JP2020518641A - Methods of treating eye diseases with APLNR antagonists and VEGF inhibitors - Google Patents

Abstract

The present disclosure provides methods for treating, preventing, or lessening the severity of eye diseases. The methods of the present disclosure include administering to a subject in need thereof a therapeutic composition comprising an APLNR antagonist, such as an anti-APLNR antibody in combination with a vascular endothelial growth factor (VEGF) antagonist (eg, aflibercept). .. [Selection diagram] Fig. 9A

Description

Sequence Listing This application was created on May 4, 2018 and contains file 10354WO01-Sequence. The sequence listings submitted in computer readable form as txt are incorporated by reference.

The present disclosure relates to a method of treating vascular eye disease, particularly by administering an apelin receptor antagonist and a vascular endothelial growth factor (VEGF) antagonist to a subject in need thereof.

Vascular eye disease is the leading cause of blindness in the elderly population of today. These diseases may be characterized by abnormal "leaky" blood vessels growing within the retina. The two largest causes of this target population are diabetic retinopathy and wet age-related macular degeneration.

Diabetic retinopathy (DR) is a major cause of visual impairment in the United States (Non-Patent Documents 1 and 2). Diabetic retinopathy results from decompensation of microvessels that begins with thickening of the basement membrane (Non-Patent Document 3), and finally progresses to vascular occlusion and angiogenesis (Non-Patent Document 4). It has been estimated that about 28% of subjects over the age of 40 with diabetes have DR and 4.4% have vision- threatening DR (Non-Patent Document 5). Diabetic macular edema (DME) is a symptom of DR and is the most frequent cause of visual loss in young and middle-aged adults (Non-Patent Documents 1 and 2).

Age-related macular degeneration (AMD) is the leading cause of severe visual loss in people over the age of 50 in developed countries. In recent years, the introduction of anti-angiogenic agents has provided tremendous advances in the treatment of AMD, providing a hope for significant visual recovery in subjects with neovascular AMD (Non-Patent Document 6).

Preproapelin is a 77 amino acid protein expressed in the human CNS and peripheral tissues such as lung, heart, and mammary gland. Peptides containing C-terminal fragments of various sizes of apelin peptides have been shown to activate the G protein-coupled receptor, APJ receptor (now known as APLNR) (Non-Patent Document 7, Patent document 8, non-patent document 9, non-patent document 10). Numerous studies show that apelin peptides and analogs mediate cardiovascular and angiogenic effects through interactions with APJ receptors (APLNR) such as endothelium-dependent vasodilation (11).

The apelin system emerges to play a role in pathophysiological angiogenesis. Studies have shown that apelin may be involved in hypoxia-induced retinal angiogenesis (12). In some reports, certain compositions have been shown to inhibit the apelin/APJ pathway (see, eg, US Pat. No. 6,037,049), such as with APLNR inhibitors capable of blocking pathological angiogenesis. It can inhibit angiogenesis and is therefore useful in inhibiting angiogenesis in the retina (13). Therefore, the interference of signal transduction via apelin may be beneficial in the early prevention of proliferative diabetic retinopathy (Non-patent Document 14, Non-patent Document 15, Non-patent Document 16). More recently, apelin has been implicated in the mechanism of retinopathy of prematurity (Non-Patent Document 17).

Anti-vascular endothelial growth factor (VEGF) therapy (eg aflibercept) is the standard of care treatment for neovascular AMD and DME. The efficacy and safety of aflibercept in these target populations is well characterized (Non-Patent Document 18). However, in AMD, approximately 95% of the subjects retained their visual acuity, but only approximately 30% of the subjects achieved an improvement in best corrected visual acuity (BCVA) of 15 letters or more at 1 year. There is also the potential for improving therapeutic outcomes in DME. As seen with aflibercept and ranibizumab, less than 50% of subjects with visual loss due to DME achieve an improvement of 15 letters or more over 1 year and 2 years. Clinical evidence of proliferative retinopathy in the ranibizumab study occurs in up to 7.2% of subjects treated monthly with ranibizumab for 3 years, and up to 3.2% of subjects may be blind. Panretinal photocoagulation, which is a promising treatment modality (Non-Patent Document 19), was required.

Both apelin/APLNR and VEGF are known factors in angiogenesis and vascular development, but the mechanism by which the two signaling pathways interact in promoting angiogenesis is not fully understood. In particular, these pathways are known to be involved in the formation of retinal blood vessels, and various studies have reported that apelin and VEGF have positive and negative feedback effects, the expression of either The increase can contribute to the expression of the other, or the antagonism of one suppresses the expression of the other (Non-Patent Document 20).

Intravitreal (IVT) delivery of anti-VEGF therapies such as ranibizumab and aflibercept has demonstrated efficacy and safety against choroidal retinal disease. However, there are numerous additional factors that contribute to vascular permeability, angiogenesis, and other vascular dysfunction.

US Pat. No. 7,736,646

Klein et al. , 1984, Ophthalmology 91:1464-1474. Moss et al. , 1998, Ophthalmology 105:998-1003. Ruggiero et al. , 1997, Diabetes Metab. 23:30-42 Porta et al. , 2002, Diabetologia 45: 1617-1634. Zhang et al. , 2010, JAMA 304: 649-656. Keane et al. , 2012, Surv Ophthalmol. 57:389-414 Habata, et al. , 1999, Biochem Biophys Acta 1452:25-35. Hosoya, et al. , 2000, JBC, 275(28): 21061-67. Lee, et al. , 2000, J Neurochem 74:34-41. Medhurst, et al. , 2003, J Neurochem 84:1162-1172. Tatemoto et al. , 2001, Regul Pept 99:87-92. Kasai et al. , 2010, Arterioscler Thromb Vasc Bioi 30:2182-2187. Kojima, Y. and Quatermous, T.S. , 2008, Arterioscler Thromb Vasc Biol 28:1687-1688. Tao et al. , 2010, Invest Optamol Visual Science 51:4237-4242. Du, JH et al. , Int J Ophthalmol. 2014 Dec 18;7(6):968-73. Lu, Q. et al. , 2013, PLoS One 8(7):e69703. Ali YF et al. , Clin Ophthalmol. 2017 Feb 21; 11:387-392. Dixon et al. , 2009; Expert Opin. Investig. Drugs 18: 1573-80 Brown et al. , 2013 Ophthalmology 10:2013-22. Lu et al. , 2014, Molecular Vision, 20: 1122-1131.

In one aspect, the 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 method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist. In certain embodiments, the APLNR antagonist is administered in combination with a vascular endothelial growth factor (VEGF) antagonist, eg, by administration of a therapeutically effective amount of a pharmaceutical composition comprising the VEGF antagonist.

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

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

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

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

In another aspect, the invention provides methods for improving vascular regeneration and reducing aberrant angiogenesis in a subject. The method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist.

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

In another aspect, the invention provides methods for doing so in a subject in need of promoting uniform regrowth of retinal blood vessels. The method comprises administering to a subject in need thereof a therapeutically effective amount of an APLNR antagonist and administering to the subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. Including.

In another aspect, the invention provides methods for doing so in a subject in need of improved vascular elongation in the retina. The method comprises administering to a subject in need thereof a therapeutically effective amount of an APLNR antagonist and administering to the subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. Including.

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

In another aspect, the invention provides methods for doing so in a subject in need of improved retinal vascular organization. The method comprises administering to a subject in need thereof a therapeutically effective amount of an APLNR antagonist and administering to the subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. Including.

In any of the methods discussed above or herein, the subject can be an individual who has been diagnosed with an eye disease or disorder. In some cases, the eye disease or disorder is diabetic retinopathy, proliferative diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, It is selected from the group consisting of polypoidal choroidal vasculopathy, choroidal neovascularization (CNV), degenerative myopia (myopia CNV), neovascular glaucoma, and retinopathy of prematurity. In some cases, the eye disease or disorder is age-related macular degeneration. In some cases, the eye disease or disorder is diabetic macular edema. In some cases, the eye disease or disorder is retinopathy of prematurity. In some cases, the eye disease or disorder is proliferative diabetic retinopathy.

Exemplary APLNR antagonists that can be used in the context of the methods or compositions of the present disclosure include small molecule chemical inhibitors of APLNR or biology targeting APLNR such as peptides, peptidomimetics, and antibodies. Formulations are included. In certain embodiments, the APLNR antagonist blocks the interaction of APLNR and apelin.

According to a particular embodiment, the APLNR antagonist is an antibody or antigen binding protein that binds to the APLNR antagonist and inhibits APLNR signaling. In certain embodiments, the anti-APLNR antibody or antigen binding protein comprises a heavy chain complementarity determining region (HCDR) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:2 or 13, and a heavy chain complementarity determining region (HCDR) of SEQ ID NO:7 or 18. A light chain CDR of a light chain variable region (LCVR) comprising an amino acid sequence. In certain embodiments, the anti-APLNR antibody or antigen binding protein comprises a complementarity determining region (CDR) of a heavy chain variable region (HCVR) of an anti-APLNR antibody selected from the group consisting of H2aM9232N (or H4H9232N) and H1M9207N. Including.

In any of the methods or compositions discussed above or herein, the APLNR antagonist can include an anti-APLNR antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof specifically binds human APLNR. To do. In some cases, the antibody or antigen-binding fragment thereof binds to a 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. And the antibody or antigen-binding fragment thereof specifically binds to human APLNR. In some cases, the antibody or antigen-binding fragment thereof binds to the same epitope on the APLNR as the reference antibody comprising an HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2/7 and 13/18, and the antibody Alternatively, the antigen-binding fragment thereof specifically binds to human APLNR. In some cases, the antibody or antigen-binding fragment comprises (a) a complementarity determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 and 13, ( b) a CDR of a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 18. In some cases, the antibody or antigen-binding fragment is HCDR1-HCDR2 selected from the group consisting of SEQ ID NOS: 3-4-5-8-9-10 and 14-15-16-19-20-20, respectively. -Includes the HCDR3-LCDR1-LCDR2-LCDR3 domains. In some cases, 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 ID NOS: 2 and 13, and (b) SEQ ID NOS: 7 and 18. A light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of: In some cases, the antibody or antigen-binding fragment comprises the heavy and light chain CDRs of an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/7 and 13/18. In some cases, the antibody or antigen-binding fragment comprises an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/7 and 13/18.

In one embodiment of any one of the methods or compositions described above or herein, the antibody or antigen-binding fragment thereof comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 2/7.

In one embodiment of any one of the methods or compositions described above or herein, the antibody or antigen-binding fragment thereof comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 13/18.

In any one of the methods or compositions described above or herein, the antibody or antigen-binding fragment can be a human antibody having an IgG1 or IgG4 heavy chain constant region. In one embodiment, the heavy chain constant region is human IgG1. In one embodiment, the heavy chain constant region is human IgG4.

In various embodiments of any one of the methods or compositions described above or herein, the antibody or antigen-binding fragment thereof blocks the interaction of APLNR with apelin. In some cases, the antibody or antigen-binding fragment thereof blocks the interaction of APLNR with apelin and exhibits 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 with apelin.

VEGF antagonists that may be used in combination with APLNR antagonists in the compositions and methods of the present disclosure include anti-VEGF antibodies (eg, ranibizumab), small molecule VEGF inhibitors (eg, sunetinib), and VEGF inhibitor fusion proteins (“VEGF trap”). ")including. An example of a VEGF antagonist that may be used in combination with an APLNR antagonist in the methods of treatment of the present disclosure is the VEGF inhibitory fusion protein, aflibercept (see, eg, US Pat. No. 7,087,411). ).

In any one of the methods or compositions discussed above or herein, the VEGF antagonist comprises a VEGF receptor system chimeric molecule (VEGF trap). In some cases, the VEGF trap comprises one or more immunoglobulin (Ig)-like domains of VEGFR1, one or more Ig-like domains of VEGFR2, and a multimerization domain. In some cases, the VEGF trap comprises the Ig-like domain 2 of VEGFR1, the Ig-like domain 3 of VEGFR2, and the multimerization domain. In some cases, the VEGF trap is aflibercept or its biosimilar molecule. In some cases, the VEGF antagonist consists of a dimer of two polypeptides consisting of amino acids 27-457 of SEQ ID NO:23.

In another aspect, 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.

In various embodiments, the disclosure provides the use of an APLNR antagonist in combination with a VEGF antagonist in the manufacture of a medicament for treating an eye disease or disorder in a subject, including a human, or of the methods discussed above or herein. To serve any other purpose. All methods above or discussed herein can be embodied as the use of APLNR antagonists and VEGF antagonists for the treatment of eye diseases or disorders, or other purposes of the recited methods. Other embodiments include APLNR antagonists and VEGF antagonists for use in the methods described above or herein.

In another aspect, the invention provides a composition for treating a vascular eye disease or disorder, the composition comprising a therapeutically effective amount of an APLNR antagonist and a therapeutically effective amount of vascular endothelium. Includes a growth factor (VEGF) antagonist and a suitable carrier, excipient or diluent. In various embodiments of the composition, the APLNR antagonist or VEGF antagonist can be as described above or herein.

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

Figure 3 is a representative photomicrograph of the retinas of mice systemically (IP) treated with P2-P5, and a graph of calculated vessel area. The residual vascular area was significantly smaller in retinas treated with alpha apelin R (anti-APLNR antibody H2aM9232N) (23% at 25 mg/kg, p<0.05) 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 performed by Student's T test. Selective inhibition of apelin R via systemic infusion delayed normal vascular outgrowth in the developing retina of P5 pups. At the 25 mg/kg dose, blocking Apelin R slightly inhibits vascular elongation. Figure 3 is a representative photomicrograph of the retinas of mice systemically (IP) treated with P2-P5, and a graph of calculated vessel area. The residual vascular area was significantly smaller in retinas treated with alpha apelin R (anti-APLNR antibody H2aM9232N) (23% at 25 mg/kg, p<0.05) 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 performed by Student's T test. Selective inhibition of apelin R via systemic infusion delayed normal vascular outgrowth in the developing retina of P5 pups. At the 25 mg/kg dose, blocking Apelin R slightly inhibits vascular elongation. Figure 3 is a representative photomicrograph of the retinas of mice systemically (IP) treated with P2-P5, and a graph of calculated vessel area. The extra vessel area was significantly smaller in retinas treated with α-Apelin R (35% at 50 mg/kg, p<0.005) 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 performed by Student's T test. Selective inhibition of apelin R via systemic infusion delayed normal vascular outgrowth in the developing retina of P5 pups. By increasing the dose to 50 mg/kg, the targeted apelin R further delays retinal vessel elongation. Figure 3 is a representative photomicrograph of the retinas of mice systemically (IP) treated with P2-P5, and a graph of calculated vessel area. The extra vessel area was significantly smaller in retinas treated with α-Apelin R (35% at 50 mg/kg, p<0.005) 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 performed by Student's T test. Selective inhibition of apelin R via systemic infusion delayed normal vascular outgrowth in the developing retina of P5 pups. By increasing the dose to 50 mg/kg, the targeted apelin R further delays retinal vessel elongation. Figure 3 is a representative photomicrograph of the retinas of mice systemically (IP) treated with P2-P5, and a graph of calculated vessel area. In this blinded study, 50 mg/kg of alpha apelin R reduced vessel 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 performed by Student's T test. Selective inhibition of apelin R via systemic infusion delays normal vascular outgrowth in the developing retina of P5 pups. Figure 3 is a representative photomicrograph of the retinas of mice systemically (IP) treated with P2-P5, and a graph of calculated vessel area. In this blinded study, 50 mg/kg of alpha apelin R reduced vessel 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 performed by Student's T test. Selective inhibition of apelin R via systemic infusion delays normal vascular outgrowth in the developing retina of P5 pups. Figure 3 is a representative photomicrograph of the retinas of mice systemically (IP) treated with P2-P5, and a graph of calculated vessel area. Surplus vascular area was compared to α-apelin R (31% at 50 mg/kg, p<0.001) or aflibercept alone (43% at 50 mg/kg, p<0.005) with single reagent And was significantly lower in the combination (α-Apelin R + aflibercept) (62% at 50 mg/kg, p<0.0001). Retinal endothelial cells were stained with GS lectin I. The effects on dose and correlative vascularized area are recorded. Images were taken at 20x (for quantification) and 40x. Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test. Both systemically and intravitreally (see FIGS. 5A and 5B), the combination therapy of alpha apelin R and aflibercept resulted in the regression of normal developing retinal vasculature. Blocking both apelin R and VEGFA via systemic infusion is more effective in blocking vascular elongation compared to blocking apelin R or VEGFA alone. In this model, combination treatment further reduced vascular area by 46% (p<0.0005) compared to α-apelin R and 32% (p<0.001) compared to aflibercept. Figure 3 is a representative photomicrograph of the retinas of mice systemically (IP) treated with P2-P5, and a graph of calculated vessel area. Surplus vascular area was compared to α-apelin R (31% at 50 mg/kg, p<0.001) or aflibercept alone (43% at 50 mg/kg, p<0.005) with single reagent And was significantly lower in the combination (α-Apelin R + aflibercept) (62% at 50 mg/kg, p<0.0001). Retinal endothelial cells were stained with GS lectin I. The effects on dose and correlative vascularized area are recorded. Images were taken at 20x (for quantification) and 40x. Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test. Both systemically and intravitreally (see FIGS. 5A and 5B), the combination therapy of alpha apelin R and aflibercept resulted in the regression of normal developing retinal vasculature. Blocking both apelin R and VEGFA via systemic infusion is more effective in blocking vascular elongation compared to blocking apelin R or VEGFA alone. In this model, combination treatment further reduced vascular area by 46% (p<0.0005) compared to α-apelin R and 32% (p<0.001) compared to aflibercept. Figure 6 is a representative photomicrograph of the retina of a mouse treated via intravitreal (IVT) injection at P4-P6, and a graph of calculated vessel area. The surplus vascular area was compared to α-ApelinR (43% at 5 μg, p<0.0001) or aflibercept alone (65% at 5 μg, p<0.0001) with a single reagent in combination ( α apelin R + aflibercept) (68% at 5 μg, p<0.0001). Retinal endothelial cells were stained with GS lectin I. The effects on dose and correlative vascularized area are recorded. Images were taken at 20x (for quantification) and 40x. Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test. Both systemic and intravitreal administration, the combined therapy of alpha apelin R and aflibercept results in the regression of normal developing retinal vasculature. Blocking both apelin R and VEGFA via intravitreal injection is more effective in blocking vasodilation compared to blocking apelin R or VEGFA alone. In IVT, combined treatment further reduced vascular area by 50% (p<0.0005) compared to α-apelin R and 34% (p<0.001) compared to aflibercept. Figure 6 is a representative photomicrograph of the retina of a mouse treated via intravitreal (IVT) injection at P4-P6, and a graph of calculated vessel area. The surplus vascular area was compared to α-ApelinR (43% at 5 μg, p<0.0001) or aflibercept alone (65% at 5 μg, p<0.0001) with a single reagent in combination ( α apelin R + aflibercept) (68% at 5 μg, p<0.0001). Retinal endothelial cells were stained with GS lectin I. The effects on dose and correlative vascularized area are recorded. Images were taken at 20x (for quantification) and 40x. Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test. Both systemic and intravitreal administration, the combined therapy of alpha apelin R and aflibercept results in the regression of normal developing retinal vasculature. Blocking both apelin R and VEGFA via intravitreal injection is more effective in blocking vasodilation compared to blocking apelin R or VEGFA alone. In IVT, combined treatment further reduced vascular area by 50% (p<0.0005) compared to α-apelin R and 34% (p<0.001) compared to aflibercept. Figure 6 is a representative photomicrograph of the retina of OIR mice systemically (IP) treated with P12-P16, and a graph of calculated avascular area. Residual avascular area was observed in retinas with α-apelin R (29%, p<0.05) and aflibercept (27.5%, p<0.01) compared to Fc (control) retinas. It was significantly smaller. Significantly reduced angiogenic chambers compared to Fc in samples treated with alpha apelin R (67%, p<0.0001) and aflibercept (94%, p<0.0005) Please pay attention to. Retinal endothelial cells were stained with GS lectin I. Images were taken at 20x (quantification of avascular areas) and 40x (quantification of abnormal neovascularization areas). Statistical analysis was performed by Student's T test. Both inhibition of Apelin R and VEGFA via systemic infusion promotes retinal blood vessel remodeling and reduces abnormal angiogenesis. Figure 6 is a representative photomicrograph of the retina of OIR mice systemically (IP) treated with P12-P16, and a graph of calculated avascular area. Residual avascular area was observed in retinas with α-apelin R (29%, p<0.05) and aflibercept (27.5%, p<0.01) compared to Fc (control) retinas. It was significantly smaller. Significantly reduced angiogenic chambers compared to Fc in samples treated with alpha apelin R (67%, p<0.0001) and aflibercept (94%, p<0.0005) Please pay attention to. Retinal endothelial cells were stained with GS lectin I. Images were taken at 20x (quantification of avascular areas) and 40x (quantification of abnormal neovascularization areas). Statistical analysis was performed by Student's T test. Both inhibition of Apelin R and VEGFA via systemic infusion promotes retinal blood vessel remodeling and reduces abnormal angiogenesis. Figure 6 is a representative photomicrograph of the retina of an oxygen-induced retinopathy (OIR) mouse treated intravitreally with P12-P16, and a graph of calculated avascular area. Residual avascular area was reduced in alpha apelin R (27.5%, p<0.05) and increased in aflibercept (32%, p<0.0001) compared to Fc controls. .. Note the significant reduction of neovascular chambers in α-Apelin R (60%, p<0.0001), and the complete loss of chambers in the aflibercept sample. Retinal endothelial cells were stained with GS lectin I. The effects on dose and correlative vascularized area are recorded. Images were taken at 20x (quantification of avascular areas) and 40x (quantification of abnormal neovascularization areas). Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test. Both systemically (see FIGS. 6A and 6B) and intravitreal administration, selective inhibition of apelin R promotes retinal vascular regeneration and reduces angiogenesis in OIR mice. Inhibition of apelin R via intravitreal injection improves vascular regeneration and reduces abnormal angiogenesis, while VEGFA inhibition suppresses vascular regeneration and completely stops angiogenesis. Figure 6 is a representative photomicrograph of the retina of an oxygen-induced retinopathy (OIR) mouse treated intravitreally with P12-P16, and a graph of calculated avascular area. Residual avascular area was reduced in alpha apelin R (27.5%, p<0.05) and increased in aflibercept (32%, p<0.0001) compared to Fc controls. .. Note the significant reduction of neovascular chambers in α-Apelin R (60%, p<0.0001), and the complete loss of chambers in the aflibercept sample. Retinal endothelial cells were stained with GS lectin I. The effects on dose and correlative vascularized area are recorded. Images were taken at 20x (quantification of avascular areas) and 40x (quantification of abnormal neovascularization areas). Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test. Both systemically (see Figures 6A and 6B) and intravitreal administration, selective inhibition of apelin R promotes retinal vascular regeneration and reduces angiogenesis in OIR mice. Inhibition of apelin R via intravitreal injection improves vascular regeneration and reduces abnormal angiogenesis, while VEGFA inhibition suppresses vascular regeneration and completely stops angiogenesis. Figure 6 is a representative photomicrograph (20x) of the retina of OIR mice systemically treated with P12-P16, and a graph of calculated avascular area. Regeneration rates with combination treatment were similar to each agent alone, vascular regeneration was improved compared to hFc-treated control retinas, and significant changes in avascular regions between treatment groups (p<0). .0005) existed. Figure 6 is a representative photomicrograph (20x) of the retina of OIR mice systemically treated with P12-P16, and a graph of calculated avascular area. Regeneration rates with combination treatment were similar to each agent alone, vascular regeneration was improved compared to hFc-treated control retinas, and significant changes in avascular regions between treatment groups (p<0). .0005) existed. Figure 6 is a representative photomicrograph (40x) of the retina of OIR mice systemically treated with P12-P16, and a graph of calculated abnormal vascular area. The combination treatment shows fewer pruned blood vessels compared to aflibercept and less abnormal neovascularization compared to anti-APLNR and Fc controls (FIG. 9A). FIG. 9B shows that there was a significant change in abnormal vascular area between treatment groups (p<0.0005). Abnormal vascular area was compared with Fc control with anti-APLNR (***, p<0.0005) and with aflibercept (***p<0.005). , The combination was significantly reduced (***, p <0.0005), and the abnormal vascular area was further significantly reduced in the combination treatment group as compared with the anti-APLNR treatment group. (*, p<0.05) Figure 6 is a representative photomicrograph (40x) of the retina of OIR mice systemically treated with P12-P16, and a graph of calculated abnormal vascular area. The combination treatment shows fewer pruned blood vessels compared to aflibercept and less abnormal neovascularization compared to anti-APLNR and Fc controls (FIG. 9A). FIG. 9B shows that there was a significant change in abnormal vascular area between treatment groups (p<0.0005). Abnormal vascular area was compared with Fc control with anti-APLNR (***, p<0.0005) and with aflibercept (***p<0.005). , The combination was significantly reduced (***, p <0.0005), and the abnormal vascular area was further significantly reduced in the combination treatment group as compared with the anti-APLNR treatment group. (*, p<0.05) 9 is a representation of the image processing and measurement techniques used to calculate vessel area and vessel length in Example 7. The “image of origin” is a 40× photomicrograph shown in FIG. 9A. Representative processed images showing vessel organization and homogeneity from photomicrographs (40×) of retinas of OIR mice systemically treated with P12-P16 and graphs of calculated vessel area. As shown in FIG. 11A, the combination of anti-APLNR antibody and aflibercept produced more organized and uniform retinal vessels as compared to anti-APLNR alone, and was more interspersed than aflibercept alone. Fewer (less pruned vessels). FIG. 11B shows that there was a significant change in blood vessel area between treatment groups (p<0.0005). Vascular area was significantly increased with anti-APLNR compared to Fc controls (***, p<0.0005), aflibercept (***, p<0.005), and It was reduced with the combination treatment (*, p<0.05). Blood vessel area was compared with anti-APLNR with aflibercept (####, p<0.0005) and with combination (####, p<0.0005). Significantly reduced. In contrast, vascular area was significantly increased with combination treatment (&&&, p<0.005) compared to aflibercept. Representative processed images showing vessel organization and homogeneity from photomicrographs (40×) of retinas of OIR mice systemically treated with P12-P16 and graphs of calculated vessel area. As shown in FIG. 11A, the combination of anti-APLNR antibody and aflibercept produced more organized and uniform retinal vessels as compared to anti-APLNR alone, and was more interspersed than aflibercept alone. Fewer (less pruned vessels). FIG. 11B shows that there was a significant change in blood vessel area between treatment groups (p<0.0005). Vascular area was significantly increased with anti-APLNR compared to Fc controls (***, p<0.0005), aflibercept (***, p<0.005), and It was reduced with the combination treatment (*, p<0.05). Blood vessel area was compared with anti-APLNR with aflibercept (####, p<0.0005) and with combination (####, p<0.0005). Significantly reduced. In contrast, vascular area was significantly increased with combination treatment (&&&, p<0.005) compared to aflibercept. Figure 6 is a representative processed image showing vessel density and calculated vessel length from photomicrographs (40X) of retinas of OIR mice systemically treated with P12-P16. As shown in FIG. 12A, the combination of anti-APLNR antibody and aflibercept produced intermediate density retinal vessels as compared to anti-APLNR (higher density) or aflibercept (lower density) alone. .. FIG. 12B shows that there was a significant change in vessel length between treatment groups (p<0.0005). Blood vessel length was compared with anti-APLNR with aflibercept (####, p<0.0005) and with combination (####, p<0.0005). Significantly reduced. In contrast, vessel length was significantly increased with combined treatment (&&&, p<0.005) compared to aflibercept. Figure 6 is a representative processed image showing vessel density and calculated vessel length from photomicrographs (40X) of retinas of OIR mice systemically treated with P12-P16. As shown in FIG. 12A, the combination of anti-APLNR antibody and aflibercept produced intermediate density retinal vessels as compared to anti-APLNR (higher density) or aflibercept (lower density) alone. .. FIG. 12B shows that there was a significant change in vessel length between treatment groups (p<0.0005). Blood vessel length was compared with anti-APLNR with aflibercept (####, p<0.0005) and with combination (####, p<0.0005). Significantly reduced. In contrast, vessel length was significantly increased with combined treatment (&&&, p<0.005) compared to aflibercept.

Before the present disclosure is described, it should be understood that the present disclosure is not limited to such methods and conditions, as the particular methods and experimental conditions described can vary. The terminology used herein is for the purpose of describing only particular embodiments and is not intended to be limiting as the scope of the invention is limited only by the appended claims. It should also be understood that it does not.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by 1% or less. For example, as used herein, the expression "about 100" includes 99 and 101, and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All patents, applications and non-patent publications referred to herein are incorporated herein by reference in their entirety.

The present invention and the present disclosure contemplate, in part, the unexpected discovery of the combination of an APLNR antagonist with a VEGF antagonist (eg, an anti-APLNR antibody with a VEGF trap) observed with either the APLNR antagonist or the VEGF antagonist alone. Can produce more organized and orderly medium density revascularization in the control retina.

Definitions As used herein, "apelin receptor", expressions such as "APLNR", "apelin R", "APJ receptor" includes humans having the amino acid sequence represented as EmuiijijidiefudienuwaiwaijieidienukyuesuishiiwaitididaburyukeiesuesujieieruaiPieiaiwaiemuerubuiefueruerujititijienujierubuierudaburyutibuiefuaruesuesuaruikeiaruaruesueidiaiefuaieiesuerueibuieidierutiefubuibuitieruPierudaburyueitiwaitiwaiarudiwaididaburyuPiefujitiefuefushikeieruesuesuwaieruaiefubuienuemuwaieiesubuiefushierutijieruesuefudiaruwaierueiaibuiaruPibuieienueiarueruarueruarubuiesujieibuieitieibuierudaburyubuierueieieruerueiemuPibuiemubuieruarutitijidieruienutitikeibuikyushiwaiemudiwaiesuemubuieitibuiesuesuidaburyueidaburyuibuijierujibuiesuesutitibuijiefubuibuiPiefutiaiemuerutishiwaiefuefuaieikyutiaieijieichiefuarukeiiaruaiijieruarukeiaruaruaruerueruesuaiaibuibuierubuibuitiefueierushidaburyuemuPiwaieichierubuikeitieruwaiemuerujiesueruerueichidaburyuPishidiefudieruefueruemuenuaiefuPiwaishitishiaiesuwaibuienuesushieruenuPiefueruwaieiefuefudiPiaruefuarukyueishitiesuemuerushishijikyuesuarushieijitiesueichiesuesuesujiikeiesueiesuwaiesuesujieichiesukyujipijiPNMGKGGEQMHEKSIPYSQETLVVD (SEQ ID NO: 22) APLNR protein, or an amino acid sequence substantially similar to SEQ ID NO:22. All references herein to proteins, polypeptides, and protein fragments unless otherwise clearly identified as being from a non-human species (eg, "mouse APLNR", "monkey APLNR", etc.), It is intended to refer to the human form of a polypeptide, or protein fragment.

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

The term “antagonist” as used herein refers, in part, to a moiety that binds to a receptor at or near the same site as an agonist (eg, an endogenous ligand), which is typically a receptor. It does not activate the intracellular response, but thereby inhibits or neutralizes the intracellular response by agonists or partial agonists. The term antagonist may also refer in part to a moiety that binds a receptor agonist, thereby sequestering the agonist from its interaction with its cognate receptor. An example of an antagonist that binds an agonist is a ligand trap such as the VEGF trap. In some cases, the antagonist does not reduce the basal intracellular response in the absence of agonist or partial agonist. Antagonists do not necessarily have to act as competitive binding inhibitors, but do function by sequestering the agonist or indirectly regulating downstream effects.

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

The term "immunoglobulin" (Ig) refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, a pair of light (L) chains and a pair of heavy (H) chains, All four can be interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. For example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

As used herein, the term “antibody” includes any at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (eg, APLNR). Antigen-binding molecule or molecular complex of. The term "antibody" refers to an immunoglobulin molecule that comprises four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as described herein. An immunoglobulin molecule comprising said multimer (eg IgM) and one or more heavy chain fragments or one or more light chain fragments (eg Fab, F(ab′) ₂ or scFv fragments), including. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V _H) and a heavy chain constant region. The heavy chain constant region comprises three _domains, a C H _{1, C} H 2, and _{C H} 3. 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 contains one domain, C _L 1. The _VH and _VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). it can. Each _VH and _VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of the anti-APLNR antibody (or antigen binding portion thereof) can be identical to the human germline sequence, or can be naturally or artificially modified. Amino acid consensus sequences can be defined based on a parallel analysis of two or more CDRs.

The term "antibody" as used herein also includes antigen binding fragments of whole antibody molecules. As used herein, the term "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like refers to any naturally occurring, enzymatically accessible, synthetic, or gene. It includes an engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antibody-binding fragments of antibodies are prepared using any suitable standard technique, such as, for example, protein digestion techniques or recombinant gene manipulation techniques related to the manipulation and expression of DNA encoding antibody variable domains and optionally constant domains, It can be derived from a whole antibody molecule. Such DNAs are known and/or are readily available, eg, from commercially available sources, DNA libraries (eg including phage-antibody libraries), or can be synthesized. The DNA may, for example, place one or more variable and/or constant domains in a suitable arrangement, or introduce codons, create cysteine residues, modify, add or delete amino acids, etc. For that purpose, it can be sequenced and manipulated chemically or by using molecular biology techniques. Such techniques can also be used to synthesize any antibody fusion molecule that contains an antigen binding fragment derived from an entire antibody molecule.

Non-limiting examples of antibody binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (Vi) a dAb fragment, and (vii) an amino acid residue that mimics an antibody hypervariable region (eg, an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide Contains the smallest recognition unit. Domain-specific antibody, single domain antibody, domain deleted antibody, chimeric antibody, CDR-grafted antibody, diabodies, triabodies, tetrabodies, minibodies, Nanobodies (eg monovalent Nanobodies, divalent Nanobodies, etc.), small modules Other engineered molecules such as type immunopharmaceuticals (SMIPs) and shark variable IgNAR domains are also encompassed by the expression "antigen-binding fragment" as used herein.

An antigen-binding fragment of an antibody will typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and generally comprises at least one CDR that is adjacent to or in frame with one or more framework sequences. In an antibody-binding fragment that has a _VH domain linked to a _VL domain, the _VH domain and the _VL domain may be arranged with respect to each other in any suitable arrangement. For example, the variable region is a dimer and may include a _VH - _VH , _VH - _VL or _VL - _VL dimer. Alternatively, the antigen-binding fragment of an antibody may comprise a monomeric _VH or _VL domain.

In certain embodiments, antigen-binding fragments of antibodies may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configuration of the variable and constant domains that can be found in the antigen-binding fragment of an antibody of this _{disclosure, (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-C H 2-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 -C _{H 1-C H 2,} including _{(xii) V L -C H 1} -C H 2-C H 3, (xiii) V L -C H 2-C H 3, and _(xiv) V _L -C L .. In any arrangement of variable and constant domains, including any of the exemplary arrangements listed above, the variable and constant domains may be directly linked to each other or may be a complete hinge region or a partial hinge region. Alternatively, they can either be linked by a linker region. The hinge region is at least two (eg 5, 10, 15, 20, 40, resulting in flexible or semi-flexible association between adjacent variable and/or constant domains in a single polypeptide molecule. Amino acids (60 or more). Moreover, antigen-binding fragments of the antibodies of the present disclosure are non-covalently associated with each other and/or with one or more monomeric V _H or V _L domains (eg, by disulfide bond(s)). ), and may include homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above.

Like whole antibody molecules, antibody-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, each variable domain specifically binding to a distinct antigen or to different epitopes on the same antigen. can do. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be prepared using conventional techniques available in the art using antigen-binding fragments of the antibodies of the present disclosure. May be adapted for use in connection with.

The phrase "antibody fusion protein" includes recombinant polypeptides and proteins derived from the antibodies of this disclosure that have been engineered to include the antibodies or antigen-binding fragments described herein. The peptide component can be fused to the anti-APLNR antibody or antigen binding fragment at either the N-terminus or C-terminus of the antibody light or heavy chain, with or without a peptide linker. As used herein, the phrase "fused with" refers to, but is not limited to, a polypeptide formed by the expression of a chimeric gene made by combining two or more sequences. ), usually one gene is cloned into an expression vector in frame with the two genes such that the two genes encode one continuous polypeptide. Recombinant cloning techniques such as polymerase chain reaction (PCR) and cloning of restriction endonucleases are well known in the art. In addition to being made by recombinant techniques, some of the polypeptides are fused to each other by chemical reactions or other means known in the art for making custom polypeptides. Can be done.

In some embodiments, the components or amino acids of the antibody fusion protein are separated by a linker (or "spacer") peptide. Such peptide linkers are well known in the art (eg, polyglycine or Gly-Ser linkers) and typically allow proper folding of one or both components of an antibody fusion protein. The linker provides a flexible junction region for the components of the fusion protein, allowing independent migration of the two ends of the molecule and may play an important role in retaining the proper function of each of the two moieties. Thus, the junction region may act as a linker that joins the two parts together, and as a spacer allows each of the two parts to form its own biological structure and not interfere with other parts. To Furthermore, the junction region must create an epitope that will not be recognized as foreign by the subject's immune system, in other words, not considered immunogenic. The choice of linker can have an effect on the binding activity of the fusion protein and thus on the biological activity. (Huston, et al, 1988, PNAS, 85:16:5879-83, Robinson & Bates, 1998, PNAS 95(11):5929-34, and Arai, et al. 2001, PEDS, 14(8):529. -32, Chen, X. et al., 2013, Advanced Drug Delivery Reviews 65:1357-1369). In one embodiment, the apelin peptide is connected to the C-terminus or N-terminus of the light 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 via complement dependent cytotoxicity (CDC) or antibody dependent cell mediated cytotoxicity (ADCC). "Complement dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of this disclosure in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) is targeted to non-specific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils and macrophages. Refers to a cell-mediated reaction that recognizes bound antibody on cells, thereby leading to lysis of target cells. CDC and ADCC can be measured using assays well known and available in the art. (See, eg, US Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al. 1998 Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important in the antibody's ability to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody can be selected based on whether it is desired that the antibody mediate cytotoxicity.

In another embodiment, the antibody can be engineered with an Fc domain such that it does not activate all, some, or none of the normal Fc effector functions without affecting the desired pharmacokinetic properties of the antibody. Thus, antibodies with engineered Fc domains with altered Fc receptor binding may have reduced side effects. Thus, in one embodiment, the protein comprises a chimeric or otherwise modified Fc domain. For examples of chimeric Fc domains, see WO 2014/121087 A1, published Aug. 7, 2014.

The term “EC ₅₀ ”or “EC ₅₀ ”as used herein refers to the half-maximal effective concentration and is the response, eg, the cellular response between the baseline and the maximum after the specified exposure time. Including the concentration of the ligand that induces. The EC ₅₀ essentially represents the concentration of the ligand at which 50% of its maximal effect is observed. Thus, with respect to cell signaling, increased receptor activity is observed at reduced EC ₅₀ values, ie, at half maximal effective concentration values (less ligand required to generate a greater response).

The term “IC ₅₀ ”or “IC ₅₀ ”as used herein refers to the maximum inhibitory concentration of half the cellular response. In other words, a measure of the effectiveness of a particular moiety (eg, protein, compound, or molecule) in inhibiting biological or biochemical receptor function, where the assay inhibits certain biological processes. Quantify the amount of such an amount required to Thus, for cell signaling, greater inhibitory activity is observed with reduced IC ₅₀ values.

Methods of treating or ameliorating a vascular eye disease or disorder 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 method according to this aspect of the present disclosure comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist. In some embodiments, the APLNR antagonist is administered subcutaneously, intravenously, or intravitreally. In embodiments, the APLNR antagonist is administered in combination with the VEGF antagonist. In some embodiments, the APLNR antagonist is administered intravitreally in combination with the VEGF antagonist. In some embodiments, the APLNR antagonist is administered as a single combination dosage formulation with the VEGF antagonist. In some embodiments, the APLNR antagonist is administered in combination with a VEGF antagonist, the APLNR antagonist is administered intravenously, and the VEGF antagonist is administered intravitreally. The VEGF antagonist may be administered before, after or simultaneously with the APLNR antagonist.

The present invention proposes, in part, that the combination of antagonism directed against both the VEGF and apelin/APLNR pathways can beneficially affect unwanted pathological neovascularization of the eye. Based on the discovery of. In particular, Applicants have discovered that such combinations can be used to treat or prevent conditions such as proliferative diabetic retinopathy, retinopathy of prematurity, and diabetic retinopathy including age-related macular degeneration. Addition of an antagonist of APLNR can improve the anti-angiogenic effect of VEGF antagonism when VEGF pathway antagonism is saturated.

The terms "treatment," "treat," and the like, as used herein, eliminate the cause of symptoms either transiently or permanently, or the appearance of symptoms of angiogenic eye diseases or disorders. Preventing or delaying means reducing symptoms. In certain embodiments, the method comprises retinal angiogenesis, neovascularization, vascular leakage, retinal thickening within 500 μm of the foveal center, hard yellow exudation within 500 μm of the foveal center with adjacent retinal thickening. Objects, and at least one disc area of retinal thickening, within one disc diameter from the center of the fovea, astigmatism, fly mosquitoes, loss of contrast, diplopia, and eventual loss of vision. Are useful in the treatment or amelioration of at least one symptom or indication, including but not limited to some of any of the following: In the context of a method for treating a vascular eye disease such as AMD or DME, the term refers to one or more (eg, from the initiation of treatment, the control in the Early Treatment Diabetic Retinopathy Study (EDTRS) visual acuity chart. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) characters are acquired. In certain embodiments, the term means that from the beginning of treatment, loss of vision of 15 characters or more is prevented in the subject.

As used herein, the terms “prevention”, “prevent” and the like mean preventing the development of symptoms, signs or complications of vascular eye disease. In the context of methods for treating vascular eye diseases such as AMD or DME, the term means that from the start of treatment, moderate or severe loss of vision is prevented in the subject.

As used herein, "vascular eye disease or disorder" refers to an eye disease or disorder that affects blood vessels in the eye. The disease may be due to abnormal angiogenesis (formation of new blood vessels) or blockage or obstruction of blood vessels. The term as used herein includes eye diseases or disorders associated with angiogenesis. The terms include diabetic retinopathy (including proliferative diabetic retinopathy), diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, polypoidal choroidal vasculopathy, It includes, but is not limited to, an ocular disease or disorder selected from the group consisting of choroidal neovascularization (CNV), degenerative myopia (myopia CNV), neovascular glaucoma, and retinopathy of prematurity. In certain embodiments, the term "angiogenic eye disease or disorder" may be used interchangeably with "angiogenesis-related eye disease or disorder".

In certain embodiments, the disclosure includes a method of treating, preventing, or ameliorating at least one symptom or sign of an ocular disease or disorder associated with angiogenesis in a subject, wherein the disease or disorder is pathological neovascularization. , Diabetic retinopathy (including proliferative diabetic retinopathy) diabetic macular edema, age-related macular degeneration, retinal neovascularization, polypoidal choroidal angiopathy, choroidal neovascularization (CNV), degenerative myopia (myopia CNV), blood vessel Selected from the group consisting of neonatal glaucoma, and retinopathy of prematurity. In certain embodiments, administration of the APLNR antagonist also promotes normal retinal revascularization, eg, in oxygen-induced retinopathy (OIR).

As used herein, "diabetic macular edema" (DME) refers to a serious ocular condition that affects people with diabetes (type 1 or type 2). Macular edema is a blood vessel in the retina that leaks into the macula and causes fluid and protein deposits to collect above or below the macula of the eye (the yellow central region of the retina), causing thickening and swelling (edema). Occurs when. Swelling can distort a person's central visual field because the macula is near the center of the retina behind the eyeball. Tumorous symptoms of DME include, but are not limited to, blurred vision, fly mosquito disease, loss of contrast, double vision, and eventual loss of vision. The pathology of DME is usually characterized by disruption of water within the retina, disruption of the blood-retinal barrier that allows fluid to accumulate in retinal tissue, and the presence of retinal thickening. DME is currently the smallest letter that can be read on a standardized chart, images such as mydriatics to identify signs of disease, optical coherence tomography (OCT) or fluorescein angiography (FA) and tonometry Diagnosis is made during an eye examination, which consists of an examination, a visual acuity test that is determined by an instrument that measures pressure inside the eye. The following studies, optical coherence tomography (OCT), fluorescein angiography, and color stereo fundus photography are further performed to determine treatment modalities. DME can be generally characterized into two main categories: localized and diffuse. Localized DME is characterized by specific areas of discrete, distinct leaks with sufficient macular blood flow within the macula. Diffuse DME results from leakage of the entire capillary bed surrounding the macula and results from disruption of the blood-retinal barrier inside the eye. In addition to localized and diffuse, DME has clinically significant macular edema (CSME), non-CSME and CSME with central lesions with fovea (CSME-CI) based on clinical trial findings. are categorized. The present disclosure includes methods of treating the above-mentioned categories of DME.

Age-related macular degeneration (AMD), as used herein, refers to a serious ocular condition when the small central portion of the retina, known as the macula, deteriorates. Wet AMD is characterized by abnormal blood vessel growth from the lower macular choroid. This is called choroidal neovascularization (CNV). These blood vessels leak blood and fluid to the retina, causing visual distortions that appear wavy in straight lines, as well as blind spots and loss of central vision. These abnormal blood vessels eventually scar and lead to permanent loss of central vision. Symptoms of AMD include dark, blurry areas within the center of vision and reduced or altered color vision. AMD can be detected by routine eye examinations. One of the most common early signs of macular degeneration is the presence of drusen, which are very small yellow deposits under the retina or pigment aggregates.

As used herein, "retinopathy of prematurity" (ROP), also known as post lens fibroplasia and Terry syndrome, refers to a condition that affects premature infants of low birth weight and younger gestational age. Retinopathy of prematurity occurs when normal retinal blood vessel development is interrupted by birth before full gestation, leading to abnormal development of retinal blood vessels. If the condition progresses, scar tissue growth can lead to retinal detachment and vision loss or loss. The pathogenesis of ROP involves two distinct stages, the vaso-occlusive phase and the vascular proliferative phase. Normal retinal blood vessel growth is delayed in the first phase as a result of exposure to hyperoxic environments, while the second phase involves a rapid increase in angiogenesis followed by retinal detachment. Removal of the avascular retina by cold therapy or laser photocoagulation is considered the primary treatment for ROP, and anti-VEGF therapy (eg, anti-VEGF antibody) is being investigated. Despite these treatment options, the condition remains a major cause of lifelong visual impairment and the incidence remains relatively constant for over 20 years.

As used herein, diabetic retinopathy (DR) is a chronic progressive disease of the retinal microvasculature associated with long-term hyperglycemia. DR is the most common microvascular complication of diabetes and is a rapidly increasing problem worldwide as the prevalence of diabetes continues to increase worldwide. Proliferative DR (PDR), as used herein, is a complication of DR that impairs vision and is characterized by abnormal new blood vessel development in the retina, optic disc, or anterior segment of the eye. PDR is an advanced stage of DR, driven by hypoxia and expression of pro-angiogenic growth factors, to stimulate the abnormal formation of new blood vessels protruding into the anterior space of the retina. Retinal neovascularization can lead to discontinuous loss of vision if it causes vitreous hemorrhage or tractional retinal detachment. Laser photocoagulation has been the standard for treatment of PDRs for many years, and more recent treatments include intraocular treatment with anti-VEGF (eg, anti-VEGF antibody) and steroids, and vitreoretinal surgery. Including. Despite these therapeutic advances, unmet therapeutic needs remain, particularly non-invasive, non-destructive and longer lasting treatment options.

As used herein, the phrase "subject in need thereof" refers to a human or non-human mammal exhibiting and/or diagnosed with one or more symptoms or signs of an eye disease or disorder associated with angiogenesis. Means an animal. The term "subject in need thereof" refers to, for example, retinal angiogenesis, angiogenesis, vascular leakage, thickening of the retina within 500 μm of the fovea center, thickening of the adjacent retina prior to treatment. Hard yellow exudates within 500 μm from the center of the fossa, and at least one disc area of retinal thickening, within one disc diameter from the center of the fovea, blurred vision, flying mosquito disease, loss of contrast, Subjects further exhibiting (or exhibiting) one or more signs of angiogenic eye disease such as dual focus and eventual loss of vision may be further included.

In the context of the present disclosure, “a subject in need thereof” is diabetic retinopathy (including proliferative diabetic retinopathy), diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein. Vascular eye disease or disorder selected from the group consisting of occlusion, branch retinal vein occlusion, polypoidal choroidal vasculopathy, choroidal neovascularization (CNV), degenerative myopia (myopia CNV), neovascular glaucoma, and retinopathy of prematurity Further including a human or non-human mammal having

In the context of the present disclosure, a “subject in need thereof” may include a subset of the population that is more susceptible to DME or AMD, or is associated with a DME- or AMD-related biomarker, or ROP or PDR. Can indicate increased levels of biomarkers that For example, a “subject in need thereof” can include a subject that has been suffering from diabetes for 10 years or more, or who has frequent high blood glucose levels or high fasting blood glucose levels. In certain embodiments, the term "subject in need thereof" includes subjects who have been or have been diagnosed with diabetes before or at the time of administration of the APLNR antagonist and/or VEGF antagonist. In certain embodiments, the term "subject in need thereof" includes subjects who are 51 years or older prior to or at the time of administration of the APLNR antagonist and/or VEGF antagonist. In some embodiments, the term "subject in need thereof" includes subjects who are smokers or who have high blood pressure or high cholesterol.

The present disclosure treats the severity of vascular eye disease, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist, Including methods for preventing or alleviating, the pharmaceutical composition is administered to a subject in multiple doses, eg, as part of a particular therapeutic dosing regimen. For example, a therapeutic dosing regimen is 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 every week. Once every two weeks, once every three weeks, once every four weeks, once every one month, once every two months, once every three months, once every four months , Or less frequently, may be administered to the subject at multiple doses of the pharmaceutical composition. In certain embodiments, the therapeutic dosing regimen can include administering multiple doses of the pharmaceutical composition to the subject once a day, or twice a day, or more times.

The present disclosure includes methods for inhibiting, reducing, or suppressing vascular leakage in a subject. In certain embodiments, methods according to this aspect of the disclosure are directed to a single dose of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist to reduce or inhibit vascular leakage in the eye of the subject. Administration to. In certain embodiments, vascular leakage is inhibited at greater than 3 weeks, greater than 4 weeks, greater than 8 weeks, or greater than 10 weeks as compared to a subject receiving a VEGF antagonist alone.

The methods of the present disclosure include administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist in combination with a VEGF antagonist, according to certain embodiments. In certain embodiments, the APLNR antagonist may be administered in combination with therapy, including laser therapy to prevent leakage into the macula. The phrase "in combination with" as used herein means that the pharmaceutical composition comprising the APLNR antagonist is administered to the subject at the same time, shortly before, or shortly after the administration of the VEGF antagonist. As used herein, the phrase "in combination with" means that a pharmaceutical composition comprising an APLNR antagonist is administered to a subject at the same time, shortly before, or shortly after administration of the VEGF antagonist. .. In certain embodiments, VEGF antagonists are administered as a co-formulation with an APLNR antagonist. In a related aspect, the present disclosure provides that a therapeutically effective amount of a pharmaceutical composition comprising an APLNR antagonist is administered to a subject to produce a greater therapeutic or synergistic effect as compared to administration of a VEGF antagonist alone. Including methods, including providing. The subject may be on a therapeutic regimen of a VEGF antagonist administered intravitreally. In some embodiments, the APLNR antagonist may be added to this therapeutic regimen to reduce one or more intravitreal infusions of the VEGF antagonist or increase the interval between consecutive intravitreal infusions. obtain.

The methods of the present disclosure are useful for treating or preventing vascular eye disease in a subject who has been diagnosed with vascular eye disease or is at risk of suffering from vascular eye disease. In general, the methods of the present disclosure may be performed within 36 weeks (at the primary dose administered at "week 0") of the treatment regimen, eg, by the end of week 6, by the end of week 12, by week 18. Demonstrate efficacy by the end of the eye, by the end of the 24th week, etc. In the context of methods of treating angiogenic eye diseases such as AMD and DME, "efficacy" means that from the beginning of treatment, the subject is 10 or less in the early treatment diabetic retinopathy study (ETDRS) visual acuity chart. Means to lose the letter. In certain embodiments, “efficacy” refers to one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more of the ETDRS table from the time of initiation of treatment. ) Means the acquisition of the character.

For example, a therapeutic dosing regimen is 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 every week. Once every two weeks, once every three weeks, once every four weeks, once every one month, once every two months, once every three months, once every four months , Or less frequently, may be administered to the subject at multiple doses of the pharmaceutical composition. In certain embodiments, the therapeutic dosing regimen can include administering multiple doses of the pharmaceutical composition to the subject once a day, or twice a day, or more times.

APLNR Antagonist The present disclosure discloses at least one symptom of a vascular eye disease or disorder in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the APLNR antagonist. Or a method for treating, preventing or ameliorating the symptoms. 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. US2014/000518 and US2015/0125459, as well as International Patent Publications WO2004081198 and WO2015140296. Exemplary peptide inhibitors or antagonists of APLNR can be found in US Pat. Nos. 9,593,153 and 7,736,646, as well as International Patent Publication No. Other APLNR antagonists are MM54, MM07, N-alpha-acetyl-nona-D-argininamide acetate (ALX40-4C), ML221, mutant apelin-13 (F13A), 4-oxo-6-((pyrimidine- 2-ylthio)methyl)-4H-pyran-3-yl-4-nitrobenzoate (Ml221), and E339-3D6.

APLNR antagonists include antibodies or antigen-binding fragments thereof selected from the group of antibodies disclosed in International Patent Publication No. WO2015/077741, published May 5, 2015, the entirety of which and US Pat. , 493,554. In some embodiments, the antibody is H2aM9232N, H4H9232N, or H1M9207N. In one embodiment, the anti-APLNR antibody is H4H9232N.

The APLNR antagonist has a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 13, or sequence identity of at least 90%, at least 95%, at least 98%, or at least 99%. Antibody or antigen-binding fragment thereof selected from the group comprising sequences substantially similar thereto.

The APLNR antagonist has a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 18, or at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. An antibody or antigen-binding fragment thereof comprising a sequence substantially similar thereto.

According to certain embodiments, the antibody or antigen-binding fragment thereof comprises heavy and light chains encoded by the nucleic acid sequences of SEQ ID NOs: 1 and 6 (eg, H1M9207N), and SEQ ID NOs: 12 and 17 (eg, H2aM9232N or H4H9232N). Contains CDR sequences.

Certain non-limiting exemplary antibodies and antigen-binding fragments of the present disclosure are SEQ ID NOs: 3-4-5-8-9-10 (eg, H1M9207N), and SEQ ID NOs: 14-15-16-19, respectively. -20-21 (eg, H2aM9232N or H4H9232N), including an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain having an amino acid sequence selected from the group consisting of: The antibodies designated H2aM9232N and H4H9232N share the same human variable regions (HCVR and LCVR). H2aM9232N contains the murine IgG2a heavy chain constant region, while H4H9232N contains the human IgG4 heavy chain constant region.

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

The APLNR antagonist has a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 16, or sequence identity of at least 90%, at least 95%, at least 98%, or at least 99%. A light chain CDR3 (LCDR3) domain having a sequence substantially similar thereto and an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 21, or at least 90%, at least 95%, at least 98%, or at least 99. Antibodies or antigen-binding fragments thereof that contain substantially similar sequences thereto with% sequence identity.

In certain embodiments, the APLNR antagonist comprises an antibody or antigen binding fragment thereof that comprises an HCDR3/LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NOs: 5/10 and 16/21.

The APLNR antagonist has a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 14, or sequence identity of at least 90%, at least 95%, at least 98%, or at least 99%. A heavy chain CDR2 (HCDR2) domain having a sequence substantially similar thereto, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 15, or at least 90%, at least 95%, at least 98%, or at least 99% A light chain CDR1 (LCDR1) domain having a sequence substantially similar thereto, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 19, or at least 90%, at least 95%, at least 98% Or a light chain CDR2 (LCDR2) domain having a sequence substantially similar thereto with at least 99% sequence identity, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 20, or at least 90%, at least Antibodies or antigen-binding fragments thereof that contain substantially similar sequences thereto with 95%, at least 98%, or at least 99% sequence identity.

Certain non-limiting exemplary anti-APLNR antibodies include antibodies or antigen-binding fragments of antibodies that specifically bind to APLNR, the antibodies or antigen-binding fragments comprising the group consisting of SEQ ID NOs: 2/7 and 13/18. A heavy and light chain CDR domain contained within a heavy and light chain variable region (HCVR/LCVR) sequence selected from

Methods and techniques for identifying CDRs within the HCVR amino acid sequence and the LCVR amino acid sequence are well known in the art and can be used to identify specific HCVR amino acid sequences and/or LCVRs disclosed herein. CDRs within the amino acid sequence can be identified. Exemplary rules that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between Kabat's and Chothia's approaches. Is. For example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991), Al-Lazikani et al. , 1997, J.; Mol. Biol. 273:927-948, and Martin et al. , 1989, Proc. Natl. Acad. Sci. USA 86:9268-9272. Public databases are also available to identify CDR sequences within antibodies.

The present disclosure provides a heavy chain (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 and 13, or to that having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. An antibody or antigen-binding fragment thereof is provided that comprises a substantially similar sequence.

The present disclosure provides a light chain (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 18, or to that having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. Further provided is an antibody or antigen-binding fragment of an antibody that comprises a substantially similar sequence.

The disclosure further provides an antibody or antigen-binding fragment thereof that comprises the HC and LC (HC/LC) amino acid sequence pairs.

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

The present disclosure includes anti-APLNR antibodies with modified glycosylation patterns. In some applications, modifications to remove undesired glycosylation sites, or antibodies lacking a fucose moiety, eg, to increase antibody-dependent cellular cytotoxicity (ADCC) function, may be useful. (See Shield et al. 2002, JBC 277:26733). In other applications, galactosylation modifications can be made to modify complement dependent cytotoxicity (CDC).

For example, the present disclosure is directed to cell-based block or inhibition bioassays using, for example, the assay formats defined in Examples 5, 8, 9 or 11 of WO2015/077491, or substantially similar assays. 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, about <300 pM, <250 pM, <200 pM, <150 pM, <100 pM, <90 pM, <80 pM, <70 pM, <60 pM, <50 pM, <40 pM, <30 pM, <20 pM , Or an APLNR antagonist thereof that blocks or inhibits apelin-mediated signaling in cells expressing human APLNR with an IC ₅₀ of less than about 10 pM.

The present disclosure provides 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, as measured in an APLNR-induced pERK assay. less 800 pM, less than about 700 pM, less than about 600 pM, comprising less than about 500 pM, a APLNR antagonist that inhibits the rate of APLNR mediated pERK in the presence apelin having an _{IC 50} of less than about less than 400 pM, or about 300 pM.

However, in other embodiments, certain APLNR antagonists of the present disclosure do not block or partially block the interaction of APLNR with apelin despite their ability to inhibit or attenuate APLNR-mediated signaling. Only shut off. Such antibodies and antigen binding fragments thereof may be referred to herein as "indirect blockers." Without being bound by theory, it is believed that the disclosed indirect blockers function by binding to APLNR at an epitope that does not overlap, or only partially overlaps, the N-terminal ligand binding domain of APLNR, but Nevertheless, it does interfere with APLNR-mediated signaling without directly blocking the APLNR/Apelin interaction.

The present disclosure includes APLNR antagonists that bind to high affinity and/or specific soluble APLNR molecules. For example, the present disclosure provides APLNR with binding ratios of greater than about 20, as measured by a fluorescence activated cell sorter (FACS) assay, using, for example, the assay format defined in Example 4 of WO2015/077491. Antibodies that bind and antigen-binding fragments of antibodies are included. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure are, for example, greater than about 15, greater than about 20, greater than about 100, greater than about 200, about 200, as measured by FACS, or a substantially similar assay. It binds to APLNR with a binding ratio of greater than 300, greater than about 400, greater than about 500, greater than about 1000, greater than about 1500, or greater than about 2000.

The present disclosure provides, for example, greater than about 10 minutes when measured by surface plasmon resonance at 25° C. or 37° C. using the well-known BIAcore™ assay format, or a substantially similar assay. It includes anti-APLNR antibodies and antigen-binding fragments thereof that specifically bind to APLNR with a dissociation half-life (t1/2).

Antibodies of the present disclosure can have one or more of the above mentioned biological characteristics, or some combination thereof. Other biological characteristics of the antibodies of the present disclosure will be apparent to those of skill in the art upon inspection of the present disclosure, including the working examples herein.

An anti-APLNR antibody has 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 sequence from which the antibody was derived. Can be included. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with, for example, germline sequences available from public antibody sequence databases. The disclosure includes antibodies and antigen-binding fragments thereof 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 derived from the antibody. To the corresponding residue(s) in the germline sequence, or to the corresponding residue(s) in another human germline sequence, or to conservative amino acid substitutions in the corresponding germline residue(s). Mutate (collectively, such sequence changes are referred to herein as "germline mutations"). One of skill in the art will readily appreciate the large number of antibodies and antibody-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. Can be produced. In certain embodiments, all of the framework and/or CDR residues within the _VH and/or _VL domain are mutated again to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back into the original germline sequence, eg, the mutated residue is within the first 8 amino acids of FR1, or the mutated residue is FR4. Residues that are found within the last 8 amino acids or mutated are found only within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) is a corresponding residue of a different germline sequence (ie, a germline sequence that differs from the germline sequence from which the antibody was originally derived). Mutate to the group(s). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and/or CDR regions, eg, where particular individual residues are specific germline sequences. While other specific residues that differ from the original germline sequence are either retained or mutated to corresponding residues in different germline sequences. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be used to improve binding specificity, increase binding affinity, improve or enhance the biological properties of an antagonist or agonist (optionally). Can be easily tested for one or more desired properties such as reduced immunogenicity. Antibodies and antigen-binding fragments obtained in this general manner are included in this disclosure.

The disclosure further includes anti-APLNR antibodies that include variants of any of the HCVR amino acid sequences, LCVR amino acid sequences, and/or CDR amino acid sequences disclosed herein that have one or more conservative substitutions. For example, the disclosure is directed to any of the HCVR amino acid sequences, LCVR amino acid sequences, and/or CDR amino acid sequences disclosed herein, eg, 10 or less, 8 or less, 6 or less or 4 or less, etc. Anti-APLNR antibodies having HCVR amino acid sequences, LCVR amino acid sequences, and/or CDR amino acid sequences with conservative amino acid substitutions of.

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

The term "substantially identical" or "substantially identical", when referring to a nucleic acid or fragment thereof, optimally aligns with the appropriate nucleotide insertion or deletion with another nucleic acid (or its complementary strand). , As determined below by at least about 95%, more preferably at least about 96%, 97% of the nucleotide bases as determined by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap. Indicates that there is%, 98%, or 99% nucleotide sequence identity. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain cases, encode a polypeptide that has the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

The term “substantially similar” or “substantially similar” when applied to a polypeptide means that the two peptide sequences are optimally aligned, such as by the program GAP or BESTFIT, with the given gap weights. Share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). .. In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, eg, Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine, (2) aliphatic-hydroxyl side chains: serine and Threonine, (3) amide-containing side chain: asparagine and glutamine, (4) aromatic side chain: phenylalanine, tyrosine, and tryptophan, (5) basic side chain: lysine, arginine, and histidine, (6) acidic side chain. : Aspartic acid and glutamic acid, and (7) Sulfur-containing side chain: cysteine and methionine. Preferred conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions may be referred to as Gonnet et al. Any change having a positive value in the PAM250 log-likelihood matrix disclosed in 1992 Science 256:1434-1445. A "moderately conservative" substitution is any change having a nonnegative value in the PAM250 log-likelihood matrix.

Sequence similarity to polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences with similar measurements assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG software may be used 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 its mutant protein. Contains programs such as Gap and Bestfit that can be used with the default parameters. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, a program in GCG version 6.1. FASTA (eg FASTA2 and FASTA3) provides alignment and percent sequence identity of the region of best overlap between the query and search sequences (Pearson (1994) supra). Another preferred algorithm for comparing the sequences of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, using the default parameters, in particular BLASTP or TBLASTN. See, for example, Altschul et al., each hereby incorporated by reference. , 1990, J.; Mol. Biol. 215:403-410 and Altschul et al. , 1997, Nucleic Acids Res. 25:3389-402.

The disclosure further includes anti-APLNR antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (eg, H1M9207N, and H2aM9232N, and H4H9232N). Similarly, the disclosure further includes anti-APLNR antibodies that compete with any of the specific exemplary antibodies described herein (eg, H1M9207N, and H2aM9232N, and H4H9232N) for binding to APLNR.

Whether the antibody binds to the same epitope as the reference anti-APLNR antibody, or competes with the reference anti-APLNR antibody for binding, using routine methods known in the art and exemplified herein. Can be easily determined. For example, the reference antibody is allowed to bind to the APLNR protein to determine if the test antibody binds to the same epitope as the reference anti-APLNR antibody of the present disclosure. Next, the ability of the test antibody to bind to APLNR is evaluated. If the test antibody is able to bind to APLNR after saturation binding to the reference anti-APLNR antibody, it can be concluded that this test antibody binds to a different epitope than the reference anti-APLNR antibody. On the other hand, if the test antibody is unable to bind to the APLNR molecule after saturation binding to the reference anti-APLNR antibody, then the test antibody may bind to the same epitope bound by the reference anti-APLNR antibody of the present disclosure. Then, the observed loss of binding of the test antibody was actually due to binding to the same epitope as the reference antibody, or a steric block (or another phenomenon) was observed. Further routine experiments (eg, peptide mutation and binding analysis) can be performed to determine if the cause is This type of experiment can be performed using ELISA, RIA, BIAcore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present disclosure, for example, a 1, 5, 10, 20, or 100-fold excess of one antibody is used in a competitive binding assay (eg, Junghans et al., 1990, Cancer Res. 50:1495). Two antibodies are identical (or overlapping) when they inhibit the binding of the other antibody by at least 50%, but preferably by 75%, 90%, or even 99% as measured by -1502). Yes) bind to the epitope. Alternatively, two antibodies are considered to bind 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 antibody. .. Two antibodies are considered to have "overlapping epitopes" if only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

To determine if the antibody competes (or cross-competes for binding) with a reference anti-APLNR antibody, the binding methodologies described above are performed in two directions. In the first orientation, the reference antibody is allowed to bind to the APLNR protein under saturating conditions and then the binding of the test antibody to the APLNR molecule is evaluated. In the second orientation, the test antibody is allowed to bind to the APLNR under saturating conditions prior to assessing the binding of the reference antibody to the APLNR. It is concluded that in both orientations the test and reference antibodies compete for binding to APLNR if only the first (saturating) antibody is able to bind to APLNR. As will be appreciated by those in the art, an antibody that competes for binding with a reference antibody does not necessarily bind the same epitope as the reference antibody, but by binding to overlapping or contiguous epitopes. , Can sterically block the binding of the reference antibody.

In certain embodiments of the present disclosure, the anti-APLNR antibody of the present disclosure is a human antibody. 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 present disclosure are encoded, for example, by human germline immunoglobulin sequences in CDRs, particularly CDR3, such as mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo. May include amino acid residues that are not. However, the term “human antibody” as used herein is intended to include antibodies in which CDR sequences derived from the germ line of another mammalian species (eg, mouse) have been grafted into human framework sequences. Not something to do. In various embodiments, the antibodies discussed herein are human antibodies with IgG heavy chain constant regions. In some cases, the antibody has a heavy chain constant region of human IgG1 or IgG4 isotype.

The antibodies of the present disclosure can be recombinant human antibodies in some embodiments. The term "recombinant human antibody", as used herein, is prepared, expressed, produced or produced by recombinant means, such as an antibody expressed using a recombinant expression vector transfected into a host cell. All isolated human antibodies (see below), antibodies isolated from recombinant combinatorial human antibody libraries (see below), antibodies isolated from animals (eg, mice) that are transgenic for human immunoglobulin genes. (Eg, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or by any other means including splicing of human immunoglobulin gene sequences into other DNA sequences. Or intended to include isolated antibodies. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis if transgenic animals are used for human Ig sequences), and thus The amino acid sequences of the _VH and _VL regions are sequences that are derived from and related to human germline _VH and _VL sequences, but that cannot occur naturally in vivo within the human antibody germline repertoire.

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

The frequency of occurrence of the second form in various intact IgG isotypes depends on, but is 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 IgG4 hinge can significantly reduce the appearance of the second form to the levels typically observed with the human IgG1 hinge (Angal et al., 1993, Molecular Immunology 30:105). The present disclosure encompasses antibodies having one or more mutations in the hinge, C _H 2 or C _H 3 regions, which may be desirable, for example in production, to improve the yield of the desired antibody form.

The antibody of the present disclosure may be an isolated antibody. As used herein, "isolated antibody" means the antibody that has been identified and that has been 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 occurs or is naturally produced is an "isolated antibody" for the purposes of this disclosure. Is. Isolated antibody further includes antibodies in situ within recombinant cells. An isolated antibody is one that has undergone at least one purification or isolation step. According to certain embodiments, the isolated antibody may be substantially free of other cellular and/or chemical agents.

The present disclosure includes neutralizing and/or blocking anti-APLNR antibodies. As used herein, a "neutralizing" or "blocking" antibody is intended to refer to an antibody that binds APLNR, and includes (i) APLNR or APLNR fragments and APLNR receptor components such as apelin peptides. A) and/or (ii) results in inhibition of at least one biological function of APLNR. Inhibition caused by neutralizing or blocking antibodies to APLNR need not be complete, as long as inhibition is detectable using a suitable assay.

VEGF Antagonist As used herein, a "VEGF antagonist" binds or interacts with VEGF, inhibits the binding of VEGF to its receptors (VEGFR1 and VEGFR2), and/or biological signaling of VEGF. And any agent that inhibits activity. VEGF antagonists include molecules that interfere with the interaction between VEGF and the native VEGF receptor, eg, bind to VEGF or the VEGF receptor and prevent or interfere with the interaction between VEGF and VEGF receptor. To do. Specific exemplary VEGF antagonists include anti-VEGF antibodies (eg, ranibizumab [LUCENTIS®]), anti-VEGF receptor antibodies (eg, anti-VEGFR1 antibody, anti-VEGFR2 antibody, etc.), small molecule inhibitors of VEGF. (Eg, sunitinib), and chimeric molecules of the VEGF receptor system such as aflibercept and dibuaflibercept or fusion proteins that inhibit VEGF (also referred to herein as “VEGF-Traps”). Other examples of VEGF traps are ALT-L9, M710, FYB203, and CHS-2020. Additional examples of VEGF traps include US Pat. Nos. 7,070,959, 7,306,799, 7,374,757, 7,374,758, 7,531,173, No. 7,608,261, No. 5,952,199, No. 6,100,071, No. 6,383,486, No. 6,897,294, and No. 7,771,721. And is specifically incorporated herein by reference. Additional VEGF inhibitors and/or antagonists include the small molecule inhibitors pazopanib, sorafenib, axitinib, ponatinib, regorafenib, cabozantinib, vandetanib, cabozantinib, and lenvatinib, and the antibodies that inhibit VEGF, bevacizumab, and Ramucirumab, or a biosimilar molecule thereof, is included.

Chimeric molecules of the VEGF receptor system include chimeric polypeptides that include two or more immunoglobulin (Ig)-like domains of the VEGF receptor, such as VEGFR1 (also called Flt1) and/or VEGFR2 (also called Flk1 or KDR). And may include a multimerization domain (eg, an Fc domain that facilitates multimerization (eg, dimerization) of two or more chimeric polypeptides). An exemplary chimeric molecule of the VEGF receptor system is the molecule referred to as VEGFR1R2-FcΔC1(a) (also known as aflibercept; marketed under the product name EYLEA®). In certain embodiments, aflibercept comprises an amino acid sequence represented as EmubuiesuwaidaburyuditijibuieruerushieierueruesushierueruerutijiesuesuesujiesuditijiaruPiefubuiiemuwaiesuiaiPiiaiaieichiemutiijiaruierubuiaipishiarubuitiesuPienuaitibuitierukeikeiefuPieruditieruaiPidijikeiaruaiaidaburyudiesuarukeijiefuaiaiesuenueitiwaikeiiaijieruerutishiieitibuienujieichieruwaikeitienuwaierutieichiarukyutienutiaiaidibuibuieruesuPiesueichijiaiieruesubuijiikeierubuieruenushitieiarutiieruenubuijiaidiefuenudaburyuiwaiPiesuesukeieichikyueichikeikeierubuienuarudierukeitikyuesujiesuiemukeikeiefueruesutierutiaidijibuitiaruesudikyujieruwaitishieieiesuesujieruemutikeikeienuesutiefubuiarubuieichiikeidikeitieichitishiPiPishiPiEipiieruerujiJipiesubuiefueruefuPiPikeiPikeiditieruemuaiesuarutiPiibuitishibuibuibuidibuiesueichiidiPiibuikeiefuenudaburyuwaibuidijibuiibuieichienueikeitikeiPiaruiikyuwaienuesutiwaiarubuibuiesubuierutibuierueichikyudidaburyueruenujikeiiwaikeishikeibuiesuenukeieieruPiEipiaiikeitiaiesukeieikeijikyuPiaruiPikyubuiwaitieruPiPiesuarudiierutikeienukyubuiesuerutishierubuikeijiefuwaiPiesudiaieibuiidaburyuiesuenujikyuPiienuenuwaikeititiPiPibuierudiesudijiesuefuefueruwaiesukeierutibuidikeiesuarudaburyukyukyujienubuiFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23).

The amount of VEGF antagonist included within the pharmaceutical formulations of the present disclosure may vary depending on the particular properties desired for the formulation as well as the particular circumstances and purpose for which the formulation is intended. In certain embodiments, the pharmaceutical formulation comprises 5±0.75 mg/mL to 150±22.5 mg/mL VEGF antagonist, 10±1.5 mg/mL to 100±15.0 mg/mL VEGF antagonist, 20. A liquid formulation comprising ±3 mg/mL to 80 ±12 mg/mL VEGF antagonist, 30 ±4.5 mg/mL to 70 ±10.5 mg/mL VEGF antagonist, or 40 ±6.0 mg/mL VEGF antagonist. is there. For example, formulations of the present disclosure include about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, or about 60 mg/mL VEGF antagonist.

The methods of the present disclosure include administering to a subject in need thereof a therapeutic composition comprising a VEGF antagonist.

Therapeutic Formulations and Administration The present disclosure provides pharmaceutical formulations comprising at least one APLNR antagonist, such as an anti-APLNR antibody, or antigen-binding fragment thereof that specifically binds human APLNR. According to certain other embodiments, the present disclosure provides pharmaceutical formulations that include an additional therapeutic agent. The disclosure further provides a pharmaceutical formulation comprising at least one VEGF antagonist, eg for use in conjunction with a pharmaceutical formulation comprising at least one APLNR antagonist.

The pharmaceutical compositions of this disclosure are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, resistance, etc. Many suitable formulations can be found in the formulation known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, lipids (cationic or anionic), DNA complexes containing powders, pastes, ointments, jellies, waxes, oils, vesicles (LIPOFECTIN™, Life Technologies, Carlsbad, CA, etc.). , Anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowaxes. Powell et al. See also "Compendium of exclusives for parental formulations" PDA, 1998, J Pharm Sci Technology 52:238-311.

The dose of an APLNR antagonist such as an anti-APLNR antibody or an antigen-binding fragment thereof that specifically binds to human APLNR administered to a subject varies depending on the subject's age and physique, target disease, condition, administration route, and the like. Preferred doses are typically calculated according to body weight or body surface area. When an APLNR antagonist is used to treat a condition or disease, it is usually beneficial to administer the antibody of the present disclosure intravenously in a single dose of about 0.01 to about 50 mg/kg body weight. obtain. In other cases, it may be beneficial to administer the APLNR antagonist intravitreally, eg, at a concentration of about 0.01 to about 50 mg/kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective doses and schedules for administering APLNR antagonists, such as anti-APLNR antibodies, can be empirically determined, for example, monitoring the subject's progress by periodic assessment and adjusting the dose accordingly. You can In addition, interspecies scaling of dose can be performed using methods well known in the art (eg, Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

The dose of VEGF antagonist may vary depending on the age and build of the subject, the target disease, the condition, the route of administration and the like. Preferred doses are typically calculated according to body weight or body surface area. When a VEGF antagonist is used to treat a condition or disease, it is usually beneficial to administer the antibody of the present disclosure intravenously in a single dose of about 0.01 to about 50 mg/kg body weight. obtain. In other cases, it may be beneficial to administer the VEGF antagonist intravitreally, eg, at a concentration of about 0.01 to about 50 mg/kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective doses and schedules for administering VEGF antagonists can be empirically determined, for example, the subject's 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 compositions of the present disclosure, eg, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, Receptor-mediated endocytosis (eg, Wu et al. 1987) J. Am. Biol. Chem. 262:4429-4432). Methods of introduction include intradermal, transdermal, intramuscular, intraperitoneal, intravenous, intravitreal, subcutaneous, intranasal, epidural and oral routes. Not limited. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucosal skin linings such as oral mucosa, rectal and intestinal mucosa, and other biologically It can be administered with the active agent. Administration can be systemic or local.

The present disclosure includes methods, including administering an APLNR antagonist to a subject, wherein the APLNR antagonist is included within a pharmaceutical composition. In certain embodiments, the pharmaceutical composition further comprises a VEGF antagonist. In an alternative embodiment, the APLNR antagonist and VEGF antagonist may each be their own separate pharmaceutical dosage formulation. The pharmaceutical compositions of this disclosure may be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, resistance, etc. A number of suitable formulations can be found in the formulation known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water. Emulsions and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowaxes are included. Powell et al. See also "Compendium of exclusives for parental formulations" PDA (1998) J Pharm Sci Technology 52:238-311.

As used herein, the phrase "pharmaceutical formulation" refers to at least an active ingredient, or at least one when combined with one or more additional inactive ingredients, which is suitable for therapeutic administration to a human or non-human animal. A combination of one active ingredient (eg, a small molecule, macromolecule, compound, etc. capable of exerting a biological effect on a human or non-human animal) and at least one inactive ingredient. The term “formulation” as used herein means “pharmaceutical formulation” unless otherwise specified.

The amount of anti-APLNR antagonist, such as anti-APLNR antibody or antigen-binding fragment thereof, included within the pharmaceutical formulations of the present disclosure will depend on the particular properties desired for the formulation, as well as the particular circumstances for which the formulation is intended. And may vary depending on the purpose. In certain embodiments, the pharmaceutical formulation is 5±0.75 mg/mL to 150±22.5 mg/mL antibody, 7.5±1.125 mg/mL to 140±21 mg/mL antibody, 10±1. 0.5 mg/mL to 130±19.5 mg/mL antibody, 10±1.5 mg/mL antibody, 20±3 mg/mL antibody, 60±9 mg/mL antibody, or 120±18 mg/mL antibody. Can be a liquid formulation. For example, the formulations of the present disclosure are 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 that specifically bind to human APLNR. , Or about 140 mg/mL of antibody or antigen-binding fragment thereof.

In certain embodiments, the pharmaceutical formulation is a liquid formulation, which can include 5±0.75 mg/mL to 100±15 mg/mL VEGF antagonist. For example, formulations of the present disclosure include about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 50 mg. /ML, about 60 mg/mL, about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, or about 100 mg/mL of a VEGF antagonist such as aflibercept.

In certain embodiments, the pharmaceutical formulation is a stable liquid co-formulation comprising about 5 mg/mL to about 150 mg/mL APLNR antagonist and about 5-100 mg/mL VEGF antagonist.

The pharmaceutical formulations of the present disclosure include one or more excipients. The term "excipient" as used herein means any non-therapeutic agent added to the formulation to provide the desired consistency, viscosity or stabilizing effect.

Exemplary formulations containing VEGF antagonists that can be used in the context of the present disclosure are disclosed, for example, in US Pat. Nos. 7,531,173 and 7,608,261. Exemplary pharmaceutical compositions containing an APLNR antagonist that can be used in the context of the present disclosure are disclosed, for example, in US Patent Application Publication No. 2013/0186797. Exemplary pharmaceutical compositions containing an APLNR antagonist that can be used in the context of the present disclosure are disclosed, for example, in International Patent Publication No. WO 2016/085750 and US Patent Application Publication No. US 2011/0027286.

Combination Therapy The methods of the present disclosure include administering an APLNR antagonist to a subject in combination with a VEGF antagonist, according to certain embodiments. The expression "in combination with" as used herein means that the VEGF antagonist is administered before, after, or at the same time as the pharmaceutical composition comprising the APLNR antagonist. The term "in combination with" further includes sequential or simultaneous administration of the anti-APLNR antibody and VEGF antagonist. For example, when administered "before" a pharmaceutical composition comprising an APLNR antagonist, about 72 hours, about 72 hours, about 60 hours, about 72 hours prior to administration of a pharmaceutical composition comprising a VEGF antagonist. 48 hours ago, about 36 hours ago, about 24 hours ago, about 12 hours ago, about 10 hours ago, about 8 hours ago, about 6 hours ago, about 4 hours ago, about 2 hours ago, about 1 hour ago, about It may be administered 30 minutes, about 15 minutes, or about 10 minutes. When administered "after" a pharmaceutical composition comprising an APLNR antagonist, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour after administration of the pharmaceutical composition comprising a VEGF antagonist. After 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 It may be administered after 72 hours, or after about 72 hours. Administering “simultaneously” with a pharmaceutical composition comprising an APLNR antagonist means that the VEGF antagonist is administered separately (before, after, or at the same time) less than 5 minutes after administration of the pharmaceutical composition comprising the APLNR antagonist. Means to be administered to a subject in a dosage form or as a single combination dosage formulation comprising an APLNR antagonist and a VEGF antagonist.

Combination therapies include APLNR antagonists and VEGF antagonists (eg, aflibercept, VEGF traps, eg, US Pat. No. 7,087,411 (also referred to herein as “VEGF inhibitory fusion proteins”), anti-VEGF antibodies. (Eg ranibizumab), small molecule kinase inhibitors of VEGF receptors (eg sunitinib, sorafenib or pazopanib) and the like.

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

Vessels and Methods of Administration Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, eg, capable of expressing liposomes, microparticles, encapsulation in microcapsules, mutant viruses. Recombinant cells, which are receptor-mediated endocytosis (eg, Wu et al. 1987)J. Biol. Chem. 262:4429-4432). Administration methods include intradermal, transdermal, intramuscular, intraperitoneal, intravenous, intravitreal, subcutaneous, intranasal, epidural and oral routes. Not limited. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucosal skin linings such as oral mucosa, rectal and intestinal mucosa, and other biologically It can be administered with the active agent.

For the treatment of eye diseases, the pharmaceutical formulations of the present disclosure are administered by, for example, eye drops, subconjunctival injection, subconjunctival implant, intravitreal injection, intravitreal implant, sub-Tenon injection, or sub-Tenon implant. obtain.

The pharmaceutical compositions of this disclosure can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, for subcutaneous delivery, pen delivery devices readily have application in delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize replaceable cartridges containing a pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, this empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. .. The pen delivery device can then be reused. There are no replaceable cartridges in a disposable pen delivery device. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied for pharmaceutical composition, the entire device is discarded.

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

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular, infusion, and the like. These injectable preparations can be prepared by known methods. For example, injectable preparations may be prepared, for example, by dissolving, suspending or emulsifying the above-mentioned antibody or salt thereof in a sterile aqueous or oily medium conventionally used for injection. Injectable aqueous media include, for example, saline, glucose-containing isotonic solutions, and other adjuvants, which include alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), It may be used in combination with a suitable solubilizing agent such as a nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. As the oily medium, for example, sesame oil, soybean oil and the like are used, and may be used in combination with a solubilizing agent such as benzyl benzoate and benzyl alcohol. The injection thus prepared is preferably filled in suitable ampoules.

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into unit dose dosage forms suitable for matching the dose of active ingredient. Such dosage forms in unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like.

The pharmaceutical formulations of the present disclosure can be contained within any container suitable for storage or administration of pharmaceuticals and other therapeutic compositions. For example, the pharmaceutical formulation can be contained in a sealed and sterilized plastic or glass container having a defined volume, such as a vial, ampoule, syringe, cartridge, bottle, or IV bag. For example, different types of vials can be used to house the formulations of the present disclosure, including clear and opaque (eg, amber) glass or plastic vials. Similarly, any type of syringe can be used to house or administer the pharmaceutical formulations of the present disclosure.

The pharmaceutical formulations of the present disclosure can be contained within a "normal tungsten" syringe or a "low tungsten" syringe. As will be appreciated by those skilled in the art, the process of manufacturing glass syringes generally involves piercing the glass and using a high temperature that functions to create a hole that allows liquid to be drawn from the syringe and drained. Includes the use of tungsten rods. This process results in the deposition of trace amounts of tungsten on the inner surface of the syringe. Subsequent cleaning and other processing steps can be used to reduce the amount of tungsten in the syringe. The term "normal tungsten" as used herein means that the syringe contains more than 500 parts per billion (ppb) of tungsten. The term "low tungsten" means that the syringe contains less than 500 ppb tungsten. For example, a low-tungsten syringe according to the present disclosure may include 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, 20, or less or less ppb of tungsten.

The rubber plunger used in the syringe and the rubber stopper used to close the opening of the vial are coated to prevent contamination of the pharmaceutical contents of the syringe or vial or to maintain their stability. Can be done. Thus, the pharmaceutical formulations of the present disclosure may be contained within a syringe that includes a coated plunger, or within a syringe that is sealed with a coated rubber stopper, according to certain embodiments. For example, the plunger or stopper can 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 include, for example, US Pat. Nos. 4,997,423, 5,908,686, 6 , 286,699, 6,645,635, 7,226,554, the contents of which are incorporated herein by reference in their entirety. Certain exemplary coated rubber stoppers and plungers that can be used in the context of the present disclosure are available from West Pharmaceutical Services, Inc. (Lionville, Pa.) under the trade name "FluroTec®". FluroTec® is an example of a fluorocarbon coating used to minimize or prevent the drug from adhering to the rubber surface.

According to certain embodiments of the present disclosure, the pharmaceutical formulation may be contained within a low tungsten syringe that includes a fluorocarbon coated plunger.

The pharmaceutical formulation can be administered to the subject by infusion (eg, subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or by parenteral routes such as transdermal, mucosal, nasal, pulmonary or oral administration. A number of reusable pen or auto-injector delivery devices can be used to deliver the pharmaceutical formulations of the present disclosure subcutaneously. Examples include AUTOPEN(TM) (Owen Mumford, Inc., Woodstock, UK), DISETRONIC(TM) pen (Distronic Medical Systems, Bergdorf, Switzerland), HUMALOG MUPEN(TM) 75/HUMLOGH MIX 75/75. , HUMALLIN 70/30™ (pen) (Eli Lilly and Co., Indianapolis, Ind), NOVOOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark, Norvoen JUNIVenor Co), NOVOTEN JUNIVORN (Novenor). Denmark), BD(TM) pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN(TM), OPTIPEN PRO(TM), OPTIPEN STARLET(TM), and OPTICLIK(TM) (sanofi-fent). Germany), but is not limited thereto. Examples of disposable pen or auto-injector delivery devices for use in subcutaneous delivery of the pharmaceutical compositions of the present disclosure include SOLOSTAR™ pen (sanofi-eventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ auto-injector (Amgen, Thousand Oaks, Calif.), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (RAY, U.P., L.H.P.). (Trademark) pen (Abbott Labs, Abbott Park, Ill), but is not limited thereto.

The use of microinjectors to deliver the pharmaceutical formulations of the present disclosure is also contemplated herein. As used herein, the term "microinjector" refers to a therapeutic formulation that is in volume (eg, up to about 2.5 mL or more) over a long period of time (eg, about 10, 15, 20, 25, 30 minutes or more). Means a subcutaneous delivery device designed to be administered slowly. For example, US Pat. No. 6,629,949, US Pat. No. 6,659,982, and Meehan et al. , J. See Controlled Release 46:107-116 (1996). Microinjectors are particularly useful for delivering high concentrations of therapeutic proteins in high concentrations (eg, about 100, 125, 150, 175, 200, or higher mg/mL) or in viscous solutions. ..

In one embodiment, the pharmaceutical formulation is administered via IV infusion such that the formulation is diluted in an IV bag containing a physiologically acceptable solution. In one embodiment, the pharmaceutical composition is a formulated sterile formulation in an intravenous infusion bag, wherein the single dose of drug is 100 mL, 250 mL (or other similar volume suitable for intravenous drip delivery). ) In physiological buffer (eg 0.9% saline). In some embodiments, the infusion bag is made of polyvinyl chloride (eg, VIAFLEX, Baxter, Deerfield, Ill.). In some embodiments, the infusion bag is made of polyolefin (EXCEL IV Bags, Braun Medical Inc., Bethlehem, Pa.).

Dosing Regimens The present disclosure discloses about four times a week, about twice a week, about once a week, about every two weeks, about every three weeks, about every four weeks. Once every 5 weeks, once every 6 weeks, once every 8 weeks, once every 12 weeks, or as often as less therapeutic response is achieved. At a dosing frequency of 3 to the subject, the method comprising administering to the subject a pharmaceutical composition comprising an APLNR antagonist, such as an anti-APLNR antibody or antigen-binding fragment thereof. In certain embodiments, the method comprises about 4 times a week, about 2 times a week, about once a week, about every 2 weeks, about every 3 weeks, about 4 times. Once a week, about every 5 weeks, about every 6 weeks, about every 8 weeks, about every 9 weeks, about every 12 weeks, or more Administration of a pharmaceutical composition comprising an APLNR antagonist and a VEGF antagonist, such as an anti-APLNR antibody or antigen-binding fragment thereof, at a frequency of administration as often as a low therapeutic response is achieved.

According to certain embodiments of the present disclosure, multiple doses of APLNR and VEGF antagonists, such as anti-APLNR antibodies, or antigen binding fragments thereof, may be administered to a subject over a defined time course. The method according to this aspect of the disclosure comprises sequentially administering to a subject multiple doses of an APLNR antagonist and a VEGF antagonist, such as an anti-APLNR antibody or antigen binding fragment thereof. As used herein, “consecutively administering” means that each dose of APLNR antagonist and VEGF antagonist, such as an anti-APLNR antibody or antigen-binding fragment thereof, is at a different time, eg, at predetermined intervals (eg, a number). Means to be administered to the subject on different days separated by time, days, weeks or months). The present disclosure discloses a single primary dose of an APLNR antagonist, such as an anti-APLNR antibody or antigen binding fragment thereof, and a VEGF antagonist, followed by an APLNR antagonist, such as an anti-APLNR antibody or antigen binding fragment thereof, and a single dose of the VEGF antagonist. A method comprising sequentially administering to a subject a second dose as above, and optionally, followed by a single third dose of an APLNR antagonist such as an anti-APLNR antibody or antigen binding fragment thereof, and a VEGF antagonist. Including.

According to certain embodiments of the present disclosure, 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 may be administered to the subject over a defined time course. The method according to this aspect of the disclosure comprises sequentially administering to the subject multiple doses of a co-formulation comprising an APLNR antagonist and a VEGF antagonist, such as an anti-APLNR antibody or antigen binding fragment thereof. As used herein, “continuously administering” means that each dose of the APLNR antagonist in combination with the VEGF antagonist is separated by a predetermined interval (eg, hours, days, weeks or months). It means that the subject is administered at different times, for example, different days. The present disclosure discloses a single primary 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 VEGF antagonist, and optionally followed. , Sequentially administering to the subject one or more third doses of a co-formulated APLNR antagonist and a VEGF antagonist.

The terms "primary dose", "secondary dose", and "tertiary dose" refer to a time series of administration. Thus, a "primary dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "base dose"), a "second dose" is a dose administered after the first dose, and a "tertiary dose". "Is the dose administered after the second dose. The first dose, the second dose, and the third dose may all contain the same amount of APLNR antagonist (or co-formulation comprising APLNR antagonist and VEGF antagonist), but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amounts included in the primary, secondary and/or tertiary doses are different (eg, adjusted up or down as appropriate) from one another during the course of treatment. In certain embodiments, one or more (eg, 1, 2, 3, 4, or 5) doses are administered as a “loading dose” at the beginning of the treatment regimen, followed by lower Subsequent doses (eg, "maintenance doses") that are administered on a frequency basis are administered. For example, an APLNR antagonist (or a co-formulation comprising an APLNR antagonist and a VEGF antagonist) can be administered to a subject having an eye disease or disorder at a loading dose of about 6 mg, followed by one or more maintenance doses.

In one exemplary embodiment of the disclosure, each secondary and/or tertiary dose comprises 1-14 of the immediately preceding dose (eg 1, 1 and 1/2, 2, 2 and 1/2, 3, 3 and 1/2, 4, 4 and 1/2, 5, 5 and 1/2, 6, 6 and 1/2, 7, 7 and 1/2, 8, 8 and 1/2, 9, 9, and 1/2, 10, 10 and 1/2, 11, 11 and 1/2, 12, 12 and 1/2, 13, 13 and 1/2, 14, 14 and 1/2, or more) weeks later Is administered. As used herein, the phrase "immediate dose" refers to an APLNR antagonist (or APLNR) that is administered to a subject in a multiple dose series prior to the administration of the next dose in the series without dose intervention. Antagonist and VEGF antagonist).

The method according to this aspect of the disclosure may include administering to the subject any number of secondary and/or tertiary doses of the anti-APLNR antagonist (or co-formulation comprising the APLNR antagonist and the VEGF antagonist). For example, in certain embodiments, only a single secondary dose is administered to the subject. In other embodiments, two or more (eg, two, three, four, five, six, seven, eight or more) secondary doses are administered to the subject. Similarly, in certain embodiments, only a single tertiary dose is administered to the subject. In other embodiments, two or more (eg, two, three, four, five, six, seven, eight or more) tertiary doses are administered to the subject.

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

The disclosure includes methods that include sequential administration of an APLNR antagonist and a VEGF antagonist to a subject to treat DME, AMD, ROP, or PDR. In some embodiments, the method comprises administering one or more doses of the APLNR antagonist, followed by one or more doses of the VEGF antagonist. In certain embodiments, the method comprises administering a single dose of VEGF antagonist, followed by one or more doses of the APLNR antagonist.

The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present disclosure, which the present inventors It is not intended to limit the scope of what is considered. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1
To evaluate the in vivo properties of selected anti-APLNR antibodies of the present disclosure, their ability to block APLNR-mediated angiogenesis in ocular vasculature was measured.

Using the retinal vascular development (RVD) model, a mixed background strain (75% C57BL6 and 25% Sv129) of mouse pups (humanized APLNR mice) homozygous for expression of human APLNR instead of mouse APLNR was used. The effect of antagonistic anti-APLNR antibodies on vascular elongation in the normal developing retina was evaluated.

Humanized (Hu) Apelin R mice were systemically (IP) injected 25 days after birth (P) with 25 mg/kg and 50 mg/kg of anti-apelin receptor antibody (αAR; H2aM9232N). Reagents were blinded to prevent experimenter prejudice and labeled as Solution A and Solution B. Tissue samples were collected 5 days after birth and fixed with PBS containing 4% paraformaldehyde. Fixed tissue samples (retinal endothelial cells) were washed with PBS followed by GS Lectin I (Vector Laboratories, #FL-1101) diluted 1:200 in 1×PBS with 0.25% Triton-X100 containing 1% BSA. ) And visualized retinal vasculature at 25° C. overnight. The next day, the stained samples were rinsed several times with PBS, laid flat on slides, followed by cover slips using Prolong Gold ((Invitrogen, #P36930). Images were taken with an epifluorescence microscope (Nikon Eclipse). 80) was used and photographed at 20× magnification.The vascularized area in the retina was measured from the images obtained from this assay using the expanded Adobe Photoshop CS6. Only after the measurement and statistical analysis were completed, the identity of the sample was revealed.

1A and 1B are representative photomicrographs of retinas of mice systemically treated with P2-P5 and graphs of calculated vessel area (images are 20x and 40x for quantification). And statistical analysis was performed with Student's T test).

The residual vascular area was significantly smaller in the retinas of α-apelin R (23% at 25 mg/kg, p<0.05) compared to untreated retinas. Selective inhibition of apelin R via systemic infusion delayed normal vascular outgrowth in the developing retina of P5 pups. At the 25 mg/kg dose, blocking Apelin R slightly inhibits vascular elongation.

Example 2
Subsequent experiments in the RVD model were performed similarly to Example 1 at the highest dose and were analyzed by a blinded reviewer. Briefly, pups were IP injected with 50 mg/kg Fc (control) or αAR. 2A and 2B are representative photomicrographs of retinas of mice systemically treated with P2-P5, and graphs of calculated vessel area. The residual vascular area was significantly smaller in the α-apelin R (35% at 50 mg/kg, p<0.005) retina compared to the untreated retina. Retinal endothelial cells were stained with GS lectin I. Images were taken at 20x (for quantification) and 40x. Statistical analysis was performed by Student's T test. Selective inhibition of apelin R via systemic infusion delayed normal vascular outgrowth in the developing retina of P5 pups. By increasing the dose to 50 mg/kg, the targeted apelin R further delays retinal vessel elongation.

Example 3
In a subsequent experiment with the RVD model (performed similarly to Example 1), P2 Hu pups received 50 mg/kg of Fc, αAR, or aflibercept, or a combination (αAR and aflibercept). Injected and collected at P5. 3A and 3B are representative photomicrographs of retinas of mice systemically treated with P2-P5 and graphs of calculated vascular area. In this blinded study, 50 mg/kg of alpha apelin R reduced blood vessel 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 performed by Student's T test. Selective inhibition of apelin R via systemic infusion delays normal vascular outgrowth in the developing retina of P5 pups.

Example 4
In another experiment with the RVD model (performed similarly to Example 1), P4 Hu pups received 5 μg of Fc, αAR, or aflibercept, or the combination (αAR and aflibercept) intravitreally. (IVT) injected and collected at P6. 4A and 4B are representative photomicrographs of retinas of mice systemically treated with P2-P5 and graphs of calculated vascular area. Surplus vascular area was compared to α-apelin R (31% at 50 mg/kg, p<0.001) or aflibercept alone (43% at 50 mg/kg, p<0.005) with single reagent And was significantly lower in the combination (α-Apelin R + aflibercept) (62% at 50 mg/kg, p<0.0001). Retinal endothelial cells were stained with GS lectin I. The effects on dose and correlative vascularized area are recorded. Images were taken at 20x (for quantification) and 40x. Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test. Both systemic and intravitreal administration (see Example 5 below), the combined therapy of alpha apelin R and aflibercept resulted in regression of normal developing retinal vasculature. Blocking both apelin R and VEGFA via systemic infusion is more effective in blocking vascular elongation compared to blocking apelin R alone or VEGFA alone.

By injecting αAR into P2, the blood vessel elongation was 23% at 25 mg/kg at P5 (n=6 eyes/group, p<0.05) and 35% at 50 mg/kg (n=6 eyes/group). , P<0.005). In a blinded study it was possible to confirm an observation with a 29% (n=5 eyes per group, p<0.0001) reduction in vessel elongation compared to the Fc control.

Combined inhibition of apelin R and VEGFA after IP and IVT injection resulted in 62% vascular elongation at 50 mg/kg (n=4 eyes per group, p<0.0001) and 68% at 5 μg (n=4). Eyes/group, p<0.0001), significantly restricted. Aflibercept alone reduced 43% in blood vessel elongation at 50 mg/kg (n=4 eyes/group, p<0.001) and 65% at 5 μg (n=3 eyes/group, p<0.001). 0.005). Also, with αAR alone, there was a 31% decrease in blood vessel elongation at 50 mg/kg (n=4 eyes/group p<0.001) and a 43% decrease at 5 μg (n=4 eyes/group, p<0). 0.005).

Example 5
In another RVD experiment, mice were treated with alpha apelin R (H2aM9232N) (5 μg), aflibercept (5 μg), or a combination of alpha apelin R and aflibercept (mixture) in an IVT. 5A and 5B are representative micrographs of mouse retinas injected intravitreally at P4-P6 and graphs of calculated vessel area. The surplus vascular area was compared to α-ApelinR (43% at 5 μg, p<0.0001) or aflibercept alone (65% at 5 μg, p<0.0001) with a single reagent in combination ( α apelin R + aflibercept) (68% at 5 μg, p<0.0001). The effects on dose and correlative vascularized area are recorded. Images were taken at 20x (for quantification) and 40x. Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test. Both systemic and intravitreal administration, the combined therapy of alpha apelin R and aflibercept results in the regression of normal developing retinal vasculature. Blocking both apelin R and VEGFA via intravitreal injection is more effective in blocking vasodilation compared to blocking apelin R or VEGFA alone.

Example 6
A murine model of oxygen-induced ischemic retinopathy (OIR) is VEGF (Smith et al. 1994 Invest Ophthalmol Vis Sci 35:101-111, Neery et al. 1998 AmJ. Pathol 153), which is an important angiogenic factor. : 665-670, Saint-Geniez et al. 2004 Int J Dev Biol 48:1045-1058), a well-characterized model of pathological angiogenesis associated with elevated expression and thus diverse disease states. (Ferrarra et al. 2005 Nature 438:967-974) and is associated with pathological angiogenesis.

Studies were attempted to determine the effects of anti-APLNR antibodies on retinal vessel growth, vascular abnormal body formation, and retinal perfusion in oxygen-induced ischemic retinopathy (OIR).

The OIR model was utilized to determine if apelin/APLNR signaling plays a role in normal development as well as in pathological angiogenesis. In the OIR model, exposure of mouse pups to hyperoxia at P6 results in rapid loss of central retinal capillaries. After returning to room air at P11, the avascular region becomes severely hypoxic, which is extensive and abnormal characterized by ectopic growth of blood vessels into the vitreous and abnormal arteriovenous shunt formation. It induces neovascularization (supraretinal blood vessel “tufts”), and the central part of the retina remains largely avascular for long periods of time.

C57/B16 mice (Taconic) are used to study the effect of VEGF traps or neutralizing APLNR antibodies on retinal neovascularization in OIR. OIR is described by Smith et al. Produced according to the method developed by 1994 (supra). Briefly, humanized mouse pups were placed in a hyperoxic environment (75% O ₂ ) at P6 and returned to room air at P11. Exposure to high oxygen induces rapid vascular occlusion in the central retina. When the mice are returned to room air (P11), loss of blood vessels from the central part of the retina leads to severe hypoxia/ischemia, which stimulates pathological vascular changes. Pups were systemically infused with 50 mg/kg Fc (control), α-Apelin R, or aflibercept at P12 and collected at P16. Retinal endothelial cells were stained with GS lectin I.

6A and 6B are representative micrographs of retinas of OIR mice systemically treated with P12-P16, and graphs of calculated avascular area. Residual avascular area was observed in retinas with α-apelin R (29%, p<0.05) and aflibercept (27.5%, p<0.01) compared to Fc (control) retinas. It was significantly smaller. Significantly reduced angiogenic chambers compared to Fc in samples treated with alpha apelin R (67%, p<0.0001) and aflibercept (94%, p<0.0005) Please pay attention to. Retinal endothelial cells were stained with GS lectin I. Images were taken at 20x (quantification of avascular areas) and 40x (quantification of abnormal neovascularization areas). Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test. Both systemically and intravitreally, selective inhibition of apelin R promotes retinal blood vessel regeneration and reduces angiogenesis in OIR mice. Inhibition of apelin R and VEGFA via systemic infusion promotes retinal blood vessel remodeling and reduces abnormal angiogenesis.

OIR mice were treated similarly to the study above, except that OIR mice were IVT injected with 5 μg Fc, αAR, or aflibercept at P12 and collected at P16. 7A and 7B are representative photomicrographs of the retinas of OIR mice intravitreally treated with P12-P16, and graphs of calculated avascular area. Avascular area was reduced in α-apelin R (27.5%, p<0.05) and increased in aflibercept (32%, p<0.0001) compared to Fc controls. Note the significant reduction of neovascular chambers in α-Apelin R (60%, p<0.0001), and the complete loss of chambers in the aflibercept sample. Retinal endothelial cells were stained with GS lectin I. Images were taken at 20x (quantification of avascular areas) and 40x (quantification of abnormal neovascularization areas). Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test. Both systemically and intravitreally, selective inhibition of apelin R promotes retinal blood vessel regeneration and reduces angiogenesis in OIR mice. Inhibition of apelin R via intravitreal injection improves vascular regeneration and reduces abnormal angiogenesis, while VEGFA inhibition suppresses vascular regeneration and completely stops angiogenesis.

In the OIR model, αAR and aflibercept were able to promote blood vessel regrowth. When pups were IP-injected with OIR P12, αAR and aflibercept showed vascular area of 29% and 27% (n=6 eyes/group, p<0.05), angiogenesis of 69% and P16, respectively. 94% (n=6 eyes/group, p<0.0001). Upon administration of IVT, aflibercept reduced vascular regrowth by 30% (n=4 eyes/group, p<0.0001), eliminating all angiogenesis, while αAR reduced vascular regrowth by 30%. % (N=4 eyes/group, p<0.05) and angiogenesis could be reduced by 60% (n=4 eyes/group, p<0.0001).

As discussed in Example 7, the combination of an APLNR antagonist with a VEGF trap, such as aflibercept, also abrogated pathological angiogenesis and promoted normal revascularization in the OIR model. Therefore, this combination is expected to treat the pathological angiogenesis found in various eye diseases including retinopathy of prematurity.

Example 7
In a further in vivo experiment, the OIR model described above in Example 6 is used to assess the organization and density of revascularization. Briefly, humanized mouse pups were placed in a hyperoxic environment (75% O ₂ ) at P6 and returned to room air at P11. Exposure to high oxygen induces rapid vascular occlusion in the central retina. When the mice are returned to room air (P11), loss of blood vessels from the central part of the retina leads to severe hypoxia/ischemia, which stimulates pathological vascular changes. Pups were systemically infused with 50 mg/kg Fc (control), αAPLNR (H4H9232N), or aflibercept (VEGF trap), or 25 mg/kg combination of αAPLNR and aflibercept at P12 at P16. Was collected. Retinal endothelial cells were stained with GS lectin I.

8A and 8B are representative photomicrographs (20×) of retinas of OIR mice systemically treated with P12-P16, and graphs of calculated avascular area. Regeneration rates with combination therapy were similar to each agent alone, so vascular remodeling was improved in all treatment conditions compared to control retinas treated with hFc, and combination therapy had an average avascular area. Is slightly reduced. FIG. 8B shows that there were significant changes in avascular areas between treatment groups (p<0.0005). Vessel area was determined by anti-APLNR antibody (**, p<0.05), VEGF trap (***, p<0.005), and combination (***, p<0. It was significantly reduced in 05). Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test.

9A and 9B are representative photomicrographs (40×) of retinas of OIR mice systemically treated with P12-P16 and graphs of calculated abnormal vascular area. The combination treatment shows fewer pruned vessels compared to the VEGF trap and less abnormal angiogenesis compared to the anti-APLNR and Fc controls (FIG. 9A). FIG. 9B shows that there was a significant change in abnormal vascular area between treatment groups (p<0.0005). Abnormal vascular area was compared to Fc control by anti-APLNR (***, p<0.0005), VEGF trap (***, p<0.005), and combination (***, p<0.005). 0.0005). Abnormal vascular area was further significantly reduced in the combination treatment group compared to the anti-APLNR treatment group (*, p<0.05). Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test.

FIG. 10 is representative of the image processing and measurement techniques used to calculate vessel area and vessel length. The “image of origin” is a 40× photomicrograph shown in FIG. 9A. “Threshold image” and “binary image” are discussed in more detail in connection with FIGS. 11A, 11B, 12A, and 12B below.

11A and 11B are representative treatments showing vessel organization and homogeneity from photomicrographs (40×) of retinas of OIR mice systemically treated with P12-P16 and graphs of calculated vessel area. It is the image. As shown in FIG. 11A, the combination of anti-APLNR antibody and VEGF trap produced more organized and uniform retinal blood vessels compared to anti-APLNR alone and was less scattered than VEGF trap alone. (Few pruned vessels). FIG. 11B shows that there was a significant change in blood vessel area between treatment groups (p<0.0005). Vessel area was significantly increased with anti-APLNR compared to Fc controls (***, p<0.0005), VEGF trap (***, p<0.005), and combination Reduced with treatment (*, p<0.05). Vessel area was significant with VEGF trap (#####, p<0.0005) and with combination (####, p<0.0005) compared to anti-APLNR. Was reduced to. In contrast, vascular area was significantly increased with combination treatment (&&&, p<0.005) compared to VEGF trap. Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test.

12A and 12B are representative processed images showing vascular density and calculated vessel length from photomicrographs (40×) of retinas of OIR mice systemically treated with P12-P16. .. As shown in FIG. 12A, the combination of anti-APLNR antibody and VEGF trap produced intermediate density retinal vessels as compared to anti-APLNR (higher density) or VEGF trap (lower density) alone. FIG. 12B shows that there was a significant change in vessel length between treatment groups (p<0.0005). Vessel length is significant with VEGF trap (####, p<0.0005) and with combination (####, p<0.0005) compared to anti-APLNR. Was reduced to. In contrast, vessel length was significantly increased with combination treatment (&&&, p<0.005) compared to VEGF trap. Statistical analysis was performed using a one-way analysis of variance with post hoc Tukey test.

As shown in FIGS. 8A-12B and discussed above, the combination of anti-APLNR antibody and VEGF trap promotes revascularization and results in a reduction in avascular area compared to Fc controls. Further, the combination treatment inhibits pathological angiogenesis and produces an intermediate effect on vessel area and vessel length compared to anti-APLNR antibody or VEGF trap alone treatment, in particular the combination treatment Promotes microvessel growth between major blood vessels compared to treatment and improves vessel organization and homogeneity compared to anti-APLNR antibody treatment.

The present disclosure should not be limited in scope by the particular embodiments described herein. Indeed, various modifications of the present disclosure in addition to those described herein will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims (53)

A method for treating a vascular eye disease or disorder, comprising:
Administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof,
Administering to said subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. The eye disease or disorder is diabetic retinopathy, proliferative diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, polypoidal choroidal vasculopathy. The method of claim 1, wherein the method is selected from the group consisting of: choroidal neovascularization (CNV), degenerative myopia (myopia CNV), neovascular glaucoma, and retinopathy of prematurity. The method of claim 2, wherein the eye disease or disorder is age-related macular degeneration. The method of claim 2, wherein the eye disease or disorder is diabetic macular edema. A method of inhibiting retinal angiogenesis, comprising:
Administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof,
Administering to said subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. A method of inhibiting retinal neovascularization, comprising:
Administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof,
Administering to said subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. A method of inhibiting choroidal neovascularization, comprising administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof.
Administering to said subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. A method of improving blood vessel regeneration and reducing abnormal angiogenesis, comprising:
Administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof,
Administering to said subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. A method of promoting retinal revascularization, comprising:
Administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof,
Administering to said subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. A method of promoting uniform regeneration of retinal blood vessels, the method comprising:
Administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof,
Administering to said subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. A method of improving the growth of blood vessels in the retina, comprising:
Administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof,
Administering to said subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. A method of promoting uniform blood vessel growth in the retina, comprising:
Administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof,
Administering to said subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. A method of improving the organization of blood vessels in the retina, comprising:
Administering a therapeutically effective amount of an APLNR antagonist to a subject in need thereof,
Administering to said subject a therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist. The subject is diabetic retinopathy, proliferative diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, polypoidal choroidal vasculopathy, choroidal blood vessel 14. Any one of claims 5-13 diagnosed with an eye disease or disorder selected from the group consisting of neonatal (CNV), degenerative myopia (myopia CNV), neovascular glaucoma, and retinopathy of prematurity. The method described. 15. The method of claim 14, wherein the eye disease or disorder is age-related macular degeneration. 15. The method of claim 14, wherein the eye disease or disorder is diabetic macular edema. The method of claim 1, wherein the APLNR antagonist comprises an anti-APLNR antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof specifically binds human APLNR. Said antibody or antigen-binding fragment thereof competes for binding to human apelin receptor (APLNR) with a reference antibody comprising a HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2/7 and 13/18, The method of claim 2, wherein the antibody or antigen-binding fragment thereof specifically binds to human APLNR. The antibody or antigen-binding fragment thereof binds to the same epitope on the APLNR as the reference antibody comprising the HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2/7 and 13/18, The method of claim 2 or 3, wherein the fragment specifically binds to human APLNR. The antibody or antigen-binding fragment comprises (a) a complementarity determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 13, and (b) SEQ ID NO: 7 And a CDR of a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of: and 18, and the method of any one of claims 2 to 4. The antibody or antigen-binding fragment is HCDR1-HCDR2-HCDR3-LCDR1- selected from the group consisting of SEQ ID NOs: 3-4-5-8-9-10 and 14-15-16-19-20-21, respectively. 6. The method according to any one of claims 2-5, comprising LCDR2-LCDR3 domains. The antibody or antigen-binding fragment is selected from the group consisting of (a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 13, and (b) consisting of SEQ ID NOs: 7 and 18. A light chain variable region (LCVR) having an amino acid sequence according to any one of claims 2 to 6. 8. Any of claims 2-7, wherein the antibody or antigen binding fragment comprises the CDRs of the heavy and light chains of an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/7 and 13/18. The method according to paragraph 1. 9. The method of any one of claims 2-8, wherein the antibody or antigen binding fragment comprises HCVR/LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NOs: 2/7 and 13/18. 10. The method of claim 9, wherein the antibody or antigen binding fragment thereof comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 2/7. 10. The method of claim 9, wherein the antibody or antigen binding fragment thereof comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 13/18. The method according to claim 10 or 11, wherein the antibody or antigen-binding fragment is a human antibody having an IgG1 or IgG4 heavy chain constant region. 13. The method of any of claims 2-12, wherein the antibody or antigen binding fragment thereof blocks the interaction of APLNR with apelin. 14. The method of any of claims 2-13, wherein the antibody or antigen binding fragment thereof blocks the interaction of APLNR with apelin and exhibits at least 50% binding inhibition in a competitive binding assay. 15. The method of any one of claims 2-14, wherein the antibody or antigen binding fragment thereof does not block or only partially blocks the interaction of APLNR with apelin. 16. The method of any one of claims 1-15, wherein the VEGF antagonist comprises a VEGF receptor system chimeric molecule (VEGF trap). 17. The method of claim 16, wherein the VEGF trap comprises one or more immunoglobulin (Ig)-like domains of VEGFRl, one or more Ig-like domains of VEGFR2, and a multimerization domain. 18. The method of claim 17, wherein the VEGF trap comprises an Ig-like domain 2 of VEGFR1, an Ig-like domain 3 of VEGFR2, and a multimerization domain. 19. The method of claim 18, wherein the VEGF trap is aflibercept. 20. The method of any one of claims 1-19, wherein the VEGF antagonist consists of a dimer of two polypeptides consisting of amino acids 27-457 of SEQ ID NO:23. A composition for treating a vascular eye disease or disorder, comprising:
A therapeutically effective amount of an APLNR antagonist,
A therapeutically effective amount of a vascular endothelial growth factor (VEGF) antagonist,
A composition comprising a suitable carrier, excipient or diluent. 75. The composition of claim 74, wherein the APLNR antagonist comprises an anti-APLNR antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof specifically binds human APLNR. Said antibody or antigen-binding fragment thereof competes for binding to human apelin receptor (APLNR) with a reference antibody comprising a HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2/7 and 13/18, 76. The composition of claim 75, 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 the APLNR as the reference antibody comprising the HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2/7 and 13/18, 77. The composition of claim 75 or 76, wherein the fragment specifically binds human APLNR. The antibody or antigen-binding fragment comprises (a) a complementarity determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 13, and (b) SEQ ID NO: 7 78. The composition of any one of claims 75-77 comprising a CDR of a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of: The antibody or antigen-binding fragment is HCDR1-HCDR2-HCDR3-LCDR1- selected from the group consisting of SEQ ID NOs: 3-4-5-8-9-10 and 14-15-16-19-20-21, respectively. 79. The composition of any one of claims 75-78, which comprises a LCDR2-LCDR3 domain. The antibody or antigen-binding fragment is selected from the group consisting of (a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 13, and (b) consisting of SEQ ID NOs: 7 and 18. A light chain variable region (LCVR) having an amino acid sequence according to claim 75. 81. Any of claims 75-80, wherein said antibody or antigen binding fragment comprises the CDRs of said heavy and light chains of an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/7 and 13/18. The composition according to one paragraph. 82. The composition of any one of claims 75-81, wherein 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. 83. The composition of claim 82, wherein the antibody or antigen binding fragment thereof comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 2/7. 83. The composition of claim 82, wherein the antibody or antigen binding fragment thereof comprises the HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 13/18. 85. The composition of claim 83 or 84, wherein the antibody or antigen binding fragment thereof is a human antibody having an IgG1 or IgG4 heavy chain constant region. 86. The composition of any one of claims 74-85, wherein the VEGF antagonist comprises a VEGF receptor system chimeric molecule (VEGF trap). 87. The composition of claim 86, wherein the VEGF trap comprises one or more immunoglobulin (Ig)-like domains of VEGFRl, one or more Ig-like domains of VEGFR2, and a multimerization domain. 88. The composition of claim 87, wherein the VEGF trap comprises Ig-like domain 2 of VEGFRl, Ig-like domain 3 of VEGFR2, and a multimerization domain. 89. The composition of claim 88, wherein the VEGF trap is aflibercept. 90. The composition of any one of claims 74-89, wherein the VEGF antagonist comprises a dimer of two polypeptides consisting of amino acids 27-457 of SEQ ID NO:23. The eye disease or disorder is diabetic retinopathy, proliferative diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branch retinal vein occlusion, polypoidal choroidal vasculopathy. 91. The composition of any one of claims 74-90, selected from the group consisting of: choroidal neovascularization (CNV), degenerative myopia (myopia CNV), neovascular glaucoma, and retinopathy of prematurity.

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