • a sterile aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g.
  • polysorbate 80 polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • oily medium there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients.
  • dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
  • the pharmaceutical formulations of the present disclosure may be contained within any container suitable for storage or administration of medicines and other therapeutic compositions.
  • the pharmaceutical formulations may be contained within a sealed and sterilized plastic or glass container having a defined volume such as a vial, ampule, syringe, cartridge, bottle, or IV bag.
  • a vial e.g., clear and opaque (e.g., amber) glass or plastic vials.
  • any type of syringe can be used to contain or administer the pharmaceutical formulations of the present disclosure.
  • the pharmaceutical formulations of the present disclosure may be contained within "normal tungsten" syringes or "low tungsten” syringes.
  • the process of making glass syringes generally involves the use of a hot tungsten rod which functions to pierce the glass thereby creating a hole from which liquids can be drawn and expelled from the syringe. This process results in the deposition of trace amounts of tungsten on the interior surface of the syringe. Subsequent washing and other processing steps can be used to reduce the amount of tungsten in the syringe.
  • normal tungsten means that the syringe contains greater than or equal to 500 parts per billion (ppb) of tungsten.
  • low tungsten means that the syringe contains less than 500 ppb of tungsten.
  • a low tungsten syringe can 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 fewer ppb of tungsten.
  • the rubber plungers used in syringes, and the rubber stoppers used to close the openings of vials may be coated to prevent contamination of the medicinal contents of the syringe or vial, or to preserve their stability.
  • pharmaceutical formulations of the present disclosure may be contained within a syringe that comprises a coated plunger, or within a vial that is sealed with a coated rubber stopper.
  • the plunger or stopper may be coated with a fluorocarbon film. Examples of coated stoppers or plungers suitable for use with vials and syringes containing the pharmaceutical formulations of the present disclosure are mentioned in, e.g., U.S. Patent Nos.
  • FluroTec® is an example of a flurocarbon coating used to minimize or prevent drug product from adhering to the rubber surfaces.
  • compositions may be contained within a low tungsten syringe that comprises a fluorocarbon-coated plunger.
  • the pharmaceutical formulations can be administered to a subject by parenteral routes such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or percutaneous, mucosal, nasal, pulmonary or oral routes.
  • parenteral routes such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or percutaneous, mucosal, nasal, pulmonary or oral
  • Numerous reusable pen or autoinjector delivery devices can be used to subcutaneously deliver the pharmaceutical formulations of the present disclosure. Examples include, but are not limited to AUTOPENTM (Owen Mumford, Inc.,
  • OPTIPEN PROTM OPTIPEN STARLETTM
  • OPTICLIKTM OPTI-aventis, Frankfurt, Germany
  • disposable pen or autoinjector delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present disclosure include, but are not limited to the SOLOSTARTM pen (sanofi- aventis), the FLEXPENTM (Novo Nordisk), and the KWIKPENTM (Eli Lilly), the SURECLICKTM Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLETTM
  • microinfusor 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 a prolonged period of time (e.g., about 10, 15, 20, 25, 30 or more minutes). See, e.g., U.S. Pat. No. 6,629,949; U.S. Pat. No. 6,659,982; and Meehan et al., J. Controlled Release 46: 107-1 16 (1996). Microinfusors are particularly useful for the delivery of large doses of therapeutic proteins contained within high concentration (e.g., about 100, 125, 150, 175, 200 or more mg/mL) or viscous solutions.
  • high concentration e.g., about 100, 125, 150, 175, 200 or more mg/mL
  • the pharmaceutical formulation is administered via an IV drip, such that the formulation is diluted in an IV bag containing a physiologically acceptable solution.
  • pharmaceutical composition is a
  • the infusion bag is made of a polyvinyl chloride (e.g., VIAFLEX, Baxter, Deerfield, III.). In some embodiments, the infusion bag is made of a polyolefin (EXCEL IV Bags, Braun Medical Inc., Bethlehem, Pa.).
  • the present disclosure includes methods comprising administering to a subject a pharmaceutical composition comprising an APLNR antagonist, such as an anti-APLNR antibody, or antigen-binding fragment thereof, at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently so long as a therapeutic response is achieved.
  • the methods involve the administration of a pharmaceutical composition comprising an APLNR antagonist, such as an anti-APLNR antibody, or antigen-binding fragment thereof, at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently so long as a therapeutic response is achieved.
  • the methods involve the administration of a pharmaceutical composition comprising an APLNR
  • a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every nine weeks, once every twelve weeks, or less frequently so long as a therapeutic response is achieved.
  • multiple doses of an APLNR antagonist such as an anti-APLNR antibody, or antigen-binding fragment thereof, and a VEGF antagonist
  • the methods according to this aspect of the disclosure comprise 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.
  • sequentialially administering 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 the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months).
  • an APLNR antagonist such as an anti-APLNR antibody, or antigen- binding fragment thereof, and a VEGF antagonist
  • the present disclosure includes methods which comprise sequentially administering to the subject a single initial dose of an APLNR antagonist, such as an anti-APLNR antibody, or antigen-binding fragment thereof, and a VEGF antagonist, followed by one or more secondary doses of the APLNR antagonist, such as an anti-APLNR antibody, or antigen-binding fragment thereof, and the VEGF antagonist, and optionally followed by one or more tertiary doses of the APLNR antagonist, such as an anti-APLNR antibody, or antigen-binding fragment thereof, and the VEGF antagonist.
  • an APLNR antagonist such as an anti-APLNR antibody, or antigen-binding fragment thereof
  • a VEGF antagonist sequentially administering to the subject a single initial dose of an APLNR antagonist, such as an anti-APLNR antibody, or antigen-binding fragment thereof, and a VEGF antagonist
  • secondary doses of the APLNR antagonist such as an anti-APLNR antibody, or antigen-binding fragment thereof
  • 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
  • a co-formulation comprising an APLNR antagonist such as an anti-APLNR antibody, or antigen-binding fragment thereof, and a VEGF antagonist
  • the methods according to this aspect of the disclosure comprise 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.
  • sequentially administering means that each dose of the APLNR antagonist in combination with the VEGF antagonist is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months).
  • the present disclosure includes methods which comprise sequentially administering to the subject a single initial dose of a co- formulation comprising an APLNR antagonist and a VEGF antagonist, followed by one or more secondary doses of the co-formulated APLNR antagonist and a VEGF antagonist, and optionally followed by one or more tertiary doses of the co- formulated APLNR antagonist and a VEGF antagonist.
  • the terms "initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration.
  • the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”);
  • the “secondary doses” are the doses which are administered after the initial dose;
  • the “tertiary doses” are the doses which are administered after the secondary doses.
  • the initial, secondary, and tertiary doses may all contain the same amount of APLNR antagonist (or a co-formulation comprising APLNR antagonist and VEGF antagonist), but generally may differ from one another in terms of frequency of administration.
  • the amount contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment.
  • one or more (e.g., 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 on a less frequent basis (e.g., "maintenance doses").
  • an APLNR antagonist or a co-formulation comprising APLNR antagonist and VEGF antagonist
  • each secondary and/or tertiary dose is administered 1 to 14 (e.g. , 1 , 1 1 ⁇ 2, 2, 2 1 / 2 , 3, 3 1 / 2 , 4, 4 1 / 2 , 5, 51 ⁇ 2, 6, 6 1 / 2 , 7, 71 ⁇ 2, 8, 81 ⁇ 2, 9, 91 ⁇ 2, 10, 101 ⁇ 2, 1 1 , 1 1 1 ⁇ 2, 12, 121 ⁇ 2, 13, 131 ⁇ 2, 14, 141 ⁇ 2, or more) weeks after the immediately preceding dose.
  • 1 to 14 e.g. , 1 , 1 1 ⁇ 2, 2, 2 1 / 2 , 3, 3 1 / 2 , 4, 4 1 / 2 , 5, 51 ⁇ 2, 6, 6 1 / 2 , 7, 71 ⁇ 2, 8, 81 ⁇ 2, 9, 91 ⁇ 2, 10, 101 ⁇ 2, 1 1 , 1 1 1 ⁇ 2, 12, 121 ⁇ 2, 13, 131 ⁇ 2, 14, 141 ⁇ 2, or more
  • the immediately preceding dose means, in a sequence of multiple administrations, the dose of APLNR antagonist (or a co-formulation comprising APLNR antagonist and VEGF antagonist) which is administered to a subject prior to the administration of the very next dose in the sequence with no intervening doses.
  • the methods according to this aspect of the disclosure may comprise administering to a subject any number of secondary and/or tertiary doses of an anti- APLNR antagonist (or a co-formulation comprising APLNR antagonist and VEGF antagonist). For example, in certain embodiments, only a single secondary dose is administered to the subject.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the subject.
  • only a single tertiary dose is administered to the subject.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the subject.
  • each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the subject 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the subject 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a subject can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual subject following clinical examination.
  • the present disclosure includes methods comprising sequential
  • the present methods comprise administering one or more doses of an APLNR antagonist followed by one or more doses of a VEGF antagonist. In certain embodiments, the present methods comprise administering a single dose of a VEGF antagonist followed by one or more doses of an APLNR antagonist.
  • a retinal vascular development (RVD) model was used to evaluate the effects of an antagonistic anti-APLNR antibody on blood vessel outgrowth in the normal developing retina of mouse pups that were of a mixed background strain (75% C57BL6 and 25% Sv129) and homozygous for expression of human APLNR in place of mouse APLNR (humanized APLNR mice).
  • Humanized (Hu) ApelinR mice were systemically (I P) injected with 25 mg/kg and 50 mg/kg anti-Apelin Receptor antibody (aAR; H2aM9232N) at postnatal day (P)2. Reagents were masked and labeled as Solution A and Solution B to prevent experimenter bias. At postnatal day 5, tissue samples were collected and then fixed in PBS containing 4% paraformaldehyde.
  • aAR anti-Apelin Receptor antibody
  • Figures 1A and 1 B are representative photomicrographs of mouse retinas treated systemically at P2 to P5 and a graph of the calculated vascular area (images at 20x, for quantification, and 40x; statistical analysis was done with Student T-test).
  • Residual vascular area was significantly smaller in aApelinR (23% at 25 mg/kg, p ⁇ 0.05) retinas compared to untreated retinas.
  • Selective inhibition of ApelinR via systemic injection delayed normal vascular outgrowth in the developing retina in P5 pups.
  • blocking ApelinR slightly inhibits vascular outgrowth.
  • FIGS. 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 smaller in aApelinR (35% at 50 mg/kg, p ⁇ 0.005) retinas compared to untreated retinas.
  • Retinal endothelial cells were stained with GS Lectin I. Images were taken at 20x (for quantification) and 40x. Statistical analysis was done with Student T-test. Selective inhibition of ApelinR via systemic injection delayed normal vascular outgrowth in the developing retina in P5 pups. By increasing the dose to 50mg/kg Dose, targeting ApelinR further delays retinal vascular outgrowth.
  • FIGS. 3A and 3B are representative photomicrographs of mouse retinas treated systemically at P2 to P5 and a graph of the calculated vascular area.
  • 50 mg/kg aApelinR decreased vascular growth by 29.8% (p ⁇ 0.0001 ) compared to Fc treated controls.
  • Retinal endothelial cells were stained with GS Lectin I. Images were taken at 20x (for quantification) and 40x. Statistical analysis was done with Student T-test. Selective inhibition of ApelinR via systemic injection delays normal vascular outgrowth in the developing retina in P5 pups.
  • FIGS. 4A and 4B 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 smaller in combination (aApelinR + aflibercept) (62% at 50 mg/kg, p ⁇ 0.0001 ) compared to single reagents, aApelinR (31 % at 50mg/kg, p ⁇ 0.001 ) or aflibercept (43% at 50mg/kg, p ⁇ 0.005) alone.
  • Retinal endothelial cells were stained with GS Lectin I. Note the effects on dose and relational vascularized area. Images were taken at 20x (for quantification) and 40x. Statistical analysis was done with one-way ANOVA with post-hoc Tukey test.
  • mice were treated IVT with aApelinR
  • FIGS. 5A and 5B are representative photomicrographs of mouse retinas treated intravitreally injected at P4 to P6 and a graph of the calculated vascular area. Residual vascular area was significantly smaller in combination (aApelinR + aflibercept) (68% at 5 ⁇ g, p ⁇ 0.0001 ) compared to single reagents, aApelinR (43% at 5 ⁇ g, p ⁇ 0.0001 ) or aflibercept (65% at 5ug, p ⁇ 0.0001 ) alone.
  • the murine model of oxygen-induced ischemic retinopathy is a well characterized model of pathological neovascularization associated with elevated expression of the essential pro-angiogenic factor, VEGF (Smith et al. 1994 Invest Ophthalmol Vis Sci 35: 101 -1 1 1 ; Neely et al. 1998 Am J. Pathol 153:665-670; Saint- Geniez et al. 2004 Int J Dev Biol 48: 1045-1058) and thus relevant to pathological angiogenesis associated with diverse disease conditions (Ferrarra et al. 2005 Nature 438:967-974).
  • VEGF essential pro-angiogenic factor
  • C57/BI6 mice (Taconic) were used to study the effect of VEGF trap or neutralizing APLNR antibody on retinal neovascularization in OIR.
  • OIR was produced following the method developed by Smith et al. 1994 (supra). Briefly, humanized mouse pups were placed in a hyperoxic environment (75% O2) at P6 and returned to room air at P1 1 . Exposure to hyperoxia induces rapid
  • FIGS. 6A and 6B are representative photomicrographs of OIR mouse retinas treated systemically at P12 to P16 and graphs 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 to Fc (control) retinas. Notice the significant decrease in neovascular tufts in aApelinR (67%, p ⁇ 0.0001 ) and aflibercept (94%, p ⁇ 0.0005) treated samples compared to Fc. Retinal endothelial cells were stained with GS Lectin I. Images were taken at 20x (Avascular area quantification) and 40x (Abnormal neovascular area quantification). Statistical analysis was done with one-way ANOVA with post-hoc Tukey test.
  • ApelinR In both systemic and intravitreal administration, selective inhibition of ApelinR promotes retinal vascular regrowth and reduces neovascularizations in OIR mice.
  • ApelinR and VEGFA inhibition via systemic injection promotes retinal vascular regrowth and reduces abnormal neovascularizations.
  • FIGS. 7A and 7B are representative photomicrographs of OIR mouse retinas treated intravitreally at P12 to P16 and graphs of calculated avascular area.
  • Avascular area was significantly decreased in aApelinR (27.5% p ⁇ 0.05) and increased in aflibercept (32%, p ⁇ 0.0001 ) conditions compared to Fc controls. Notice the significant decrease 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 taken at 20x (Avascular area
  • ApelinR inhibition promotes retinal vascular regrowth and reduces neovascularizations in OIR mice.
  • ApelinR inhibition via intravitreal injection improves vascular regrowth and decreases abnormal neovascularizations, while VEGFA inhibition arrests vascular regrowth and completely halts any neovascularizations.
  • a combination of APLNR antagonist and VEGF trap e.g. aflibercept, also regressed pathological angiogenesis and promoted normal vascular regrowth in an OIR model, as discussed in Example 7. This combination is therefore expected to treat pathological angiogenesis as seen in various eye diseases, including
  • mice When mice are returned to room air (P1 1 ), the loss of vessels from the central retina results in severe hypoxia / ischemia which in turn stimulates the pathological vascular changes.
  • Pups were injected systemically with 50 mg/kg Fc (control), aAPLNR (H4H9232N), or aflibercept (VEGF-Trap), or 25 mg/kg of a combination of aAPLNR and aflibercept at P12 and collected at P16.
  • Retinal endothelial cells were stained with GS Lectin I .
  • Figures 8A and 8B are representative photomicrographs (20x) of OIR mouse retinas treated systemically at P12 to P16 and graphs of the calculated avascular area.
  • the rate of regrowth with the combination treatment is similar to each agent alone, and to that end, vascular regrowth is improved in all treatment conditions compared to hFc-treated control retinas, with combination treatment showing a slightly more decreased average avascular area.
  • Figure 8B shows that there were significant changes in avascular area between treatment groups (p ⁇ 0.0005). Vessel area was significantly decreased with anti-APLNR antibody (**, p ⁇ 0.05), with VEGF Trap (***, p ⁇ 0.005), and with the combination (***, p ⁇ 0.05) compared to Fc control.
  • Statistical analysis was done with One-way ANOVA with post-hoc Tukey test.
  • Figures 9A and 9B are representative photomicrographs (40x) of OIR mouse retinas treated systemically at P12 to P16 and graphs of the calculated abnormal vascular area.
  • the combination treatment shows fewer pruned vessels compared to VEGF Trap, and fewer abnormal neovascularizations compared to anti-APLNR and Fc control (Figure 9A).
  • Figure 9B shows that there were significant changes in abnormal vascular area between treatment groups (p ⁇ 0.0005).
  • Abnormal vascular area was significantly decreased with anti-APLNR (***, p ⁇ 0.0005), with VEGF Trap (*** p ⁇ 0.005), and with the combination (***, p ⁇ 0.0005) compared to Fc control.
  • the abnormal vascular area was also significantly decreased in the combination treatment group (*, p ⁇ 0.05) compared to the anti-APLNR treatment group.
  • Figure 10 is representative of the image processing and measurement techniques used to calculate vessel area and vessel length.
  • the "original images” are the 40x photomicrographs shown in Figure 9A.
  • the "threshold images” and the “binary images” are discussed more specifically in connection with Figures 1 1 A, 1 1 B, 12A and 12B, below.
  • Figures 1 1A and 1 1 B are representative processed images showing the organization and uniformity of vessels from the photomicrographs (40x) of OIR mouse retinas treated systemically at P12 to P16 and graphs of the calculated vessel area.
  • the combination of anti-APLNR antibody and VEGF Trap produced retinal vessels that are more organized and uniform compared to anti-APLNR alone, and less sparse (with fewer pruned vessels) compared to VEGF Trap alone.
  • Figure 1 1 B shows that there were significant changes in vessel area between the 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 the combination treatment (*, p ⁇ 0.05) compared to Fc control.
  • Vessel area was significantly decreased with VEGF Trap (####, p ⁇ 0.0005) and with the combination (####, p ⁇ 0.0005) compared to anti-APLNR.
  • vessel area was significantly increased with the combination treatment (&&&, p ⁇ 0.005) compared to VEGF Trap.
  • Statistical analysis was done with One-way ANOVA with post-hoc Tukey test.
  • FIGs 12A and 12B are representative processed images showing the density of vessels from the photomicrographs (40x) of OIR mouse retinas treated systemically at P12 to P16 and graphs of the calculated vessel length.
  • the combination of anti-APLNR antibody and VEGF Trap produced retinal vessels of intermediate density compared to anti-APLNR (greater density) or VEGF Trap (lesser density) alone.
  • Figure 12B shows that there were significant changes in vessel length between the treatment groups (p ⁇ 0.0005). Vessel length was significantly decreased with VEGF Trap (####, p ⁇ 0.0005) and with the combination (####, p ⁇ 0.0005) compared to anti-APLNR. In contrast, vessel length was significantly increased with the combination treatment (&&&, p ⁇ 0.005) compared to VEGF Trap.
  • Statistical analysis was done with One-way ANOVA with post-hoc Tukey test.
  • the combination of an anti-APLNR antibody and a VEGF Trap promotes revascularization, leading to a decrease in avascular area compared to Fc controls.
  • the combination treatment inhibits pathological neovascularization, and produces an intermediate effect on vessel area and vessel length, compared to anti-APLNR antibody or VEGF Trap treatments alone.
  • the combination treatment promotes
  • microvessel growth between the major blood vessels compared to VEGF Trap treatment, and improves blood vessel organization and uniformity compared to anti- APLNR antibody treatment.

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Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4997423A (en) 1986-10-15 1991-03-05 Daikyo Gomu Seiko Ltd. Laminated sliding stopper for a syringe
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US5908686A (en) 1992-01-23 1999-06-01 Daikyo Gomu Seiko, Ltd Modified polysiloxane composition and a sanitary rubber article coated with the composition
US5952199A (en) 1996-05-07 1999-09-14 Genentech, Inc. Chimeric receptors as inhibitors of vascular endothelial growth factor activity, and processes for their production
US6286699B1 (en) 1995-04-05 2001-09-11 Daikyo Seiko, Ltd. Laminated rubber stopper
US6629949B1 (en) 2000-05-08 2003-10-07 Sterling Medivations, Inc. Micro infusion drug delivery device
US6645635B2 (en) 2001-01-19 2003-11-11 Daikyo Seiko, Ltd. Laminated rubber stopper for a medicament vial
US6659982B2 (en) 2000-05-08 2003-12-09 Sterling Medivations, Inc. Micro infusion drug delivery device
WO2004081198A2 (en) 2003-03-12 2004-09-23 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods for modulating angiogenesis with apelin compositions
US7070959B1 (en) 1999-06-08 2006-07-04 Regeneron Pharmaceuticals, Inc. Modified chimeric polypeptides with improved pharmacokinetic properties
US7087411B2 (en) 1999-06-08 2006-08-08 Regeneron Pharmaceuticals, Inc. Fusion protein capable of binding VEGF
US7226554B2 (en) 1999-01-29 2007-06-05 Daikyo Seiko, Ltd. Molding die assembly for rubber members and rubber member produced thereby
US7306799B2 (en) 1999-06-08 2007-12-11 Regeneron Pharmaceuticals, Inc. Use of VEGF inhibitors for treatment of eye disorders
US7531173B2 (en) 2005-02-02 2009-05-12 Regeneron Pharmaceuticals, Inc. Ophthalmic composition of a VEGF antagonist
US7608261B2 (en) 2006-06-16 2009-10-27 Regeneron Pharmacuticals, Inc. VEGF antagonist formulations suitable for intravitreal administration
US20110027286A1 (en) 2009-07-29 2011-02-03 Regeneron Pharmaceuticals, Inc. High Affinity Human Antibodies to Human Angiopoietin-2
US20130186797A1 (en) 2012-01-23 2013-07-25 Regeneron Pharmaceuticals, Inc. Stabilized Formulations Containing Anti-Ang2 Antibodies
US20140000518A1 (en) 2012-06-27 2014-01-02 Tokyo Electron Limited Coating apparatus and method for filling coating liquid
WO2014121087A1 (en) 2013-02-01 2014-08-07 Regeneron Pharmaceuticals, Inc. Antibodies comprising chimeric constant domains
US20150125459A1 (en) 2009-02-27 2015-05-07 The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv Apelin peptides, antibodies thereto, and methods of use
WO2015077491A1 (en) 2013-11-20 2015-05-28 Regeneron Pharmaceuticals, Inc. Aplnr modulators and uses thereof
WO2015140296A2 (en) 2014-03-20 2015-09-24 Centre National De La Recherche Scientifique (Cnrs) Use of compounds inhibiting apelin / apj / gp130 signaling for treating cancer
US20160144025A1 (en) * 2014-11-25 2016-05-26 Regeneron Pharmaceuticals, Inc. Methods and formulations for treating vascular eye diseases
US9493554B2 (en) 2011-07-18 2016-11-15 Amgen Inc. Methods of treating retinal disorders with anti-apelin antibodies
US9593153B2 (en) 2012-04-11 2017-03-14 Inserm (Institut National De La Sante Et De La Recherche Medicale) Modified apelin polypeptides

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EA201591700A1 (ru) * 2013-03-14 2015-12-30 Ридженерон Фармасьютикалз, Инк. Гибридные белки апелина и их применение
BR112018005737A2 (pt) * 2015-09-23 2018-10-09 Genentech Inc anticorpos, polinucleotídeo, vetor, célula hospedeira, método para produzir o anticorpo, para reduzir ou inibir a angiogênese, para tratar um distúrbio associado à angiogênese, para inibir a permeabilidade vascular, composição, conjugado de anticorpo, proteína de fusão, para identificar uma alteração de resíduos, utilização do anticorpo, utilização do conjugado e utilização da proteína

Patent Citations (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4997423A (en) 1986-10-15 1991-03-05 Daikyo Gomu Seiko Ltd. Laminated sliding stopper for a syringe
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US5908686A (en) 1992-01-23 1999-06-01 Daikyo Gomu Seiko, Ltd Modified polysiloxane composition and a sanitary rubber article coated with the composition
US6286699B1 (en) 1995-04-05 2001-09-11 Daikyo Seiko, Ltd. Laminated rubber stopper
US5952199A (en) 1996-05-07 1999-09-14 Genentech, Inc. Chimeric receptors as inhibitors of vascular endothelial growth factor activity, and processes for their production
US6100071A (en) 1996-05-07 2000-08-08 Genentech, Inc. Receptors as novel inhibitors of vascular endothelial growth factor activity and processes for their production
US6383486B1 (en) 1996-05-07 2002-05-07 Genentech, Inc. Inhibitors of vascular endothelial growth factor activity, their uses and processes for their production
US7771721B2 (en) 1996-05-07 2010-08-10 Genentech, Inc. Methods for using chimeric vascular endothelial growth factor receptor proteins
US6897294B2 (en) 1996-05-07 2005-05-24 Genentech, Inc. Inhibitors of vascular endothelial growth factor activity, their uses and processes for their production
US7226554B2 (en) 1999-01-29 2007-06-05 Daikyo Seiko, Ltd. Molding die assembly for rubber members and rubber member produced thereby
US7306799B2 (en) 1999-06-08 2007-12-11 Regeneron Pharmaceuticals, Inc. Use of VEGF inhibitors for treatment of eye disorders
US7374758B2 (en) 1999-06-08 2008-05-20 Regeneron Pharmaceuticals, Inc. Modified chimeric polypeptides with improved pharmacokinetic properties and methods of using thereof
US7374757B2 (en) 1999-06-08 2008-05-20 Regeneron Pharmaceuticals, Inc. Modified chimeric polypeptides with improved pharmacokinetic properties
US7070959B1 (en) 1999-06-08 2006-07-04 Regeneron Pharmaceuticals, Inc. Modified chimeric polypeptides with improved pharmacokinetic properties
US7087411B2 (en) 1999-06-08 2006-08-08 Regeneron Pharmaceuticals, Inc. Fusion protein capable of binding VEGF
US6629949B1 (en) 2000-05-08 2003-10-07 Sterling Medivations, Inc. Micro infusion drug delivery device
US6659982B2 (en) 2000-05-08 2003-12-09 Sterling Medivations, Inc. Micro infusion drug delivery device
US6645635B2 (en) 2001-01-19 2003-11-11 Daikyo Seiko, Ltd. Laminated rubber stopper for a medicament vial
US7736646B2 (en) 2003-03-12 2010-06-15 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods for modulating angiogenesis with apelin compositions
WO2004081198A2 (en) 2003-03-12 2004-09-23 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods for modulating angiogenesis with apelin compositions
US7531173B2 (en) 2005-02-02 2009-05-12 Regeneron Pharmaceuticals, Inc. Ophthalmic composition of a VEGF antagonist
US7608261B2 (en) 2006-06-16 2009-10-27 Regeneron Pharmacuticals, Inc. VEGF antagonist formulations suitable for intravitreal administration
US20150125459A1 (en) 2009-02-27 2015-05-07 The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv Apelin peptides, antibodies thereto, and methods of use
US20110027286A1 (en) 2009-07-29 2011-02-03 Regeneron Pharmaceuticals, Inc. High Affinity Human Antibodies to Human Angiopoietin-2
US9493554B2 (en) 2011-07-18 2016-11-15 Amgen Inc. Methods of treating retinal disorders with anti-apelin antibodies
US20130186797A1 (en) 2012-01-23 2013-07-25 Regeneron Pharmaceuticals, Inc. Stabilized Formulations Containing Anti-Ang2 Antibodies
US9593153B2 (en) 2012-04-11 2017-03-14 Inserm (Institut National De La Sante Et De La Recherche Medicale) Modified apelin polypeptides
US20140000518A1 (en) 2012-06-27 2014-01-02 Tokyo Electron Limited Coating apparatus and method for filling coating liquid
WO2014121087A1 (en) 2013-02-01 2014-08-07 Regeneron Pharmaceuticals, Inc. Antibodies comprising chimeric constant domains
US20150252107A1 (en) * 2013-11-20 2015-09-10 Regeneron Pharmaceuticals, Inc. APLNR Modulators and Uses Thereof
WO2015077491A1 (en) 2013-11-20 2015-05-28 Regeneron Pharmaceuticals, Inc. Aplnr modulators and uses thereof
WO2015140296A2 (en) 2014-03-20 2015-09-24 Centre National De La Recherche Scientifique (Cnrs) Use of compounds inhibiting apelin / apj / gp130 signaling for treating cancer
US20160144025A1 (en) * 2014-11-25 2016-05-26 Regeneron Pharmaceuticals, Inc. Methods and formulations for treating vascular eye diseases
WO2016085750A1 (en) 2014-11-25 2016-06-02 Regeneron Pharmaceuticals, Inc. Methods and formulations for treating vascular eye diseases

Non-Patent Citations (52)

* Cited by examiner, † Cited by third party
Title
"Fundamental Immunology", 1989, RAVEN PRESS
"Medical Applications of Controlled Release", 1974, CRC PRES
"Remington's Pharmaceutical Sciences", MACK PUBLISHING COMPANY
AI-LAZIKANI ET AL., J. MOL. BIOL., vol. 273, 1997, pages 927 - 948
ALI YF ET AL., CLIN OPHTHALMOL., vol. 11, 21 February 2017 (2017-02-21), pages 387 - 392
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 402
ANGAL ET AL., MOLECULAR IMMUNOLOGY, vol. 30, 1993, pages 105
ARAI ET AL., PEDS, vol. 14, no. 8, 2001, pages 529 - 32
BROWN ET AL., OPHTHALMOLOGY, vol. 10, 2013, pages 2013 - 22
CHEN, X. ET AL., ADVANCED DRUG DELIVERY REVIEWS, vol. 65, 2013, pages 1357 - 1369
CLYNES ET AL., PROC. NATL. ACAD. SCI. (USA, vol. 95, 1998, pages 652 - 656
DIXON ET AL., EXPERT OPIN. INVESTIG. DRUGS, vol. 18, 2009, pages 1573 - 80
DU, JH ET AL., INT J OPHTHALMOL., vol. 7, no. 6, 18 December 2014 (2014-12-18), pages 968 - 73
ELEFTHERIADOU MARIA ET AL: "Long-Term Outcomes of Aflibercept Treatment for Neovascular Age-Related Macular Degeneration in a Clinical Setting", AMERICAN JOURNAL OF OPHTHALMOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 174, 14 October 2016 (2016-10-14), pages 160 - 168, XP029890555, ISSN: 0002-9394, DOI: 10.1016/J.AJO.2016.09.038 *
FERRARRA ET AL., NATURE, vol. 438, 2005, pages 967 - 974
GONNET ET AL., SCIENCE, vol. 256, 1992, pages 1443 - 1445
GOODSON, MEDICAL APPLICATIONS OF CONTROLLED RELEASE, vol. 2, 1984, pages 115 - 138
GREENWOOD JOHN ET AL: "Apelin Is Required For Non-sprouting Vascular Remodelling In The Developing And Pathogenic Retina", ARVO ANNUAL MEETING ABSTRACT SEARCH AND PROGRAM PLANNER,, vol. 2011, 1 May 2011 (2011-05-01), pages 4823, XP009507405 *
HABATA ET AL., BIOCHEM BIOPHYS ACTA, vol. 1452, 1999, pages 25 - 35
HOSOYA ET AL., JBC, vol. 275, no. 28, 2000, pages 21061 - 67
HUSTON ET AL., PNAS, vol. 85, no. 16, 1988, pages 5879 - 83
JUNGHANS ET AL., CANCER RES., vol. 50, 1990, pages 1495 - 1502
KABAT: "Sequences of Proteins of Immunological Interest", 1991, NATIONAL INSTITUTES OF HEALTH
KASAI ET AL., ARTERIOSCLER THROMB VASC BIOI, vol. 30, 2010, pages 2182 - 2187
KEANE ET AL., SURV OPHTHALMOL., vol. 57, 2012, pages 389 - 414
KLEIN ET AL., OPHTHALMOLOGY, vol. 91, 1984, pages 1464 - 1474
KOJIMA, Y.; QUERTERMOUS, T., ARTERIOSCLER THROMB VASC BIOL, vol. 28, 2008, pages 1687 - 1688
LANGER, SCIENCE, vol. 249, 1990, pages 1527 - 1533
LEE ET AL., J NEUROCHEM, vol. 74, 2000, pages 34 - 41
LU ET AL., MOLECULAR VISION, vol. 20, 2014, pages 1122 - 1131
LU, Q. ET AL., PLOS ONE, vol. 8, no. 7, 2013, pages e69703
MARTIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 9268 - 9272
MEDHURST ET AL., J NEUROCHEM, vol. 84, 2003, pages 1162 - 1172
MEEHAN ET AL., J. CONTROLLED RELEASE, vol. 46, 1996, pages 107 - 116
MORDENTI ET AL., PHARMACEUT. RES., vol. 8, 1991, pages 1351
MOSS ET AL., OPHTHALMOLOGY, vol. 105, 1998, pages 998 - 1003
NEELY ET AL., AM J. PATHOL, vol. 153, 1998, pages 665 - 670
PEARSON, METHODS MOL. BIOL., vol. 24, 1994, pages 307 - 331
PORTA ET AL., DIABETOLOGIA, vol. 45, 2002, pages 1617 - 1634
POWELL ET AL.: "Compendium of excipients for parenteral formulations", J PHARM SCI TECHNOL, vol. 52, 1998, pages 238 - 311, XP009119027
ROBINSON; BATES, PNAS, vol. 95, no. 11, 1998, pages 5929 - 34
RUGGIERO ET AL., DIABETES METAB., vol. 23, 1997, pages 30 - 42
SAINT-GENIEZ ET AL., INT J DEV BIOL, vol. 48, 2004, pages 1045 - 1058
SAKIMOTO SUSUMU ET AL: "A role for endothelial cells in promoting the maturation of astrocytes through the apelin/APJ system in mice", DEVELOPMENT, THE COMPANY OF BIOLOGISTS LTD, GB, vol. 139, no. 7, 1 April 2012 (2012-04-01), pages 1327 - 1335, XP009507404, ISSN: 0950-1991, DOI: 10.1242/DEV.072330 *
SHIELD ET AL., JBC, vol. 277, 2002, pages 26733
SMITH ET AL., INVEST OPHTHALMOL VIS SCI, vol. 35, 1994, pages 101 - 111
TAO ET AL., INVEST OPTHAMOL VISUAL SCIENCE, vol. 51, 2010, pages 4237 - 4242
TATEMOTO ET AL., REGUL PEPT, vol. 99, 2001, pages 87 - 92
TAYLOR ET AL., NUCL. ACIDS RES., vol. 20, 1992, pages 6287 - 6295
WU ET AL., J. BIOL. CHEM., vol. 262, 1987, pages 4429 - 4432
ZHANG ET AL., JAMA, vol. 304, 2010, pages 649 - 656

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