WO2020146731A1 - Compositions and methods for inhibiting vascular endothelial growth factor - Google Patents

Compositions and methods for inhibiting vascular endothelial growth factor Download PDF

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Publication number
WO2020146731A1
WO2020146731A1 PCT/US2020/013083 US2020013083W WO2020146731A1 WO 2020146731 A1 WO2020146731 A1 WO 2020146731A1 US 2020013083 W US2020013083 W US 2020013083W WO 2020146731 A1 WO2020146731 A1 WO 2020146731A1
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vegf
aptamer
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aptamers
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PCT/US2020/013083
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French (fr)
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Carl ERICKSON
Christopher P. Rusconi
Matthew Levy
Keith E. MAIER
Sarah E. THACKER
Derek PARKS
Kevin G. Mclure
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Drive Therapeutics Llc
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Publication of WO2020146731A1 publication Critical patent/WO2020146731A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U

Definitions

  • Visual impairment is a national and global health concern that has a negative impact on physical and mental health.
  • the number of people with visual impairment and blindness is increasing due to an overall aging population.
  • Visual impairment and blindness can be caused by any one of a large number of eye diseases and disorders affecting people of all ages.
  • VEGF-A Vascular endothelial growth factor- A
  • VEGF-A Vascular endothelial growth factor- A
  • VEGF-A is thought to be the most significant regulator of angiogenesis in the VEGF family.
  • VEGF-A promotes growth of vascular endothelial cells which leads to the formation of capillary -like structures and may be necessary for the survival of newly formed blood vessels.
  • VEGF-A is thought to play a role in various ocular diseases and disorders. Previous attempts at developing aptamers that inhibit VEGF-A have proven difficult because such aptamers have been unable to target multiple isoforms and variants of VEGF-A.
  • pan-variant specifi c aptamers that demonstrate high specificity and potency towards multiple isoforms and variants of VEGF-A. These needs may be met by the aptamers provided in the present disclosure.
  • an aptamer comprising a nucleic acid sequence that selectively binds to and inhibits at least one of VEGF -A and VEGF-Ano, wherein less than 50% of pyrimidines present in said nucleic acid sequence comprise a C-5 modified pyrimidine. In some cases, less than 25% of pyrimidines present in said nucleic acid sequence comprise a C-5 modified pyrimidine. In some cases, less than 10% of pyrimidines present in said nucleic acid sequence comprise a C-5 modified pyrimidine. In some cases, the nucleic acid sequence does not comprise any C-5 modified pyrimidines. In some cases, the C-5 modified pyrimidine comprises a C-5 modified cytosine or a C-5 modified uridine. In some cases, the C-5 modified pyrimidine comprises a C-5 hydrophobic modification.
  • an aptamer comprising a nucleic acid sequence that selectively binds to and inhibits at least one of VEGF -A and VEGF-Ano, wherein less than 100% of uridines present in said nucleic acid sequence comprise a C-5 modified uridine. In some cases, less than 50% of uridines present in said nucleic acid sequence comprise a C-5 modified uridine. In some cases, less than 25% of uridines present in said nucleic acid sequence comprise a C-5 modified uridine. In some cases, less than 10% of uridines present in said nucleic acid sequence comprise a C-5 modified uridine.
  • no uridines present in said nucleic acid sequence comprise a C-5 modified uridine.
  • the C-5 modified uridine comprises a C-5 hydrophobic modification.
  • the C-5 modified uridine is selected from the group consisting of: 5-(N-benzylcarboxyamide)-2'-deoxyuridine (BndU), 5-(N- benzyicarboxyamide)-2'-Q-methyiuridine, 5-(N-benzylcarboxyamide)-2'-fluorouridine, 5-QNJ- phenethylcarboxyamide)-2'-deoxyuiidine (PEdU), 5-(N-thiophenylmethylcarboxyamide)-2'- deoxyuridine (ThdU), 5-(N-isobutylcarboxyamide)-2'-deoxyuridine (IBud!J), 5-(N- tyrosylcarboxyamide)-2'-deoxyuridine (TyrdU), 5-(N-3,
  • any aptamer of the preceding selectively binds to a receptor binding face or receptor binding domain of VEGF-Am or VEGF-Ano.
  • the receptor binding domain comprises at least one of residues 1 -109 of SEQ ID NO: 1.
  • the receptor binding domain comprises at least one of residues Phel7, Ile43, Ile46, Glu64, Gln79, Ile83, Lys84, Pro85, Arg82, His86, Asp63, and Glu67 of SEQ ID NO: 1
  • any aptamer of the preceding inhibits VEGF-Am, VEGF-Ano, or both, with an IC50 of less than about 50 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation
  • any aptamer of the preceding inhibits VEGF-Am, VEGF-Ano, or both, with an IC50 of less than about 25 nM as measured by a VEGF-A:KDR competition binding assay, a KDR
  • any aptamer of the preceding inhibits VEGF-Am, VEGF-Ano, or both, with an IC 50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation AlphaLISA 8, assay, or an in vitro model of VEGF-A-induced angiogenesis.
  • any aptamer of the preceding inhibits VEGF-Am, VEGF-Ano, or both, with an IC50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation Alpha! .
  • ISA assay, or an in vitro model of VEGF-A-induced angiogenesis.
  • any aptamer of the preceding inhibits VEGF-Am, VEGF-Ano, or both, with an IC50 of less than about 1 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation AlphaLISA ® assay, or an in vitro model of VEGF-A-induced angiogenesis.
  • any aptamer of the preceding binds to VEGF-A , VEGF-Ano, or both, with a K d of less than about 50 nM as measured by surface plasmon resonance assay.
  • any aptamer of the preceding binds to VEGF-Am, VEGF-Ano, or both, with a K d of less than about 25 nM as measured by surface plasmon resonance assay. In some cases, any aptamer of the preceding binds to VEGF ⁇ A , VEGF-Apo, or both, with a K d of less than about 10 nM as measured by surface plasmon resonance assay. In some cases, any aptamer of the preceding binds to VEGF-Am, VEGF-Ano, or both, with a K d of less than about 5 nM as measured by surface plasmon resonance assay.
  • any aptamer of the preceding binds to VEGF- Am, VEGF-Ano, or both, with a K of less than about 1 nM as measured by surface plasmon resonance assay. In some cases, any aptamer of the preceding selectively binds to and inhibits at least one ofVEGF-A 165 , VEGF-Ai 89 , and VEGF-A 2 o 6 . In some cases, any aptamer of the preceding inhibits or reduces an interaction of VEGF-A with KDR. In some cases, any aptamer of the preceding inhibits or reduces VEGF-A-induced KDR phosphorylation.
  • any aptamer of the preceding comprises RNA or sugar-modified RNA. In some cases, any aptamer of the preceding comprises DNA or sugar-modified DNA. In some cases, at least 50% of said nucleic acid sequence of any aptamer of the preceding comprises sugar-modified nucleotides. In some cases, 100% of said nucleic acid sequence of any aptamer of the preceding comprises sugar-modified nucleotides. In some cases, the sugar-modified nucleotides comprise a 2’F- modified nucleotide, a 2’OMe-modified nucleotide, or both.
  • the sugar-modified nucleotides are selected form the group consisting of: 2’F-G, 2’OMe-G, 2’OMe-U, 2’OMe-A, 2’OMe-C, and any combination thereof.
  • any aptamer of the preceding further comprises a 3’ terminal inverted deoxythymidine.
  • any aptamer of the preceding comprises a nuclease-stabilized nucleic acid backbone.
  • the nucleic acid sequence of any aptamer of the preceding comprises from about 30 to about 90 nucleotides, wherein said nucleotides are unmodified nucleotides, modified nucleotides, or a combination of modified nucleotides and unmodified nucleotides.
  • any aptamer of the preceding is conjugated to a polyethylene glycol (PEG) molecule.
  • the PEG molecule has a molecular weight selected from the group consisting of: less than about 5 kDa, less than about 10 kDa, less than about 20 kDa, less than about 40 kDa, less than about 60 kDa, and less than about 80 kDa.
  • an aptamer of any of the preceding is provided for use in treating an ocular disease or disorder in a subject in need thereof. In some cases, one or more symptoms of said ocular disease or disorder are treated.
  • a method for treating an ocular disease or disorder in a subject in need thereof comprising administering to said subject an aptamer of any of the preceding, thereby treating said ocular disease or disorder.
  • the ocular disease or disorder is selected from the group consisting of: diabetic retinopathy, retinopathy of
  • the ocular disease or disorder exhibits elevated levels of VEGF- A
  • any aptamer of the preceding is provided for use in a formulation of a medicament for treatment of an ocular disease or disorder.
  • any aptamer of the preceding is provided for use for treatment of an ocular disease or disorder.
  • a method for modulating vascular endothelial growth factor-A (VEGF-A) in a biological system comprising: administering to said biological system any aptamer of the preceding, thereby modulating VEGF-A in said biological system.
  • the biological system comprises a biological tissue or biological cells.
  • the biological system is a subject.
  • the subject is a human.
  • the modulating comprises inhibiting a function associated with VEGF-A.
  • the modulating comprises preventing or reducing an association of VEGF-A with one or more of Flt-1, KDR, or Nrp-1.
  • FIG. 1A depicts a non-limiting example of a primary sequence of an aptamer library suitable for screening for aptamers that target VEGF-A.
  • FIG. IB depicts a non-limiting example of a secondary 7 structure of the aptamer library depicted in FIG. 1A, with a reverse primer hybridized thereto.
  • FIG. 1C depicts a non-limiting example of chemical structures of 2’-modified nucleotides that may be used in the VEGF-A aptamer selection process.
  • FIG. 2A depicts a non-limiting example of flow cytometry' data demonstrating the interaction of various anti -VEGF-A aptamer selection rounds with unlabeled“negative” beads.
  • FIG. 2B depicts a non-limiting example of flow cytometry data demonstrating the interaction of various anti -VEGF-A aptamer selection rounds with VEGFm-functionalized beads.
  • FIG. 2C depicts a non-limiting example of flow cytometry 7 data demonstrating the dose- dependent interaction of various anti-VEGF-A aptamer selection rounds with VEGF no- functionalized beads.
  • FIG. 2D depicts a non-limiting example of flow cytometry data demonstrating the dose- dependent interaction of various anti-VEGF-A aptamer selection rounds with VEGFm- functionalized beads.
  • FIG, 2E depicts a non-limiting example of flow cytometry data demonstrating the interaction of various anti-VEGF-A aptamer selection rounds with VEGF -functionalized beads in the presence of a non-bead bound VEGFm decoy.
  • FIG. 3 depicts a non-limiting example of flow cytometry data demonstrating the dose- dependent interaction of an anti-VEGF-A aptamer with VEGF !65 - and VEGFm-functionalized beads.
  • FIG, 4A demonstrates a non-limiting example of the inhibition of VEGF 155 binding to KDR by an anti-VEGF-A aptamer of the disclosure. Compounds were tested in a dose- dependent fashion to determine an IC50 against VEGF 105.
  • FIG. 4B demonstrates a non-limiting example of the inhibition of VEGFm binding to KDR by an anti-VEGF-A aptamer of the disclosure.
  • Compounds were tested in a dose- dependent fashion to determine an IC50 against VEGF 121.
  • FIG. 5A demonstrates a non-limiting example of the inhibition of V EGF r.s-sti mill ated KDR phosphorylation by an anti-VEGF-A aptamer of the disclosure. Compounds were tested in a dose-dependent fashion to determine an IC50 against VEGF 155.
  • FIG, 5B demonstrates a non-limiting example of the inhibition of VEGFm-stimulated KDR phosphorylation by an anti-VEGF-A aptamer of the disclosure. Compounds were tested in a dose-dependent fashion to determine an IC50 against VEGF 121.
  • FIG. 6A demonstrates a non-limiting example of the inhibition of VEGFies-stimulated angiogenesis by an anti-VEGF-A aptamer of the disclosure. Anti-VEGF-A aptamer was tested in a dose-dependent fashion to determine an IC 50 against VEGF165.
  • FIG. 6B demonstrates a non-limiting example of the inhibition of VEGFi 2i -stimulated angiogenesis by an anti-VEGF-A aptamer of the disclosure.
  • Anti-VEGF-A aptamer was tested in a dose-dependent fashion to determine an IC50 against VEGF ⁇ .
  • FIG. 7 depicts representative images of the inhibition of VEGF165- or VEGF m- stimuiated angiogenesis with an anti-VEGF-A aptamer of the disclosure.
  • FIG. 8A depicts a non-limiting example of duration of action modeling of a 1 nig/eye dose pan-variant specific anti-VEGF-A aptamer in the vitreous for the treatment of wet age- related macular degeneration, diabetic retinopathy, diabetic macular edema, or macular edema following retinal vein occlusion
  • FIG. SB depicts a non-limiting example of duration of action modeling of a 5 mg/eye dose pan-variant specific anti-VEGF-A aptamer in the vitreous for the treatment of wet age- related macular degeneration, diabetic retinopathy, diabetic macular edema, or macular edema following retinal vein occlusion.
  • FIG. 9A depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of a high dose of pegylated anti-VEGF-A aptamer of the disclosure delivered by IVT administration for the treatment of macular edema following retinal vein occlusion.
  • FIG. 9B depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of a low dose of pegylated anti-VEGF-A aptamer of the disclosure delivered by IVT administration for the treatment of macular edema following retinal vein occlusion.
  • FIG. 9C depicts a non-limiting exampl e of potency modeling in the vi treous or in the systemic and tissue compartments of an anti-VEGF-A aptamer of the disclosure pegylated with an optimized PEG moiety and delivered by IVT administration for the treatment of macular edema following retinal vein occlusion.
  • FIG. 9D depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of an anti-VEGF-A aptamer of the disclosure that has been engineered for metabolic instability and delivered by IVT administration for the treatment of macular edema following retinal vein occlusion.
  • FIG. I0A depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of a high dose of a pegylated anti -VEGF-A aptamer of the disclosure delivered by IVT administration for the treatment of retinopathy of prematurity
  • FIG. 10B depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of a low' dose of a pegylated anti-VEGF-A aptamer of the disclosure delivered by IVT administration for the treatment of retinopathy of prematurity.
  • FIG. IOC depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of an anti-VEGF-A aptamer of the disclosure pegylated with an optimized PEG moiety and delivered by IVT administration for the treatment of retinopathy of prematurity.
  • FIG. 10D depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of an anti-VEGF-A aptamer of the disclosure that has been engineered for metabolic instability and delivered by IVT administration for the treatment of retinopathy of prematurity.
  • FIG. 11 depicts a non-limiting example of a circular dichroism (CD) spectra of Aptamer 3R02 according to embodiments of the disclosure.
  • CD circular dichroism
  • FIG. 12 depicts non-limiting examples of inhibition of VEGF-A 165 or VEGF-A - stimulated KDR phosphorylation by Aptamers Vap7, VEaP121 or 3R02, as compared to an anti- VEGF mAh according to embodiments of the disclosure.
  • the left hand bar represents stimulation with VEGF-Aies and the right hand bar represents stimulation with VEGF-A 121.
  • Data is presented as the mean ⁇ standard deviation.
  • VEGF-A vascular endothelial growth factor-A
  • an aptamer composition of the disclosure may comprise an anti -VEGF-A aptamer that binds to one or more isoforms or variants of VEGF-A.
  • an aptamer composition of the disclosure may comprise a pan-variant specific anti- VEGF-A aptamer that binds to each of VEGF-A no, VEGF-A , VEGF-Aies, VEGF-A i89 , and VEGF-A206 ⁇
  • the anti-VEGF-A aptamers may hind to the receptor binding face of VEGF-A, or a portion thereof.
  • the anti-VEGF-A aptamers may bind to the receptor binding domain (RBD) of VEGF-A, or a portion thereof. In some cases, an aptamer of the disclosure does not bind to the heparin-binding domain (HBD) of VEGF-A. Without wishing to be hound by theory, anti-VEGF-A aptamers of the disclosure may prevent or reduce binding of VEGF-A to a VEGF receptor (VEGFR).
  • VBGFR VEGF receptor
  • an anti-VEGF-A aptamer of the disclosure may prevent or reduce binding of VEGF-A to VEGFR1 (also known as Fms- reiated tyrosine kinase 1 (1 ⁇ It i )), VEGFR2 (also known as Kinase insert domain receptor (KDR) or Flk-1), Neuropilin-1 (Nrp-1), or any combination thereof.
  • VEGFR1 also known as Fms- reiated tyrosine kinase 1 (1 ⁇ It i )
  • VEGFR2 also known as Kinase insert domain receptor (KDR) or Flk-1
  • Neuropilin-1 Neuropilin-1
  • an anti-VEGF-A aptamer of the disclosure may inhibit a function associated with VEGF-A (e.g, engaging a VEGF receptor, a signaling pathway downstream of VEGF-A, or both).
  • the disclosure herein further provides methods for inhibiting VEGF-A (and/or a downstream signaling pathway of VEGF-A).
  • the methods include administering an anti-VEGF-A aptamer (or a composition comprising said aptamer) to a biological system (e.g, biological ceils, biological tissue, a subject, and the like).
  • a biological system e.g, biological ceils, biological tissue, a subject, and the like.
  • the disclosure further provides methods for treating ocular diseases or disorders including administering an anti-VEGF-A aptamer (or a pharmaceutical composition comprising said aptamer) to a subject having, suspected of having, or at risk of developing, an ocular disease or disorder.
  • the ocular disease or disorder may be diabetic retinopathy.
  • the ocular disease or disorder may be retinopathy of prematurity'. In some cases, the ocular disease or disorder may be central retinal vein occlusion. In some cases, the ocular disease or disorder may be macular edema. In some cases, the ocular disease or disorder may be choroidal neovascularization. In some cases, the ocular disease or disorder may be neovascular (or wet) age-related macular degeneration. In some cases, the ocular disease or disorder may be myopic choroidal neovascularization. In some cases, the ocular disease or disorder may be punctate inner choroidopathy.
  • the ocular disease or disorder may be presumed ocular histoplasmosis syndrome. In some cases, the ocular disease or disorder may be familial exudative vitreoretinopathy. In some cases, the ocular disease or disorder may be
  • a subject having, suspected of having, or at risk of developing, an ocular disease or disorder may exhibit elevated levels of one or more variants or isoforms of VEGF-A.
  • a subject having, suspected of having, or at risk of developing, an ocular disease or disorder may exhibit elevated levels of one or more of VEGF-A206, VEGF-A gy, VEGF-A !65 , VEGF ⁇ A m , and VEGF-A U0.
  • the methods and compositions may involve the inhibition of a function associated with VEGF-A.
  • the methods and compositions may involve preventing or reducing VEGF-A binding to or interaction with one or more VEGF receptors.
  • the methods and compositions may involve preventing or reducing VEGF-A binding to or interaction with Fit- 1 , KDR, Nrp-1, or any combination thereof.
  • the methods and compositions may involve preventing or reducing downstream signaling associated with Flt-1, KDR, Nrp-1, or any combination thereof.
  • the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of ocular diseases or disorders.
  • the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of diabetic retinopathy. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of retinopathy of prematurity. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of central retinal vein occlusion. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of macular edema. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of choroidal neovascularization.
  • the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of neovascular (or wet) age-related macular degeneration. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of myopic choroidal neovascularization. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of punctate inner choroidopathy. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of presumed ocular histoplasmosis syndrome.
  • the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of familial exudative vitreoretinopathy. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of retinoblastoma. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of an ocular disease or disorder exhibiting elevated levels of one or more isoforms or variants of VEGF-A [0044] In various aspects, the compositions may include one or more aptamers that selectively bind to and inhibit a function associated with VEGF-A.
  • the compositions may include one or more aptamers that bind to the receptor binding face of VEGF-A. In some cases, the compositions may include one or more aptamers that bind to the receptor binding domain of VEGF-A. In some cases, the compositions may include one or more aptamers that bind to a region of VEGF-A other than the heparin binding domain of VEGF-A. Put another way, the compositions may include one or more aptamers that do not bind to the heparin binding domain of VEGF-A. In some cases, the compositions may include one or more aptamers that bind to one or more variants or isoforms of VEGF-A.
  • the compositions may include one or more aptamers that bind to one or more of VEGF-A206, VEGF-Aig 9 , VEGF-A 1 ⁇ 2 5, VEGF-Am, and VEGF-A io.
  • the compositions may include one or more pan-variant specific anti -VEGF-A aptamers.
  • compositions may include pan-variant specific aptamers that bind to each of VEGF-Am,, VEGF-Am, VEGF-A 165 , VEGF-A 189 , and VEGF- A206 ⁇
  • a pan-variant specific apta ner disclosed herein may bind to a structural feature of VEGF-A that is shared amongst VEGF-A U0 , VEGF-Am, VEGF-A 165 , VEGF-A 189 , and VEGF-A206.
  • the structural feature is the receptor binding face or the receptor binding domain.
  • the compositions may include one or more aptamers that prevent or reduce binding of VEGF-A to Fit- 1 , KDR, Nrp-1, or any combination thereof. In some cases, the compositions may include one or more aptamers that prevent or reduce downstream signaling pathways associated with Flt-1, KDR, Nrp-1 , or any combination thereof. In some cases, the aptamers may be RNA aptamers, DNA aptamers, modified RNA aptamers, or modified DNA aptamers.
  • the aptamers do not contain non-naturally occurring hydrophobic modifications.
  • less than 100% of the pyrimidines e.g., C, T, or U
  • the pyrimidines e.g., C, T, or U
  • the pyrimidines present in a nucleic acid sequence of an aptarner herein comprise a C-5 modified pyrimidine.
  • pyrimidines present in a nucleic acid sequence of an aptamer herein comprise a C-5 modified pyrimidine.
  • none of the pyrimidines present in a nucleic acid sequence of an aptamer herein comprise a C-5 modified pyrimidine.
  • none of the bases in an aptamer sequence herein comprise a C-5 modification.
  • less than 100% of the uridines present in a nucleic acid sequence of an aptamer herein comprise a C-5 modified uridine.
  • less than 100%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% of the uridines present in a nucleic acid sequence of an aptamer herein comprise a C-5 modified uridine.
  • none of the uridines present in a nucleic acid sequence of an aptamer herein comprise a C-5 modified uridine.
  • the C-5 modified pyrimidine or C-5 modified uridine is selected from the group consisting of: 5-(N-benzylcarboxyamide)-2'-deoxyuridine (BndU), 5-(N- benzylcarboxyamide)-2'-0-methyluridine, 5-(N-benzylcarboxyamide)-2'-fluorouridine, 5-(N- phenethylcarboxyamide)-2'-deoxyuridine (PEdU), 5-(N-thiophenylmethylcarboxyamide)-2'- deoxyuridine (ThdU), 5-(N-isobutylcarboxyamide)-2'-deoxyuridine (iBudU), 5-(N- tyrosylcarboxyamide)-2'-deoxyuridine (TyrdU), 5-(N-3,4-methylenedioxybenzyl
  • sequence identity refers to an exact nucleotide-to-nucleotide or amino acid- to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence.
  • Two or more sequences can be compared by determining their“percent identity.”
  • the percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health.
  • the BLAST program is based on the alignment method of Karlin and Aitschui, Proc. Natl. Acad. Sci USA , 87:2264-2268 (1990) and as discussed in Aitschui, et al, J Mol. Biol. , 215:403-410 (1990); Karlin And Aitschui,
  • the program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program.
  • the program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are approximately 50% to 100% and integer values therebetween.
  • this disclosure encompasses sequences with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any sequence provided herein.
  • “modification identity” refers to two polynucleotides with identical patterns of modifications on a nucleotide-to-nucleotide level. Techniques for determining modification identity may include determining the modifications of a polynucleotide and comparing these modifications to modifications of a second polynucleotide. The percent modification identity of two sequences is the number of exact modification matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Ranges of desired degrees of modification identity are generally approximately 50% to 100%, and integer values
  • this disclosure encompasses sequences with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% modification identity with any sequence provided herein.
  • aptamer refers to an oligonucleotide and/or nucleic acid analogues that can hind to a specific target molecule
  • Aptamers can include RNA, DNA, modified RNA, modified DNA, any nucleic acid analogue, and/or combinations thereof.
  • Aptamers can be single-stranded oligonucleotides.
  • aptamers may comprise more than one nucleic acid strand (e.g., two or more nucleic acid strands).
  • Aptamers may bind to a target (e.g., a protein) with high affinity and specificity through non-Watson-Crick base pairing interactions.
  • a target e.g., a protein
  • the aptamers described herein are non-naturally occurring
  • oligonucleotides ⁇ e.g, synthetically produced
  • Aptamers can bind to essentially any target molecule including, without limitation, proteins, oligonucleotides, carbohydrates, lipids, small molecules, and even bacterial cells.
  • Aptamers may be monomeric (composed of a single unit) or multimeric (composed of multiple units).
  • Multimeric aptamers can be homomeric (composed of multiple identical units) or heteromeric (composed of multiple non-identical units)
  • Aptamers herein may be described by their primary structures, meaning the linear nucleotide sequence of the aptamer.
  • aptamers herein are generally described from the 5’ end to the 3’ end, unless otherwise stated. Additionally or alternatively, aptamers herein may be described by their secondary structures which may refer to the combination of single-stranded regions and base-pairing interactions within the aptamer. Whereas many naturally occurring oligonucleotides, such as mRNA, encode information in their linear base sequences, aptamers generally do not encode information in their linear base sequences. Further, aptamers can be distinguished from naturally occurring oligonucleotides in that binding of aptamers to target molecules is dependent upon secondary' and tertiary structures of the aptamer.
  • Aptamers may be suitable as therapeutic agents and may be preferable to other therapeutic agents because: 1) aptamers may be fast and economical to produce because aptamers can be developed entirely by in vitro processes; 2) aptamers may have low toxicity and may lack an immunogenic response; 3) aptamers may have high specificity and affinity for their targets, 4) aptamers may have good solubility; 5) aptamers may have tunable pharmacokinetic properties; 6) aptamers may be amenable to site-specific conjugation of PEG and other carriers; and 7) aptamers may be stable at ambient temperatures.
  • VEGF-A includes any variant or isoform of VEGF-A.
  • “VEGF-A” may mean one or more of VEGF-Ano, VEGF-
  • pan-variant specific aptamer refers to an aptamer that selectively binds to at least VEGF-Ano, VEGF-A m , VEGF-Ai 65 , VEGF-A i 89 , and VEGF-A 206 ⁇
  • a pan-variant specific aptamer may, but not necessarily, bind to one or more additional VEGF-A isoforms or variants.
  • a pan-variant specific aptamer binds to a structural feature of VEGF-A that is common amongst VEGF-Ano, VEGF-Am, VEGF-A !65 , VEGF-A 189 , and VEGF-A206.
  • the term“or” is used nonexclusively to encompass“or” and“and.”
  • “A or B” includes“A but not B,”“B but not A,” and“A and B”, unless otherwise indicated.
  • VEGF-A Vascular endothelial growth factor- A
  • compositions that bind to vascular endothelial growth factor-A VEGF-A
  • methods of using such compositions to modulate VEGF-A signaling pathways VEGF-A
  • VEGF-A is thought to be the most significant regulator of angiogenesis in the VEGF family.
  • VEGF-A promotes growth of vascular endothelial cells which leads to the formation of capillary-like structures and may be necessary for the survival of newly formed blood vessels.
  • Vascular endothelial cells are thought to be major effectors of VEGF signaling.
  • Retinal pigment epithelial (RPE) cells may also express VEGF receptors and have been shown to proliferate and migrate upon exposure to VEGF
  • VEGF is thought to play roles beyond the vascular system.
  • VEGF may play roles in normal physiological functions, including, but not limited to, bone formation, hematopoiesis, wound healing, and development.
  • the compositions provided herein include aptamers that bind to VEGF -A, thereby inhibiting or reducing angiogenesis, e.g., by inhibiting or preventing growth of vascular endothelial cells, retinal pigment epithelial cells, or both.
  • the anti- VEGF -A aptamers provided herein may prevent or reduce binding or association of VEGF -A with a VEGF receptor (e.g., Fit- 1 , KDR, Nrp-1) expressed on vascular endothelial cells, retinal pigment epithelial cells, or both.
  • a VEGF receptor e.g., Fit- 1 , KDR, Nrp-1
  • the VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factor (PIGF).
  • the aptamers disclosed herein primarily bind to variants and isoforms of VEGF-A. In some cases, such aptamers may also bind to one or more of VEGF- B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PIGF. Transcription of VEGF mRNA may be upregulated under hypoxic conditions.
  • VEGF-A is thought to play a role in various ocular diseases and disorders such as, but not limited to, diabetic retinopathy, retinopathy of
  • retinoblastoma prematurity, central retinal vein occlusion, macular edema, choroidal neovascularization, neovascular (or wet) age-related macular degeneration, myopic choroidal neovascularization, punctate inner choroidopathy, presumed ocular histoplasmosis syndrome, familial exudative vitreoretinopathy, and retinoblastoma.
  • the aptamers provided herein may be used to treat an ocular disease or disorder involving one or more factors that upregulate VEGF-A expression and/or activity, including, but not limited to, hypoxic conditions; a growth factor such as EGF, TGF-a, TGF-b, KGF, IGF-1, FGF, or PDGF; and a cytokine such as IL-i-a, IL6, and 118.
  • a growth factor such as EGF, TGF-a, TGF-b, KGF, IGF-1, FGF, or PDGF
  • a cytokine such as IL-i-a, IL6, and 118.
  • the aptamers provided herein may be used to treat an ocular disease or disorder selected from the group consisting of: diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, macular edema, choroidal neovascularization, neovascular (or wet) age-related macular degeneration, myopic choroidal neovascularization, punctate inner choroidopathy, presumed ocular histoplasmosis syndrome, familial exudative vitreoretinopathy, and retinoblastoma.
  • an ocular disease or disorder selected from the group consisting of: diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, macular edema, choroidal neovascularization, neovascular (or wet) age-related macular degeneration, myopic choroidal neovascularization, punctate inner choroidopathy, presumed ocular histoplasmo
  • the gene for human VEGF-A contains eight exons and encodes at least 16 isoforms.
  • the most common isoforms generated by alternative splicing mechanisms are VEGF-A 121 , VEGF- A 165 , VEGF-A189, and VEGF-A 206.
  • VEGF-A 165 , VEGF-A 189 , and VEGF-A 206 each contain a C -terminal heparin binding domain (HBD).
  • HBD C -terminal heparin binding domain
  • VEGF-A m lacks a heparin- binding domain.
  • plasmin activation may result in proteolytic cleavage of VEGF- Ai 65 , VEGF-A 189 , and VEGF-A20 & , resulting in the release of the soluble VEGF-Ano variant, which also lacks a heparin-binding domain.
  • the aptamers provided herein may bind to and inhibit a function associated with one or more VEGF-A isoforms or variants.
  • the aptamers provided herein may bind to and inhibit a function associated with one or more of VEGF-Ano, VEGF- Am, VEGF-A 165, VEGF-Ai 89 , and VEGF-A 206.
  • the aptamers provided herein may be pan-variant specific aptamers.
  • a pan-variant specific aptamer may bind to each of VEGF-Ano, VEGF-Am, VEGF-A i65 , VEGF-A 1S9 , and VEGF-A 206 .
  • the aptamers provided herein may bind to a structural feature that is common to each of VEGF-Ano, VEGF-Am, VEGF-A165, VEGF-A189, and VEGF-A206 ⁇
  • the aptamers provided herein may bind to the receptor binding face, or a portion thereof, of each of VEGF-Ano, VEGF- Am, VEGF-A 165, VEGF-Ai 89 , and VEGF-A 2 o 6 ⁇
  • the aptamers provided herein may bind to the receptor binding domain, or a portion thereof, of each of VEGF-Ano, VEGF-Am, VEGF-A 165 , VEGF-A 139 , and VEGF-A 206 .
  • the aptamers provided herein may bind to a structural feature of VEGF-A other than the heparin binding domain found in VEGF-Ai65, VEGF-Ai65, VE
  • VEGF-A is known to interact with the receptor tyrosine kinases VEGFIll (also known as Fit- 1 ), VEGFR2 (also known as KDR or Flk-1), and Neuropilin-1 (Nrp-1). Nrp- i is thought to be a co-receptor for KDR.
  • VEGF receptors have been shown to be expressed by endothelial cells, macrophages, hematopoietic cells, and smooth muscle cells.
  • KDR is a class IV receptor tyrosine kinase that binds 2: 1 to VEGF-A dimers.
  • Fit- 1 is a receptor tyrosine kinase that binds to VEGF-A with a 3-10 fold higher affinity than KDR, and has also been shown to bind to VEGF-B and P1GF Flt-1 expression may be upregulated by hypoxia, and its affinity for VEGF-A has been proposed as a negative regulator of signaling by KDR by acting as a decoy receptor.
  • An alternative splicing variant of Fit- 1 results in a soluble variant of the receptor (sFlt-1) which has been suggested to act as an anti-angiogenic sink for VEGF-A.
  • VEGF-A i65 Association of VEGF-A i65 with KDR may be enhanced by the interaction of the heparin binding domain with co-receptor Nrp-1, which may enhance downstream signaling of KDR.
  • Nrp-1 also has strong affinity for Fit- 1 , which may prevent Nrp-1 association with VEGF-A165 and may be a secondary' regulatory mechanism for VEGF-A induced angiogenesis.
  • aptarners provided herein may bind to one or more isoforms or variants of VEGF-A, and may prevent or reduce binding or association of VEGF-A with a VEGF receptor.
  • aptarners provided herein may prevent or reduce binding of one or more isoforms or variants of VEGF-A with Fit- 1 , KDR, Nrp-1, or any combination thereof.
  • aptarners provided herein may prevent or reduce binding of one or more of VEGF-Ano, VEGF-Am, VEGF-A 165 , VEGF-A 189 , and VEGF-A 206 to one or more of Fit- 1, KDR, and Nrp-1.
  • aptarners provided herein may prevent or reduce binding of one or more isoforms or variants of VEGF-A to KDR.
  • the aptarners are pan-variant specific aptarners that bind to each of VEGF-Ano, VEGF-Am, VEGF-A 165 , VEGF-A !89 , and VEGF- A 2O6 , and reduce or prevent binding or association thereof with one or more of Fit- 1, KDR, and Nrp-1.
  • an amino acid sequence of human VEGF-A206 may comprise the following sequence:
  • an amino acid sequence of human VEGF-A 8 may comprise the fol 1 owing sequence :
  • an amino acid sequence of human VEGF-Aies may comprise the following sequence: APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGG CCNDEGLECVPTEFiSNITMQiMRlKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCG PCSiiRRKHLFVQDPQTCKCSCKNTDSRGKARQLE-LNERTGRGDKPRR (SEQ ID NO: 3)
  • an amino acid sequence of human VEGF-Au may comprise the following sequence:
  • an amino acid sequence of human VEGF-Auo may comprise the following sequence:
  • the methods and compositions described herein include the use of one or more aptamers for the treatment of an ocular disease or disorder. In some cases, the methods and compositions described herein include the use of one or more aptamers for inhibiting an activity associated with VEGF-A.
  • Aptamers as described herein may include any number of modifications that can affect the function or affinity of the aptamer.
  • aptamers may be unmodified or they may contain modified nucleotides to improve stability, nuclease resistance or delivery characteristics.
  • modifications may include chemical substitutions at the sugar and/or phosphate and/or base positions, for example, at the T position of ribose, the 5 position of pyrimidines, and the 8 position of purines, various :2'-modified pyrimidines and purines and modifications with 2'-amino (2'-NH 2 ), 2 -fluoro (2 ! -F), and/or 2'-0-methyl (2'-OMe) substituents.
  • aptamers described herein comprise a 2’-OMe and/or a 2’F modification to increase in vivo stability.
  • the aptamers described herein contain modified nucleotides to improve the affinity and specificity of the aptamers for a target. Examples of modified nucleotides include those modified with guanidine, indole, amine, phenol,
  • pyrimidine nucleotide triphosphate analogs or CE-phosphoramidites may be modified at the 5 position to generate, for example, 5- benzylaminocarbonyl-2’-deoxyuridine (BndU); 5-[N-(phenyl-3-propyl)carboxamide]-2'- deoxyuridine (PPdU); 5-(N-thiophenylmethylcarboxyamide)-2'-deoxyuridine (ThdU); 5-(N-4- fluorobenzylcarboxyamide)-2'-deoxyuridine (FBndU); 5-(N-(l -naphthylmethyl)carboxamide)-2'- deoxyuridine (NapdU); 5 -(N-2-naphthylmethylcarboxyamide)-2'-deoxyuridine (2NapdU); 5-(N- l-naphthylethylcarboxyamide)-2'-deoxyuridine (BndU); 5-[N-(phenyl-3
  • Modifications of the aptamers contemplated in this disclosure include, without limitation, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and functionality to the nucleic acid aptamer bases or to the nucleic acid aptamer as a whole. Modifications to generate
  • oligonucleotide populations that are resistant to nucleases can also include one or more substitute internucleotide linkages, altered sugars, altered bases, or combinations thereof.
  • modifications include, but are not limited to, mposition sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate, phosphorodithioate, or alkyl phosphate modifications, methylations, and unusual base-pairing combinations such as the isobases isocytidine and isoguanosine.
  • Modifications can also include 3’ and 5' modifications such as capping, e.g., addition of a 3'-3'- dT cap to increase exonuclease resistance, or conjugation of a PEG to the 5’ or 3’ end to increase exonuclease and endonuclease resistance
  • Aptamers of the disclosure may generally comprise nucleotides having ribose in the b-D- ribofuranose configuration. In some cases, 100% of the nucleotides present in the aptamer have ribose in the b-D-ribofuranose configuration. In some cases, at least 50% of the nucleotides present in the aptamer have ribose in the b-D-ribofuranose configuration.
  • At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the nucleotides present in the aptamer have ribose in the b-D- ribofuranose configuration.
  • the length of the aptamer can be variable. In some cases, the length of the aptamer is less than 100 nucl eotides. In some cases, the length of the aptamer is greater than 10 nucleotides. In some cases, the length of the aptamer is between 10 and 90 nucleotides.
  • the aptamer can be, without limitation, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, or about 90 nucleotides in length.
  • a polyethylene glycol (PEG) polymer chain is covalently bound to the aptamer, referred to herein as PEGylation.
  • PEGylation may increase the half-life and stability of the aptamer in physiological conditions.
  • the PEG polymer is covalently bound to the 5' end of the aptamer.
  • the PEG polymer is covalently hound to the 3' end of the aptamer.
  • the PEG polymer is covalently bound to both the 5’ end and the 3' end of the aptamer.
  • the PEG polymer is covalently bound to a specific site on a nueleobase within the aptamer, including the 5-position of a pyrimidine or 8-position of a purine. In some cases, the PEG polymer is covalently bound to an abasic site within the aptamer.
  • an aptamer described herein may be conjugated to a PEG having the general formula, H-CQ-CH -CEBj n -OH.
  • an aptamer described herein may be conjugated to a methoxy-PEG (mPEG) of the general formula, CH 3 0-(CH 2 -CH 2 -0) n -H.
  • the aptamer is conjugated to a linear chain PEG or mPEG.
  • the linear chain PEG or mPEG may have an average molecular weight of up to about 30 kD.
  • Multiple linear chain PEGs or mPEGs can be linked to a common reactive group to form multi-arm or branched PEGs or mPEGs.
  • more than one PEG or mPEG can be linked together through an amino acid linker (e.g., lysine) or another linker, such as glycerine.
  • the aptamer is conjugated to a branched PEG or branched mPEG.
  • Branched PEGs or mPEGs may be referred to by their total mass (e.g., two linked 2QkD mPEGs have a total molecular weight of 40kD).
  • Branched PEGs or mPEGs may have more than two aims.
  • Multi -arm branched PEGs or mPEGs may he referred to by their total mass (e.g, four linked 10 kD mPEGs have a total molecular weight of 40 kD).
  • an aptamer of the present disclosure is conjugated to a PEG polymer having a total molecular weight from about 5 kD to about 200 kD, for example, about 5 kD, about 10 kD, about 20 kD, about 30 kD, about 40 kD, about 50 kD, about 60 kD, about 70 kD, about 80 kD, about 90 kD, about 100 kD, about 1 10 kD, about 120 kD, about 130 kD, about 140 kD, about 150 kD, about 160 kD, about 170 kD, about 180 kD, about 190 kD, or about 200 kD.
  • the aptamer is conjugated to a PEG having a total molecular weight of about 40 kD.
  • the reagent that may be used to generate PEGylated aptamers is a branched PEG N-Hydroxysuccinimide (mPEG-NHS) having the general formula: with a 20 kD, 40 kD or 60 kD total molecular weight (e.g., where each mPEG is about lOkD, 20 kD or about 30 kD).
  • mPEG-NHS branched PEG N-Hydroxysuccinimide
  • the branched PEGs can be linked through any appropriate reagent, such as an amino acid (e.g, lysine or glycine residues).
  • the reagent used to generate PEGylated aptamers is [N 2 - (monomethoxy 20K polyethylene glycol carbamoyl)-N°-(monomethoxy 20K polyethylene glycol carbamoyl)] -lysine N-hydroxysuccinimide having the formula:
  • the reagent used to generate PEGylated aptamers has the formula:
  • X is N-hydroxysuccinimide and the PEG arms are of approximately equivalent molecular weight.
  • PEG architecture may provide a compound with reduced viscosity compared to a similar aptamer conjugated to a two-armed or single-arm linear PEG.
  • the reagent used to generate PEGyiated aptamers has the formula:
  • X is N-hydroxysuccinimide and the PEG arms are of different molecular weights
  • a 40 kD PEG of this architecture may be composed of 2 amis of 5 kD and 4 arms of 7.5 kD.
  • Such PEG architecture may provide a compound with reduced viscosity compared to a similar aptamer conjugated to a two-armed PEG or a single-ami linear PEG.
  • the reagent that may be used to generate PEGyiated aptamers is a non- branched mPEG-Succinimidyl Propionate (mPEG-SPA), having the general formula:
  • mPEG is about 20 kD or about 30 kD.
  • the reactive ester may be
  • the reagent that may be used to generate PEGyiated aptamers may include a branched PEG linked through glycerol, such as the SUNBRIGHT® series from NOF Corporation, Japan.
  • a branched PEG linked through glycerol such as the SUNBRIGHT® series from NOF Corporation, Japan.
  • Non-limiting examples of these reagents include:
  • the reagents may include a non-branched mPEG Succinimidyl alpha-methylbutanoate (mPEG-SMB) having the general formula:
  • the reactive ester may be -O-CH2.CH2. CH(CH 3 )-C0 2 -NHS.
  • the PEG reagents may include nitrophenyl carbonate-linked PEGs, having the general formula:
  • Compounds including nitrophenyl carbonate can be conjugated to primary amine containing linkers.
  • the reagents used to generate PEGy!ated aptamers may include PEG with thiol -reactive groups that can be used with a thiol -modi tied linker.
  • PEG PEG with thiol -reactive groups that can be used with a thiol -modi tied linker.
  • One non-limiting example may include reagents having the following general structure: where mPEG is about 10 kD, about 20 kD or about 30 kD.
  • Another non-limiting example may include reagents having the following general structure:
  • Branched PEGs with thiol reactive groups that can be used with a thiol-modified linker, as described above, may include reagents in which the branched PEG has a total molecular weight of about 40 kD or about 60 kD (e.g., where each mPEG is about 20 kD or about 30 kD).
  • the reagents used to generated PEGylated aptamers may include reagents having the following structure:
  • the reaction to conjugate the PEG to the aptamer is carried out between about pH 6 and about pH 10, or between about pH 7 and pH 9 or about pH 8.
  • the reagents used to generate PEGylated aptamers may include reagents having the following structure:
  • the reagents used to generate PEGylated aptamers may include reagents having the following structure:
  • the aptamer is associated with a single PEG molecule. In other cases, the aptamer is associated with two or more PEG molecules.
  • the aptamers described herein may be bound or conjugated to one or more molecules having desired biological properties. Any number of molecules can be bound or conjugated to aptamers, non-limiting examples including antibodies, peptides, proteins, carbohydrates, enzymes, polymers, drugs, small molecules, gold nanoparticles, radiolabels, fluorescent labels, dyes, haptens (e.g., biotin), other aptamers, or nucleic acids (e.g, siRNA). In some cases, aptamers may be conjugated to molecules that increase the stability, the solubility or the bioavailability of the aptamer. Non-limiting examples include polyethylene glycol (PEG) polymers, carbohydrates and fatty acids.
  • PEG polyethylene glycol
  • molecules that improve the transport or deliver ⁇ - of the aptamer may be used, such as cell penetrating peptides.
  • cell penetrating peptides can include peptides derived from Tat, penetratin, polyarginine peptide Args sequence, Transportan, VP22 protein from Herpes Simplex Virus (HSV), antimicrobial peptides such as Buforin I and SynB, polyproline sweet arrow ' peptide molecules, Pep-1 and MPG.
  • the aptamer is conjugated to a lipophilic compound such as cholesterol, dialkyl glycerol, diacyl glycerol, or a non-immunogenic, high molecular weight compound or polymer such as polyethylene glycol (PEG) or other water-soluble pharmaceutically acceptable polymers including, but not limited to, polyaminoamines (PAMAM) and polysaccharides such as dextran, or polyoxazolines (POZ).
  • a lipophilic compound such as cholesterol, dialkyl glycerol, diacyl glycerol, or a non-immunogenic, high molecular weight compound or polymer such as polyethylene glycol (PEG) or other water-soluble pharmaceutically acceptable polymers including, but not limited to, polyaminoamines (PAMAM) and polysaccharides such as dextran, or polyoxazolines (POZ).
  • PEG polyethylene glycol
  • POZ polyoxazolines
  • the molecule to be conjugated can be covalently bonded or can be associated through non-covalent interactions with the aptamer of interest.
  • the molecule to be conjugated is covalently attached to the aptamer.
  • the covalent attachment may occur at a variety of positions on the aptamer, for example, to the exocyclic amino group on the base, the 5- position of a pyrimidine nucleotide, the 8-position of a purine nucleotide, the hydroxyl group of the phosphate, or a hydroxyl group or other group at the 5 ! or 3' terminus.
  • the covalent attachment is to the 5' or 3’ hydroxyl group of the aptamer.
  • the aptamer can be attached to another molecule directly or with the use of a spacer or linker.
  • a lipophilic compound or a non-immunogenic, high molecular weight compound can be attached to the aptamer using a linker or a spacer.
  • linkers and attachment chemistries are known in the art.
  • 6- (trifluoroacetamido)hexanol (2-cyanoethyl-N,N-diisopropyl)phosphoramidite can be used to add a hexylamino linker to the 5 ! end of the synthesized aptamer.
  • linker phosphoramidites may include: TFA-amino C4 CED phosphoramidite having the structure:
  • 5'-amino modifier 5 having the structure:
  • S'-amino modifier C 12 having the structure:
  • MMT 4-Monomethoxytrityl 5' thiol-modifier C6 having the structure:
  • the 5'-thiol modified linker may be used, for example, with PEG-maleimides, PEG- vinylsulfone, PEG-iodoacetamide and PEG-orthopyridyl-disulfide.
  • the aptamer may be bonded to the 5'-thiol through a maleimide or vinyl sulfone functionality.
  • the aptamer formulated according to the present disclosure may also be modified by encapsulation within or displayed on the surface of a liposome. In other cases, the aptamer formulated according to the present disclosure may also be modified by encapsulation within or displayed on the surface of a micelle.
  • Liposomes and micelles may be comprised of any lipids, and in some cases the lipids may be phospholipids, including phosphatidylcholine.
  • Liposomes and micelles may also contain or be comprised in part or in total of other polymers and amphipathic molecules including PEG conjugates of poly lactic acid (PLA), poly DL-lactic- co-glycolic acid (PLGA), or poly caprolactone (PCL) [0095]
  • the aptamers described herein may be designed to inhibit a function associated with VEGF-A.
  • the aptamers described herein may be designed to bind the receptor binding face of VEGF-A, or a portion thereof.
  • the aptamers described herein may be designed to bind the receptor binding domain of VEGF-A, or a portion thereof.
  • the receptor binding domain of VEGF-A may include any one or more of residues 1- 109 as described in SEQ ID NOs: 1-5.
  • the aptamers described herein may bind to a structural feature of VEGF-A other than the heparin binding domain of VEGF-A.
  • the heparin binding domain of VEGF-A may include any one or more of residues 111-165 as described in SEQ ID NOs: 1-3.
  • the aptamers described herein may block or reduce binding of one or more isoforms or variants of VEGF-A to one or more of Flk-1, KDR, and Nrp-1.
  • an aptamer is isolated or purified.
  • isolated (used interchangeably with“substantially pure” or“purified”) as used herein means an aptamer that is synthesized chemically, or has been separated from other aptamers.
  • an aptamer of the disclosure may comprise one of the following sequences described in Table 1 or Table 2.
  • an aptamer of the disclosure may have a nucleic acid sequence comprising any one of SEQ ID NOs: 6 or 7, or may have a nucleic acid sequence that shares at least 50% sequence identity to any one of SEQ ID NOs: 6 or 7.
  • an aptamer of the disclosure may have a nucleic acid sequence consisting of any one of SEQ ID NOs: 6 or 7, or may have a nucleic acid sequence that shares at least 50% sequence identity to a sequence that consists of any one of SEQ ID NOs: 6 or 7.
  • the nucleic acid sequence may comprise one or more modified nucleotides. In some cases, at least 50% of said nucleic acid sequence may comprise the one or more modified nucleotides.
  • the one or more modified nucleotides may comprise a 2'F-modified nucleotide, a 2'QMe-modified nucleotide, or a combination thereof.
  • the one or more modified nucleotides may be selected from the group consisting of: 2'F-G, 2'OMe-G, 2'QMe-U, 2'OMe-A, 2'OMe-C, an inverted deoxythymidine at the 3' terminus, and any combination thereof.
  • the aptamer may have a nucleic acid sequence comprising any one of SEQ ID NOs: 6 or 7, wherein the nucleic acid sequence comprises modified nucleotides as described in Table 2.
  • the aptamer is selected from the group consisting of: Aptamer 4.2 as described in Table 2, and Aptamer 26 as described in Table 2.
  • the aptamer may be conjugated to a polyethylene glycol (PEG) molecule.
  • the PEG molecule may have a molecular weight of 80 kDa or less (e.g., 40kX)a).
  • an aptamer of the disclosure may have at least 50%, 55%, 60%, 65%,
  • an anti-VEGF-A aptamer of the disclosure may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% >, or 99% sequence identity with any aptamer described herein.
  • an anti-VEGF-A aptamer of the disclosure may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any aptamer described in Table 1 or Table 2.
  • an anti-VEGF-A aptamer of the disclosure may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%o sequence identity with any one of SEQ ID NOs: 6 or 7
  • an anti-VEGF-A aptamer of the disclosure may have at least 50% sequence identity with any one of SEQ ID NOs: 6 or 7. In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 55% sequence identity with any one of SEQ ID NOs: 6 or 7. In some eases, an anti-VEGF-A aptamer of the disclosure may have at least 60% sequence identity with any one of S EQ ID NOs: 6 or 7. In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 65% sequence identity with any one of SEQ ID NOs: 6 or 7.
  • an anti-VEGF-A aptamer of the disclosure may have at least 70% sequence identity with any one of SEQ ID NOs: 6 or 7, In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 75% sequence identity with any one of SEQ ID NOs: 6 or 7. In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 80% sequence identity with any one of SEQ ID NOs: 6 or 7. In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 85% sequence identity with any one of SEQ ID NOs: 6 or 7.
  • an anti-VEGF- A aptamer of the disclosure may have at least 90% sequence identity with any one of SEQ ID NOs: 6 or 7. In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 95% sequence identity with any one of SEQ ID NOs: 6 or 7,
  • an aptamer of the disclosure may have a primary nucleotide sequence that shares at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 contiguous nucleotides with a nucleotide sequence described in Table 1 or Table 2
  • an aptamer of the disclosure may have a primary nucleotide sequence that shares at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least
  • nucleotide modifications have been recited, it should be understood that any number and type of nucleotide modifications may be substituted.
  • 2’OMe-G may be substituted for 2’F-G.
  • nucleotide modifi cations have been provided herein.
  • all of the nucleotides of an aptamer are modified. In some instances, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,
  • an aptamer of the disclosure has the primary nucleotide sequence of any one of SEQ ID NOS: 6 or 7 and has a modified nucleotide sequence as described in Table 2.
  • an aptamer of the disclosure may have a modified nucleotide sequence.
  • an aptamer of the disclosure may have a modified nucleotide sequence as described in Table 2
  • an aptamer of the disclosure may have a primary nucleotide sequence according to any aptamer described in Table 2, and a modified nucleotide sequence that is different than that described in Table 2.
  • an aptam er of the disclosure may have a modified nucleotide sequence that shares at least 10% modification identity with any modified nucleotide sequence described in Table 2
  • an aptamer of the disclosure may have a modified nucleotide sequence that shares at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% modification identity with any modified nucleotide sequence described in Table 2.
  • an aptamer of the disclosure may have a primary nucleotide sequence of any one of SEQ ID NOS: 6 or 7, and a modified nucleotide sequence in which at least 10% of the C nucleotides are modified (e.g., 2'OMe-C).
  • an aptamer of the disclosure may have a modified nucleotide sequence in which at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least
  • an aptamer of the disclosure may have a modified nucleotide sequence wherein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least
  • an aptamer of the disclosure may have a primary nucleotide sequence of any one of SEQ ID NOS: 6 or 7, and a modified nucleotide sequence in which at least 10% of the A nucleotides are modified (e.g, 2'OMe-A).
  • an aptamer of the disclosure may have a modified nucleotide sequence in which at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the A nucleotides are modified (e.g., 2’OMe-A).
  • an aptamer of the disclosure may have a modified nucleotide sequence wherein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the A nucleotides are modified according to Table 2.
  • an aptamer of the disclosure may have a primary nucleotide sequence of any one of SEQ ID NOS: 6 or 7, and a modified nucleotide sequence in which at least 10% of the U nucleotides are modified (e.g., 2'OMe-U).
  • an aptamer of the disclosure may have a modified nucleotide sequence in which at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least
  • an aptamer of the disclosure may have a modified nucleotide sequence wherein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least
  • an aptamer of the disclosure may have a primary nucleotide sequence of any one of SEQ ID NOS: 6 or 7, and a modified nucleotide sequence in which at least 10% of the G nucleotides are modified (e.g., 2'F-G, 2'OMe-G).
  • an aptamer of the disclosure may have a modified nucleotide sequence wherein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the G nucleotides are modified according to Table 2.
  • the disclosure provides anti -VEGF-A compositions that inhibit a function associated with VEGF-A.
  • the anti -VEGF-A compositions may include one or more anti -VEGF-A aptamers that bind to specific regions of VEGF-A with high specificity and high affinity.
  • the anti-VEGF-A compositions may include one or more anti -VEGF-A aptamers that bind to a region of VEGF-A that includes the receptor binding face of VEGF-A.
  • the anti-VEGF-A compositions may include one or more anti-VEGF-A aptamers that bind to a region of VEGF-A that includes the receptor binding domain of VEGF-A, or a portion thereof.
  • the receptor binding domain of VEGF-A may include any one or more of residues 1-109 as described in SEQ ID NOs: 1-5.
  • compositions may include one or more anti-VEGF-A aptamers that prevent or reduce binding of one or more isoforms or variants of VEGF-A with Fit- 1 , KDR, Nrp-1, or any combination thereof.
  • anti-VEGF-A aptamers of the disclosure may block or reduce the interaction of VEGF-A with Fit- 1 , may block or reduce the interaction of VEGF-A with KDR, may block or reduce the interaction of VEGF-A with Nrp-1, or any combination thereof.
  • anti-VEGF-A aptamers of the disclosure bind to structural features that are common to VEGF-A U0 , VEGF-Am, VEGF-A 165 , VEGF-A 189 , and VEGF-A 2 o 6 .
  • anti-VEGF-A aptamers of the disclosure bind to regions of VEGF-A other than the heparin binding domain present in VEGF-A i65 , VEGF-A i 89 , and VEGF-A 2 o 6 ⁇
  • the heparin binding domain may include residues 111-165 as described by SEQ ID NOs: 1-3.
  • the cationic heparin binding domain of VEGF-A is thought to be the dominant epitope for aptamer recognition due to the anionic nature of the oligonucleotide sugar phosphate backbone. Therefore, selection of aptamers to regions of VEGF-A other than the heparin binding domain have proven difficult.
  • pegaptanib brand name Macugen 8
  • pegaptanib is an oligonucleotide inhibitor of VEGF-A that binds to the heparin binding domain.
  • VEGF-Am and VEGF-Ano lack the heparin binding domain, pegaptanib does not bind to or inhibit VEGF-Am and VEGF-Auo, thereby providing inferior VEGF-A suppression as compared to an inhibitor that binds to the receptor binding domain of VEGF-A.
  • additional aptamer inhibitors of VEGF-A have been described which bind to the heparin binding domain of VEGF-A.
  • a DNA aptamer specific for VEGF-A t es, but not VEGF-Am has been described (Hasegawa, Hijiri, Koji Sode, and Kazunori Ikebukuro.
  • RNA aptamers that bind to the heparin binding domain have been described for thrombin (Jeter, Martha L., et al. "RNA aptamer to thrombin binds anionUbinding exosite P 2 and alters protease inhibition by heparin ⁇ binding serpins.” FEBS letters568.1-3 (2004): 10-14.; Long, Stephen B., et al. "Crystal structure of an RNA aptamer bound to thrombin.” Rna (2008)), basic fibroblast growth factor (Jellinek, D., et al.
  • RNA ligands to basic fibroblast growth factor inhibit receptor binding Proceedings of the National Academy of Sciences 90.23 (1993): 11227-11231.
  • interleukin-8 Sung, Ho Jin, et al. "Inhibition of human neutrophil activity by an RNA aptamer bound to interleukin-8.” Biomaterials 35.1 (2014): 578-589.
  • Plasmodium falciparum erythrocyte membrane protein 1 Barfod, Anders, Tina Persson, and Johan Lindh. "In vitro selection of RNA aptamers against a conserved region of the Plasmodium falciparum erythrocyte membrane protein 1.” Parasitology research 105.6 (2009): 1557-1566.).
  • anti -VEGF-A aptamers of the disclosure may bind to a region ofVEGF- A that includes the receptor-binding face of any isoform or variant of VEGF-A, or portions thereof.
  • the receptor-binding face of VEGF-A may include strands b2, b5, and b6, and loops b ⁇ to b2 of one monomer, and the N-terminal a helix and loop b3 to b4 of a second monomer.
  • VEGF-A The receptor-binding face of VEGF-A may be as described by Muller et al.“The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding.” Structure 5.10 (1997): 1325-1338.
  • anti-VEGF-A aptamers of the disclosure may bind to one or more amino acid residues of any isoform or variant of VEGF -A, including, without limitation, Phe17, He43, 1!e46, Glu64, Gln79, Ile83, Lys84, Pro85, Arg82, His86, Asp63, and Glu67 as described by SEQ O) NOs: 1-5.
  • anti -VEGF - A aptamers that bind to the receptor-binding face of VEGF- A, or a portion thereof, may prevent or reduce the association of VEGF- A with one or more of Fit- 1, KDR, or Nrp-1.
  • anti-VEGF-A aptamers that bind to the receptor-binding face of VEGF- A, or a portion thereof may interact with recombinant bead-bound VEGF -A es, VEGF -Am , or VEGF-Ano as measured by flow cytometry or may interact with recombinant surface-bound VEGF-A I65 , VEGF- A , or VEGF- A io as measured by surface piasmon resonance (see Examples 1 and 2, respectively).
  • anti-VEGF-A aptamers that bind to the receptor-binding face of VEGF -A, or a portion thereof may inhibit or reduce the interaction of VEGF-A 165 , VEGF -Am , or VEGF-Ano with KDR as measured by a reduction in FRET signal (see Example 3).
  • anti- VEGF-A aptamers that bind to the receptor-binding face of VEGF- A, or a portion thereof may inhibit or reduce VEGF-A105, VEGF -Am , or VEGF-Ano induced trans autophosphorylation of the intracellular domain of KDR as measured by phospho-KDR AlphaLISA ® ( see Example 4 ).
  • anti-VEGF-A aptamers that bind to the receptor-binding face of VEGF- A, or a portion thereof may inhibit or reduce VEGF-Ai 65 , VEGF-Am . or VEGF-Ano induced gene expression of tissue factor in HUVEC ceils as measured by qPCR.
  • anti-VEGF-A aptamers that bind to the receptor-binding face of VEGF -A, or a portion thereof may inhibit or reduce VEGF-Aies, VEGF-Am , or VEGF-Ano induced tube formation of GFP-HUVECs in co- culture with human dermal fibroblasts cells as measured by change in network length or network area (see Example 5).
  • anti-VEGF-A aptamers that bind to the receptor-binding face of VEGF -A, or a portion thereof may inhibit or reduce vascular leakage in a mouse, rat, rabbit, or primate eye following exogenous VEGF-A 365 , VEGF-Am , or VEGF-Ano challenge as measured by fluorescein angiography and Evans-blue albumin staining.
  • anti-VEGF-A aptamers of the disclosure may bind to a region of VEGF - A that includes the receptor binding domain of any isoform or variant of VEGF- A, or portions thereof.
  • the receptor binding domain of VEGF -A may include one or more of residues 1-109, as described in SEQ ID NOs: 1-5.
  • anti-VEGF-A aptamers of the disclosure may bind to one or more amino acid residues of any isoform or variant of VEGF-A, including, without limitation, Phel7, Tyr21, Tyr25, Ile43, Ue46, Ile83, Asp63, Glu64, Pro85, and His86, as described by SEQ ID NQs: 1-5
  • anti-VEGF-A aptamers of the disclosure may bind to a region within the receptor binding domain of VEGF-A which results in global conformational changes in VEGF-A such that it no longer binds to and activates signaling via KDR.
  • anti-VEGF-A aptamers that bind to the receptor binding domain of VEGF- A, or a portion thereof may prevent or reduce the association of VEGF-A with one or more of Flt-1, KDR, and Nrp-1.
  • anti-VEGF-A aptamers that bind to the receptor binding domain of VEGF-A, or a portion thereof may interact with recombinant bead-bound VEGF- Ai65, VEGF-A 121 . or VEGF-Ano as measured by flow cytometr 7 or may interact with
  • anti-VEGF-A aptamers that bind to the receptor-binding domain of VEGF-A, or a portion thereof, may inhibit or reduce the interaction of VEGF-A 165 , VEGF-Am , or VEGF-Ano with KDR as measured by a reduction in FRET signal (see Example 3).
  • anti-VEGF-A aptamers that bind to the receptor-binding domain of VEGF-A, or a portion thereof may inhibit or reduce VEGF-A165, VEGF-Ai2i , or VEGF-Ano induced trans autophosphorylation of the intracellular domain of KDR as measured by phospho-KDR AlphaLISA ® (see Example 4).
  • anti-VEGF- A aptamers that bind to the receptor binding domain of VEGF-A, or a portion thereof may inhibit or reduce VEGF-A es, VEGF-A , or VEGF-Ano induced gene expression of tissue factor in HUVEC cells as measured by qPCR.
  • anti-VEGF-A aptamers that bind to the receptor binding domain of VEGF-A, or a portion thereof may inhibit or reduce VEGF- Ai65, VEGF-A 121 , or VEGF-Ano induced tube formation of GFP-HUVECs in co-culture with human dermal fibroblasts cells as measured by change in network length or network area (see Example 5).
  • anti-VEGF-A aptamers that bind to the receptor binding domain of VEGF-A, or a portion thereof may inhibit or reduce vascular leakage in a mouse, rat, rabbit, or primate eye following exogenous VEGF-Ai 65 , VEGF-Ani. or VEGF-Ano challenge as measured by fluorescein angiography and Evans-blue albumin staining.
  • the dissociation constant (3 ⁇ 4) can be used to describe the affinity of an aptamer for a target (or to describe how tightly the aptamer binds to the target) or to describe the affinity of an aptamer for a specific epitope of a target.
  • the dissociation constant may be defined as the molar concentration at which half of the binding sites of a target are occupied by the aptamer.
  • the smaller the K d the tighter the binding of the aptamer to its target.
  • an anti-VEGF- A aptamer of the disclosure may have a 3 ⁇ 4 for one or more isoforms or variants of VEGF-A of less than about 1000 nM, for example, less than about 500 nM, less than about 100 nM, less than about 50 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM, as measured by a surface plasmon resonance assay (see Example 2).
  • an anti -VEGF-A aptamer may have a dissociation constant (3 ⁇ 4) for one or more isoforms or variants of VEGF-A of less than about 50 nM, as measured by a surface plasmon resonance assay (see Example 2). In some cases, an anti -VEGF-A aptamer may have a dissociation constant (3 ⁇ 4) for one or more isoforms or variants of VEGF-A of less than about 25 nM, as measured by a surface plasmon resonance assay (see Example 2).
  • an anti- VEGF-A aptamer may have a dissociation constant (3 ⁇ 4) for one or more isoforms or variants VEGF-A of less than about 10 nM, as measured by a surface plasmon resonance assay (see Example 2). In some cases, an anti -VEGF-A aptamer may have a dissociation constant (3 ⁇ 4) for one or more isoforms or variants of VEGF-A of less than about 5 nM, as measured by a surface plasmon resonance assay (see Example 2).
  • an anti -VEGF-A aptamer may have a dissociation constant (3 ⁇ 4) for one or more isoforms or variants of VEGF-A of less than about l nM, as measured by a surface plasmon resonance assay (see Example 2). In some cases, an anti- VEGF-A aptamer may have a dissociation constant (3 ⁇ 4) for one or more isoforms or variants of VEGF-A of less than about 0.5 nM, as measured by a surface plasmon resonance assay (see Example 2).
  • an anti -VEGF-A aptamer may have a dissociation constant (3 ⁇ 4) for one or more Isoforms or variants of VEGF-A of less than about 0 1 nM, as measured by a surface plasmon resonance assay (see Example 2).
  • the aptamer may be a pan-variant specific aptamer that binds to each ofVEGF-Ano, VEGF-A , VEGF-Aies, VEGF-A 89 , and VEGF-A206 with a K Q of less than about 1000 nM, for example, less than about 500 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM, as measured by a surface plasmon resonance assay (see Example 2).
  • the aptamer may bind to any region of VEGF-A described herein, or a portion thereof, with a K of less than about 1000 nM, for example, less than about 500 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM, as measured by a surface plasmon resonance assay (see Example 2).
  • the aptamer may bind to the receptor-binding face or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 1000 nM, for example, less than about 500 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM, as measured by a surface plasmon assay (see Example 2).
  • the anti-VEGF-A aptamer may bind to the receptor-binding face or the receptor binding domain of VEGF-A, or portions thereof with a I from about 0.5 nM to about 25 nM, as measured by a surface plasmon resonance assay (see Example 2).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor-binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 50 nM as measured by a surface plasmon assay (see Example 2), and may have an IC 50 of less than about 50 nM as measured by a VEGF-A :KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay ( see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor biniding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 50 nM as measured by a surface plasmon resonance assay ( see Example 2), and may have an IC50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay ( see Example 3), a KDR phosphorylation AlphaLISA 8, assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K of less than about 50 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA 8 assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 50 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF-A- induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 50 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 0.5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a 3 ⁇ 4 of less than about 50 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 0.1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF -A -induced angiogenesis (see Example 5).
  • a region of VEGF-A such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a 3 ⁇ 4 of less than about 50 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 0.1 nM as measured by
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 10 nM as measured by a surface pl asmon resonance assay (see Example 2), and may have an IC 50 of less than about 50 nM as measured by a VEGF- A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 10 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay ( see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see, Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K of less than about 10 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF- A, such as the receptor binding face of VEGF- A, or the receptor binding domain of VEGF- A, or portions thereof, with a K d of less than about 10 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF- A- induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF- A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 10 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.5 nM as measured by a VEGF-A: KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K 4 of less than about 10 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 50 nM as measured by a VEGF- A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 10 nM as measured by a VEGF-A: KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K of less than about 5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an ICso of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesi s (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a 3 ⁇ 4 of less than about 5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ⁇ assay (see Example 4), or an in vitro model of VEGF-A- induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a 3 ⁇ 4 of less than about 5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 0.5 nM: as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA' 8 ' assay (see Example 4), or an i vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K Q of less than about 5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 0.1 nM as measured by a VEGF ⁇ A:KDR competition binding assay (see Example 3), a KDR phosphorylation Alpha) .
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a 3 ⁇ 4 of less than about 1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 50 nM as measured by a VEGF- A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA 1® assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA' 4 ' assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K of less than about 1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF-A- induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K of less than about 1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 0.5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA 4’’ assay (see Example 4), or an in vitro model of VEGF -A -induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K Q of less than about 1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 0.1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® ' assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a 3 ⁇ 4 of less than about 0.5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 50 nM as measured by a VEGF- A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Ka of less than about 0.5 nM as measured by a surface plasmon resonance assay ( see Example 2), and may have an IC 50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® ' assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a ICj of less than about 0.5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a 3 ⁇ 4 of less than about 0.5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 1 nM as measured by a VEGF- A: KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF-A- induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K Q of less than about 0.5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 0.5 nM as measured by a VEGF ⁇ A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® ' assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis ( see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 0.5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 0.1 nM as measured by a VEGF-A :KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 0.1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 50 nM as measured by a VEGF- A: KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 0.1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF- A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 0.1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 0.1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC 50 of less than about 1 nM as measured by a VEGF- A: KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA ® assay (see Example 4), or an in vitro model of VEGF-A- induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 0.1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K d of less than about 0.1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.1 nM: as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA' 8 ' assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
  • the aptamers disclosed herein may have an improved half-life as compared to other therapeutics, including antibodies.
  • the aptamers may have an improved half-life in a biological fluid or solution as compared to an antibody.
  • the aptamers may have an improved half-life in vivo as compared to an antibody.
  • the aptamers may have an improved half-life when injected into the eye (intraocular half-life) as compared to an antibody.
  • the aptamers may have an improved intraocular half-life when injected into the eye of a human.
  • the aptamers may demonstrate improved stability over antibodies under physiological conditions.
  • the aptamers described herein may have an intraocular half-life of at least 7 days in a human. In some cases, the aptamers described herein may have an intraocular half-life of at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 20 days or greater in a human.
  • the aptamers described herein may have an intraocular half-life of at least 1 day in a non-human animal (e.g rodent/rabbit/monkey/chimpanzee/pig). In some cases, the aptamers described herein may have an intraocular half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days or greater in a non-human animal such as a rodent, rabbit or monkey.
  • a non-human animal e.g rodent/rabbit/monkey/chimpanzee/pig.
  • the aptamers described herein may have an intraocular half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days or greater in a non-human animal such as a rodent, rabbit
  • the aptamers described herein may have a shorter half-life as compared to other therapeutics.
  • an unmodified or unconjugated aptamer may have a lower half-life as compared to a modified or conjugated aptamer, however, the low molecular weight of the unmodified or unconjugated forms may allow for orders of magnitude greater initial concentrations, thereby achieving greater duration/efficacy.
  • the aptamer may have an intraocular half-life of less than about 7 days in a human.
  • the aptamers described herein may have an intraocular half-life of less than about 6 days, less than about 5 days or even less than about 4 days in a human
  • the aptamers disclosed herein may demonstrate high specificity for VEGF-A versus other members of the VEGF family, including VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and P1GF.
  • the aptamer may be selected such that the aptamer has high affinity for VEGF-A, but with little to no affinity for VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or P1GF.
  • the aptamers of the disclosure may bind to VEGF-A with a specificity of at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 1,000-fold, at least 5, 000-fold, at least 10,000-fold, at least 50,000-fold, or at least 100,000-fold, or greater than 100,000-fold than the aptamers bind to VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or P1GF at relative serum concentrations.
  • the activity of a therapeutic agent can be characterized by the half maximal inhibitory concentration (IC50).
  • the IC50 may be calculated as the concentration of therapeutic agent in nM at which half of the maximum inhibitory effect of the therapeutic agent is achieved.
  • the IC50 may be dependent upon the assay utilized to calculate the value.
  • the IC50 of an aptamer described herein may be less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM or less than 0.01 nM as measured by a VEGF-A :KDR competition binding assay ⁇ see Example 3).
  • the IC50 of an aptamer described herein may be less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM or less than 0.01 nM as measured by a KDR phosphorylation AlphaLISA ® assay ⁇ see Example 4).
  • the IC50 of an aptamer described herein may be less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM or less than 0.01 nM as measured by an in vitro model of VEGF-A- induced angiogenesis (see Example 5)
  • Aptamers generally have high stability at ambient temperatures for extended periods of time.
  • the aptamers described herein may demonstrate greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or greater than 99.9% activity in solution under physiological conditions at 30 days or later
  • a composition of the disclosure comprises anti-VEGF-A aptamers, wherein essentially 100%o of the anti-VEGF-A aptamers comprise nucleotides having ribose in the b-D-ribofuranose configuration.
  • a composition of the disclosure may comprise anti-VEGF-A aptamers, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90% of the anti-VEGF-A aptamers have ribose in the b-D- ribofuranose configuration.
  • the methods and compositions provided herein may be suitable for the treatment of ocular diseases or disorders. In some aspects, the methods and compositions provided herein may be suitable for the prevention of ocular diseases or disorders. In some aspects, the methods and compositions provided herein may be suitable to slow or halt the progression of ocular diseases or disorders.
  • the ocular disease or disorder is diabetic retinopathy. In some cases, the ocular disease or disorder is retinopathy of prematurity. In some cases, the ocular disease or disorder is central retinal vein occlusion. In some cases, the ocular disease or disorder is macular edema. In some cases, the ocular disease or disorder is choroidal neovascularization.
  • the ocular disease or disorder is neovascular (or wet) age-related macular degeneration. In some cases, the ocular disease or disorder is myopic choroidal neovascularization. In some cases, the ocular disease or disorder is punctate inner choroidopathy. In some cases, the ocular disease or disorder is presumed ocular histoplasmosis syndrome. In some cases, the ocular disease or disorder is familial exudative vitreoretinopathy. In some cases, the ocular disease or disorder is retinoblastoma.
  • Additional examples of ocular diseases or disorders may include, without limitation, pterygium, inflammatory conjunctivitis, including allergic and giant papillary conjunctivitis, infectious conjunctivitis, vernal keratoconjunctivitis, Stevens- Johnson disease, corneal herpetic keratitis, rhegmatogenous retinal detachment, pseudo-exfoliation syndrome, endophthalmitis, sc!eritis, corneal ulcers, dry eye syndrome, glaucoma, ischemic retinal disease, corneal transplant rejection, complications related to intraocular surgery such intraocular lens implantation and inflammation associated with cataract surgery, Behcet's disease, Stargardt disease, immune complex vasculitis, Fuch's disease, Vogt-Koyanagi-Harada disease, subretinal fibrosis, keratitis, vitreo-retinal inflammation, ocular parasit
  • blepharochalasis ptosis, xanthelasma of the eyelid, parasitic infestation of the eyelid, dermatitis of the eyelid, dacryoadenitis, epiphora, dysthyroid exophthalmos, conjunctivitis, scleritis, adenovirus keratitis, corneal ulcer, corneal abrasion, snow blindness, arc eye, Thygeson’s superficial punctate keratopathy, corneal neovascularization, Fuchs’ dystrophy, keratoconus, keratoconjunctivitis sicca, ulceris, sympathetic ophthalmia, cataracts, chorioretinal inflammation, focal chorioretinal inflammation, focal chorioretinitis, focal choroiditis, focal retinitis, focal retinochoroiditis, disseminated chorioretinal inflammation, disseminated chorioretinitis, diss
  • insufficiency choroidal thrombosis
  • neovascularization of the optic nerve diabetic macular edema, cystoid macular edema, proliferative vitreoretinopathy, and neovascularization due to penetration of the eye or ocular injury.
  • the methods and compositions provided herein are suitable for the treatment of diseases that cause one or more ocular symptoms.
  • symptoms which may be amenable to treatment with the methods disclosed herein include, but are not limited to choroidal or vitreal neovascularization, vascular leakage, reduced reading speed, reduced color vision, macular edema, increased retinal thickening, increase in central retinal volume and/or, macular sensitivity, loss of retinal cells, increase in area of retinal atrophy, reduced best corrected visual acuity such as measured by Snellen or ETDRS scales, reduced Best Corrected Visual Acuity under low luminance conditions, impaired night vision, impaired light sensitivity, impaired dark adaptation, impaired contrast sensitivity, worsened patient reported outcomes, and any combination thereof.
  • the methods and compositions provided herein may alleviate or reduce a symptom of a disease.
  • treatment with an aptamer provided herein may result in a reduction in the severity of any of the symptoms described herein.
  • treatment with an aptamer described herein may slow, halt or reverse the progression of any of the symptoms described herein.
  • treatment with an aptamer described herein may prevent the development of any of the symptoms described herein.
  • treatment with an aptamer described herein may slow, halt or reverse the progression of a disease, as measured by the number and severity of symptoms experienced.
  • Examples of symptoms and relevant endpoints where the aptamer may have a therapeutic effect include choroidal or retinal neovascularization, vascular leakage, reduced reading speed, reduced color vision, macular edema, increased retinal thickening, increase in central retinal volume and/or, macular sensitivity, loss of retinal cells, increase in area of retinal atrophy, reduced best corrected visual acuity such as measured by Snellen or ETDRS scales, reduced Best Corrected Visual Acuity under low luminance conditions, impaired night vision, impaired light sensitivity, impaired dark adaptation, impaired contrast sensitivity, and worsening patient reported outcomes.
  • treatment with an aptamer described herein may have beneficial effects as measured by clinical endpoints including reading speed, choroidal or retinal neovascularization or vascular leakage as measured by fluorescein angiography, retinal thickness as measured by Optical Coherence Tomography or other techniques, central retinal volume, number and density of retinal cells, area of retinal atrophy as measured by Fundus Photography or Fundus Autofluoresence or other techniques, best corrected visual acuity such as measured by Snellen or ETDRS scales. Best Corrected Visual Acuity under low luminance conditions, light sensitivity, dark adaptation, contrast sensitivity, and patient reported outcomes as measured by such tools as the National Eye Institute Visual Function Questionnaire and Health Related Quality of Life Questionnaires.
  • the methods and compositions provided herein may alleviate or reduce a symptom of a neovascular eye disease.
  • treatment with an aptamer provided herein may result in a reduction in the severity of any symptoms associated with a neovascular eye disease.
  • treatment with an aptamer described herein may slow, halt or reverse the progression of any symptom associated with a neovascular eye disease.
  • treatment with an aptamer described herein may prevent the development of any symptom associated with a neovascular eye disease.
  • treatment with an aptamer described herein may slow, halt or reverse the progression of a neovascular eye disease, as measured by the number and severity of symptoms experienced.
  • Non-limiting examples of symptoms associated with neovascular eye diseases where the aptamer may have a therapeutic effect include choroidal or retinal neovascularization, vascular leakage within the eye, macular edema, central retinal thickness and visual acuity.
  • the terms“subject” and“patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, rodents (e.g., mice, rats, rabbits, etc.) simians, humans, research animals (e.g., beagles, etc.), farm animals (e.g., pigs, horses, cows, llamas, alpacas, etc.), sport animals, and pets. In some cases, the methods described herein may be used on tissues or cells derived from a subject and the progeny of such tissues or cells. For example, aptamers described herein may be used to affect some function in tissues or cells of a subject.
  • rodents e.g., mice, rats, rabbits, etc.
  • research animals e.g., beagles, etc.
  • farm animals e.g., pigs, horses, cows, llamas, alpacas,
  • the tissues or cells may be obtained from a subject in vivo.
  • the tissues or cells are cultured in vitro and contacted with a composition provided herein (e.g., an aptamer).
  • a composition provided herein e.g., an aptamer.
  • the methods and compositions provided herein are used to treat a subject in need thereof.
  • the subject has, is suspected of having, or is at risk of developing, an ocular disease or disorder.
  • the subject is a human.
  • the human is a patient at a hospital or a clinic.
  • the subject is a non-human animal, for example, a non-human primate, a livestock animal, a domestic pet, or a laboratory animal.
  • a non-human animal can be an ape (e.g, a chimpanzee, a baboon, a gorilla, or an orangutan), an old world monkey (e.g., a rhesus monkey), a new' world monkey, a dog, a cat, a bison, a camel, a cow, a deer, a pig, a donkey, a horse, a mule, a lama, a sheep, a goat, a buffalo, a reindeer, a yak, a mouse, a rat, a rabbit, or any other non-human animal
  • an ape e.g, a chimpanzee, a baboon, a gorilla, or an orangutan
  • an old world monkey e.g., a rhesus monkey
  • a new' world monkey e.g., a dog, a cat, a bison, a camel
  • the subject may be of any age.
  • the subject has an age-related ocular disease or disorder (e.g., age-related macular degeneration).
  • the subject is about 50 years or older.
  • the subject is about 55 years or older.
  • the subject is about 60 years or older.
  • the subject is about 65 years or older.
  • the subject is about 70 years or older.
  • the subject is about 75 years or older.
  • the subject is about 80 years or older.
  • the subject is about 85 years or older.
  • the subject is about 90 years or older.
  • the subject is about 95 years or older.
  • the subject is about 100 years or older.
  • the subject is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
  • the subject is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater than 20 years old.
  • the methods and compositions provided herein may be used to treat a subject having, suspected of having, or at risk of developing ocular symptoms as described herein. In some aspects, the methods and compositions provided herein may be used to treat a subject having, suspected of having, or at risk of developing an ocular disease as provided herein. In some cases, the methods and compositions provided herein may be used to treat a subject having, suspected of having, or at risk of developing an ocular disease or disorder. In some cases, the ocular disease or disorder is diabetic retinopathy. In some cases, the ocular disease or disorder is retinopathy of prematurity. In some cases, the ocular disease or disorder is central retinal vein occlusion.
  • the ocular disease or disorder is macular edema. In some cases, the ocular disease or disorder is choroidal neovascularization. In some cases, the ocular disease or disorder is neovascular (or wet) age-related macular degeneration. In some cases, the ocular disease or disorder is myopic choroidal neovascularization. In some cases, the ocular disease or disorder is punctate inner choroidopathy. In some cases, the ocular disease or disorder is presumed ocular histoplasmosis syndrome. In some cases, the ocular disease or disorder is familial exudative vitreoretinopathy. In some cases, the ocular disease or disorder is retinoblastoma. In some cases, the methods and compositions provided herein may be used to treat a subject having, suspected of having, or at risk of developing an ocular disease or disorder exhibiting elevated levels of one or more isoforms or variants of VEGF-A.
  • compositions or medicaments are provided.
  • the pharmaceutical compositions can be used for the treatment of an ocular disease or disorder.
  • the pharmaceutical compositions comprise one or more anti-VEGF-A aptamers for the treatment of an ocular disease or disorder.
  • the ocular disease or disorder is diabetic retinopathy.
  • the ocular disease or disorder is retinopathy of prematurity.
  • the ocular disease or disorder is central retinal vein occlusion.
  • the ocular disease or disorder is macular edema.
  • the ocular disease or disorder is choroidal neovascularization.
  • the ocular disease or disorder is neovascular (or wet) age-related macular degeneration.
  • the ocular disease or disorder is myopic choroidal neovascularization. In some cases, the ocular disease or disorder is punctate inner choroidopathy. In some cases, the ocular disease or disorder is presumed ocular histoplasmosis syndrome. In some cases, the ocular disease or disorder is familial exudative vitreoretinopathy. In some cases, the ocular disease or disorder is retinoblastoma. In some cases, the
  • compositions can be used for the treatment of an ocular disease or disorder that exhibits elevated levels of one or more isoforms or variants of VEGF-A.
  • the one or more anti-VEGF-A aptamers may bind to one or more isoforms or variants of VEGF-A.
  • the one or more anti-VEGF-A aptamers are pan-variant specific aptamers that bind to each of VEGF-Ano, VEGF-Am, VEGF-Aies, VEGF- Ai89, and VEGF-A 2 o6 ⁇
  • the one or more anti-VEGF-A aptamers may bind to the receptor binding face of VEGF-A, or a portion thereof.
  • the receptor binding face of VEGF-A may include strands b2, b5, and b ⁇ and loop b ⁇ and b2 of a first monomer, and the N -terminal a helix and loop b3 to b4 of the second monomer (see Muller, Yves A., et al . "The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding " Structure 5.10 (1997): 1325-1338 ).
  • anti- VEGF-A aptamers that bind to the receptor binding face of VEGF-A may bind to one or more of residues Phel7, Ile43, Ile46, Glu64, Gln79, Ile83, Lys84, Pro85, Arg82, His86, Asp63, Glu67, as described in SEQ ID NOs: 1-5.
  • the one or more anti -VEGF -A aptamers may bind to the receptor binding domain of VEGF-A, or a portion thereof.
  • the receptor binding domain of VEGF-A may include any one or more of residues 1-109, as described in SEQ ID NOs: 1-5.
  • the one or more anti-VEGF-A aptamers may prevent or reduce the binding of one or more isoforms or variants of VEGF-A with Flt-1, KDR, or Nrp-1.
  • the compositions may include, e.g., an effective amount of the aptamer, alone or in combination, with one or more vehicles (e.g., pharmaceutically acceptable compositions or e.g., pharmaceutically acceptable carriers).
  • compositions as described herein may comprise a liquid formulation, a solid
  • compositions of the present disclosure may further comprise any number of excipients.
  • Excipients may include any and all solvents, coatings, flavorings, colorings, lubricants, disintegrants, preservatives, sweeteners, binders, diluents, and vehicles (or carriers). Generally, the excipient is compatible with the therapeutic compositions of the present disclosure.
  • the pharmaceutical composition may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as, for example, sodium acetate, and
  • a formulation is administered to the eye of a subject for the treatment of an ocular disease as described herein.
  • Administration to the eye can be; a) local ocular delivery; or b) systemic.
  • a topical formulation can be applied directly to the eye (e.g., eye drops, contact lens loaded with the formulation) or to the eyelid (e.g, cream, lotion, gel).
  • topical administration can be to a site remote from the eye, for example, to the skin of an extremity. This form of administration may be suitable for targets that are not produced directly by the eye.
  • a formulation of the disclosure is administered by local ocular delivery'.
  • Non-limiting examples of local ocular delivery ' include intravitreai (IVT), intracamarel, subconjunctival, subtenon, retrobulbar, posterior juxtascleral, and peribulbar.
  • IVT intravitreai
  • a formulation of the disclosure is delivered by intravitreai administration (IVT).
  • IVT intravitreai administration
  • Local ocular delivery may generally involve injection of a liquid formulation.
  • a formulation of the disclosure is administered systemically.
  • Systemic administration can involve oral administration.
  • systemic administration can be intravenous administration, subcutaneous administration, infusion, implantation, and the like.
  • formulations suitable for delivery of the pharmaceutical compositions described herein may include a sustained release gel or polymer formulations by surgical implantation of a biodegradable microsize polymer system, e.g., microdevice, microparticle, or sponge, or other slow release transscleral devices, implanted during the treatment of an ophthalmic disease, or by an ocular delivery device, e.g., polymer contact lens sustained delivery device.
  • the formulation is a polymer gel, a self-assembling gel, a durable implant, an eluting implant, a biodegradable matrix or biodegradable polymers.
  • the formulation may be administered by iontophoresis using electric current to drive the composition from the surface to the posterior of the eye.
  • the formulation may be administered by a surgically implanted port with an intravitreai reservoir, an extra-vitreal reservoir or a combination thereof.
  • implantable ocular devices can include, without limitation, the DurasertTM technology developed by Bausch & Lomb, the ODTx device developed by On Demand
  • nanotechnologies can be used to deliver the pharmaceutical compositions including nanospheres, nanoparticles, nanocapsules, liposomes, nanomicelles and dendrimers.
  • composition of the disclosure can be administered once or more than once each day.
  • the composition is administered as a single dose (i.e., one-time use).
  • the single dose may be curative.
  • the composition may be administered serially (e.g., taken every day without a break for the duration of the treatment regimen).
  • the treatment regime can be less than a week, a week, two weeks, three weeks, a month, or greater than a month.
  • the composition is administered over a period of at least 12 weeks, at least 16 weeks, at least 20 weeks, or at least 24 weeks.
  • the composition is administered for a day, at least two consecutive days, at least three consecutive days, at least four consecutive days, at least five consecutive days, at least six consecutive days, at least seven consecutive days, at least eight consecutive days, at least nine consecutive days, at least ten consecutive days, or at least greater than ten consecutive days.
  • a therapeutically effective amount can be administered one time per week, two times per week, three times per week, four times per week, five times per week, six times per week, seven times per week, eight times per w ' eek, nine times per week, 10 times per week, 11 times per week, 12 times per week,
  • a therapeutically effective amount can be administered one time per day, two times per day, three times per day, four times per day, five times per day, six times per day, seven times per day, eight times per day, nine times per day, 10 times per day, or greater than 10 times per day.
  • the composition is administered at least twice a day. In further eases, the
  • composition is administered at least every hour, at least every two hours, at least every three hours, at least ever ⁇ ' four hours, at least every five hours, at least every six hours, at least every' seven hours, at least every eight hours, at least every' nine hours, at least every' 10 hours, at least every ' 11 hours, at least every' 12 hours, at least every' 13 hours, at least every 14 hours, at least every' 15 hours, at least every 16 hours, at least every 17 hours, at least every' 18 hours, at least every 19 hours, at least every' 20 hours, at least every 21 hours, at least every' 22 hours, at least every 23 hours, or at least every day
  • Aptamers as described herein may be particularly advantageous over antibodies as they may sustain therapeutic intravitrea! concentrations of drug for longer peri ods of time, thus requiring less frequent administration.
  • the aptamers described herein may have a longer intraocular half-life, and/or sustain therapeutic intravitreal concentrations of drug for longer periods of time than an anti-VEGF-A antibody therapy and can be dosed less frequently.
  • the aptamers of the disclosure are dosed at least once every' 4 weeks (q4w), once every 5 weeks (qSw), once every 6 weeks (q6w), once every 7 weeks (q7w), once every 8 weeks (q8w), once ever ⁇ ' 9 weeks (q9w), once every 10 weeks (qlOw), once every 11 weeks (ql Iw) once every 12 weeks (ql2w), once every 13 weeks (ql3w), once every 14 weeks (ql4w), once every 15 weeks (q15w), once every 16 weeks (ql6w), once every 17 weeks (ql7w), once every 18 weeks (ql 8w), once every 19 weeks (ql9w), once every 20 weeks (q20w), once every 21 weeks (q21w), once every 22 weeks (q22w), once every 23 weeks (q23w), once every 24 weeks (q24w), or greater than once every 24 weeks.
  • a therapeutically effective amount of the aptamer may be administered.
  • A“therapeutically effective amount” or“therapeutically effective dose” are used interchangeably herein and refer to an amount of a therapeutic agent (e.g, an aptamer) that provokes a therapeutic or desired response in a subject.
  • the therapeutically effective amount of the composition may he dependent on the route of administration.
  • a therapeutically effective amount may be about 10 mg/kg to about 100 mg/kg.
  • a therapeutically effective amount may be about 10 pg/kg to about 1000 pg/kg for systemic administration.
  • a therapeutically effective amount can be about 0.01 mg to about 150 mg in about 25 pi to about 100 m ⁇ volume per eye.
  • the ocular disease or disorder may be diabetic retinopathy. In some cases, the ocular disease or disorder may be retinopathy of prematurity. In some cases, the ocular disease or disorder may be central retinal vein occlusion. In some cases, the ocular disease or disorder may be macular edema. In some cases, the ocular disease or disorder may be choroidal
  • the ocular disease or disorder may be neovascular (or wet) age-related macular degeneration. In some cases, the ocular disease or disorder may be myopic choroidal neovascularization. In some cases, the ocular disease or disorder may be punctate inner choroidopathy. In some cases, the ocular disease or disorder may be presumed ocular histoplasmosis syndrome. In some cases, the ocular disease or disorder may be familial exudative vitreoretinopathy. In some cases, the ocular disease or disorder may be
  • the ocular disease or disorder may exhibit elevated levels of one or more isoforms or variants of VEGF-A.
  • the method involves administering a therapeutically effective amount of a composition to a subject to treat an ocular disease.
  • the composition includes one or more aptamers as described herein.
  • the aptamers may bind to and inhibit a function associated with one or more isoforms or variants of VEGF-A as described herein.
  • the methods can be performed at a hospital or a clinic, for example, the pharmaceutical compositions can be administered by a health-care professional. In other cases, the pharmaceutical compositions can be self-administered by the subject. Treatment may commence with the diagnosis of a subject with an ocular disease. In the event that further treatments are necessary, follow-up appointments may be scheduled for the administration of subsequent doses of the composition, for example, administration every 8, 12, 16, 20, or 24 weeks.
  • the methods may involve administering a composition of the disclosure, including one or more anti-VEGF-A aptamers, to a biological system (e.g, biological cells, biological tissue, a subject) to inhibit a function associated with VEGF-A.
  • a biological system e.g, biological cells, biological tissue, a subject
  • the anti-VEGF-A aptamers may bind to the receptor binding face of VEGF-A, or portions thereof.
  • the anti-VEGF-A aptamers may bind to the receptor binding domain of VEGF-A.
  • the methods may be used to prevent or reduce binding of VEGF-A to Fit- 1 , KDR, Nrp-1, or any combination thereof.
  • the methods may be used to inhibit downstream signaling pathways associated with VEGF-A.
  • the aptamers described herein can be generated by any method suitable for generating aptamers.
  • the aptamers described herein are generated by a process known as Systematic Evolution of Ligands by Exponential Enrichment" ("SELEX 1M ").
  • SELEX 1M Systematic Evolution of Ligands by Exponential Enrichment
  • the SELEX f process is described in, e.g, U.S. patent application Ser. No. 07/536,428, filed Jun. 1 1, 1990, now abandoned, U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands", and U.S. Pat. No. 5,270,163 (see, also WO 91/19813) entitled "Nucleic Acid Ligands", each of which are herein incorporated by reference.
  • SELEXTM may be used to obtain aptamers with any desired level of target binding affinity.
  • the SELEX 1M method generally relies as a starting point upon a large library or pool of single stranded oligonucleotides comprising randomized sequences.
  • the oligonucleotides can be modified or unmodified DNA, RNA, or DNA/RNA hybrids.
  • the pool comprises 100% random or partially random oligonucleotides.
  • the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and/or conserved sequence incorporated within randomized sequence.
  • the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and/or conserved sequence at its 5' and/or 3' end which may comprise a sequence shared by all the molecules of the oligonucleotide pool.
  • Fixed sequences are sequences common to oligonucleotides in the pool which are incorporated for a preselected purpose such as, CpG motifs, hybridization sites for PCR primers, promoter sequences for RNA polymerases (e.g., T3, T4, T7, and SP6), sequences to form stems to present the randomized region of the library' within a defined terminal stem structure, restriction sites, or homopolymeric sequences, such as poly A or poly T tracts, catalytic cores, sites for selective binding to affinity columns, and other sequences to facilitate cloning and/or sequencing of an oligonucleotide of interest.
  • conserveed sequences are sequences, other than the previously described fixed sequences, shared by a number of aptamers that bind to the same target.
  • the oligonucleotides of the pool can include a randomized sequence portion as well as fixed sequences necessary for efficient amplification.
  • the oligonucleotides of the starting pool contain fixed 5' and 3' terminal sequences which flank an internal region of 30-50 random nucleotides.
  • the randomized nucleotides can be produced in a number of ways including chemical synthesis and size selection from randomly cleaved cellular nucleic acids. Sequence variation in test nucleic acids can also be introduced or increased by mutagenesis before or during the selection/amplification iterations.
  • the random sequence portion of the oligonucleotide can be of any length and can comprise ribonucleotides and/or deoxyribonucleotides and can include modified or non-natural nucleotides or nucleotide analogs.
  • Typical syntheses carried out on automated DNA synthesis equipment yield 10 4 -l 0 io individual molecules, a number sufficient for most SELEX‘ M experiments. Sufficiently large regions of random sequence in the sequence design increases the likelihood that each synthesized molecule is likely to represent a unique sequence.
  • the starting library of oligonucleotides may be generated by automated chemical synthesis on a DNA synthesizer.
  • random oligonucleotides comprise entirely random sequences; however, in other cases, random oligonucleotides can comprise stretches of nonrandom or partially random sequences. Partially random sequences can be created by adding the four nucleotides in different molar ratios at each addition step.
  • the starting library of oligonucleotides may be RNA, DNA, substituted RNA or DNA or combinations thereof.
  • an RNA library is typically generated by synthesizing a DNA library ' , optionally PCR amplifying, then transcribing the DNA library ' in vitro using T7 RNA polymerase or modified T7 RNA
  • nucleic acid library is then mixed with the target under conditions favorable for binding and subjected to step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity.
  • the SELEX 1M method includes steps of: (a) contacting the mixture with the target under conditions favorable for binding; (b) partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; (c) dissociating the nucleic acid-target complexes; (d) amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids; and (e) reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.
  • the SELEX iM method further comprises the steps of: (i) reverse transcribing the nucleic acids dissociated from the nucleic acid-target complexes before amplification in step (d); and (ii) transcribing the amplified nucleic acids from step (d) before restarting the process.
  • nucleic acid mixture containing a large number of possible sequences and structures, there is a wide range of binding affinities for a given target. Those which have the higher affinity (lower dissociation constants) for the target are most likely to bind to the target.
  • a second nucleic acid mixture is generated, enriched for the higher binding affinity candidates. Additional rounds of selection progressively favor the best ligands until the resulting nucleic acid mixture is predominantly composed of only one or a few sequences. These can then be cloned, sequenced and individually tested as ligands or aptamers for 1) target binding affinity; and 2) ability to effect target function.
  • Cycles of selection and amplification are repeated until a desired goal is achieved. In the most general case, selection/amplification is continued until no significant improvement in binding strength is achieved on repetition of the cycle.
  • the method is typically used to sample approximately iO 14 different nucleic acid species but may be used to sample as many as about l(3 lS different nucleic acid species.
  • nucleic acid aptamer molecules are selected in a 3 to 20 cycle procedure.
  • the aptamers of the disclosure are generated using the SELEX 1M method as described above. In other cases, the aptamers of the disclosure are generated using any modification or variant of the SELEX method.
  • pan-variant specific anti-VEGF-A aptamers bind to the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A.
  • the methods provided herein bias the selection process towards aptamers that selectively bind to the receptor binding face or receptor binding domain of VEGF-A. In some cases, such aptamers do not bind to the heparin binding domain of VEGF-A.
  • the methods may involve incubating an aptamer library with an isoform or variant of VEGF-A which contains a receptor binding domain but does not contain a heparin binding domain.
  • the isoform or variant of VEGF-A is VEGF-Am or VEGF-Ano.
  • the methods involve immobilizing the VEGF-A variant on a solid support in a manner that does not preclude access of the library to the receptor binding face or receptor binding domain of VEGF-A.
  • the methods involve performing the selection in the absence of Ca ⁇
  • methods for screening pan-variant specific anti-VEGF-A aptamers are provided.
  • the methods may involve measuring the interaction of a candidate aptamers with recombinant bead-bound VEGF-A i6 , VEGF-Am . or VEGF-Ano by flow cytometry' ⁇ see Example 1).
  • interaction with each isoform or variant would indicate binding to the receptor binding domain, while only binding to VEGF-Ai 65 would indicate recognition of the heparin binding domain.
  • the methods may involve measuring the ability of the candidate aptamer to inhibit or reduce the interaction of VEGF ⁇ A !65 , VEGF-Am , or VEGF-Ano with KDR by a reduction in FRET signal ⁇ see Example 3). In such cases, efficacy against each variant would indicate binding to the receptor binding domain, while only inhibiting VEGF-Aies would indicate binding to the heparin binding domain. In some cases, the methods may involve measuring the ability of the candidate aptamer to inhibit or reduce VEGF-Aies, VEGF-Am , or VEGF-Ano induced trans autophosphorylation of the intracellular domain of KDR by phospho-KDR AlphaLISA ⁇ ⁇ see Example 4).
  • efficacy against each variant would indicate binding to the receptor binding domain, while only inhibiting VEGF165 would indicate binding to the heparin binding domain.
  • the methods may involve measuring the ability of the candidate aptamer to inhibit or reduce VEGF- Ai65, VEGF-Am , or VEGF-Ano induced gene expression of tissue factor in HUVEC cells as measured by qPCR. In such cases, efficacy against each variant would indicate binding to the receptor binding domain, while only inhibiting VEGFies would indicate binding to the heparin binding domain.
  • the methods may involve measuring the ability of the candidate aptamer to inhibit or reduce VEGF-Aies, VEGF-Am , or VEGF-Ano induced tube formation of GFP-HUVECs in co-culture with human dermal fibroblasts ceils by change in network length or network area ⁇ see Example 5). In such cases, efficacy against each variant would indicate binding to the receptor binding domain, while only inhibiting VEGF-A i65 would indicate binding to the heparin binding domain. In some cases, the methods may involve measuring the ability of the candidate aptamer to inhibit or reduce vascular leakage in a mouse, rat, rabbit, or primate eye following exogenous VEGF-Aies, VEGF-Am .
  • VEGF-Ano challenge by fluorescein angiography and Evans-blue albumin staining efficacy against each VEGF isoform or variant would indicate binding to the receptor binding domain, while only inhibiting VEGF-Ai65 would indicate binding to the heparin binding domain.
  • Example 1A Selection of anti-VEGF-A aptamers
  • Anti-VEGF-A (VEGF) aptamers targeting the receptor binding domain (RBD) of VEGF-A were identified using an N30 library (N30S) comprised of a 30-nucleotide random region flanked by constant regions containing a built-in stem region as depicted in FIG. 1A. The sequence in italics represents the forward and reverse primer binding sites. The built-in stem region is shown in bold and underlined.
  • FIG. IB depicts a representation of the N30S library with the reverse primer hybridized. For nuclease stability, the library was composed of 2’- fluoro-G (2’F GTP) and T -O-methyl (2’OMe) A/C/TJ.
  • FIG. 1C depicts structures of modified nucleotides used to generate the N30S library for selection against target VEGF. For simplicity, the nucleosides, and not the nucleotide triphosphates are shown.
  • the starting library was transcribed from a pool of ⁇ 10 14 double-stranded DNA
  • dsDNA double-stranded DNA molecules.
  • the dsDNA library w'as generated by primer extension using Klenow exo (-) DNA polymerase, the pool forward primer (N30S.F; SEQ ID NO: 9) and a synthetic single- stranded DNA (ssDNA) molecule encoding the reverse complement of the library.
  • the dsDNA w'as subsequently converted to 100% backbone modified RNA via transcription using a mixture of 2’F GXP, 2’OMe ATP/CTP/UTP and a modified phage polymerase in buffer optimized to facilitate efficient transcription. Following transcription, RNAs were treated with DNAse to remove the template dsDNA and were purified.
  • HBD heparin binding domain
  • VEGF-A glycans were oxidized with 100 mM sodium periodate for 30 minutes, and excess periodate was quenched with 1 mM glycerol.
  • Biotinylated VEGF was immobilized on magnetic streptavidin capture beads
  • beads were washed three times with binding buffer SB 1T-Ca ++ (40 mM F1EPES, pH 7.5, 125 mM NaCi, 5 mM KC1, 1 mM MgCl 2 , 0.05% Tween-20) and were resuspended in 50 pi. of recombinant VEGF in SB 1 T Ca and incubated at room temperature for 30 minutes. The beads were subsequently blocked with excess biotin for 10 minutes. The amount of target protein varied with each round (Table 4). The beads w ? ere washed three times with SB 1 T-Ca buffer to remove any unbound protein.
  • SB 1T-Ca ++ 40 mM F1EPES, pH 7.5, 125 mM NaCi, 5 mM KC1, 1 mM MgCl 2 , 0.05% Tween-20
  • the library was added to VEGFno immobilized on beads and incubated at 37°C for 30 minutes on a tube rotary. After 30 minutes, the beads were washed three times using 0.5 ml SB1T-Ca ++ buffer to remove unbound aptamers. After washing, VEGFno-bound aptamers were eluted using 200 pL elution buffer (2M Guanidine-HCl in SBIT buffer) two times (total volume 400 pL). The eluted aptamers, in 400 pL of elution buffer, were ethanol precipitated.
  • the recovered library was converted to DNA by reverse transcription using Super Script 41 IV reverse transcriptase, and the ssDNA was subsequently amplified by PCR.
  • the resulting dsDNA library was subsequently converted back into modified RNA via transcription as described above. DNased, purified RNA was used for subsequent rounds.
  • a negative selection step was included in ail of the subsequent rounds.
  • the pool was prepared as described before and incubated with biotin-saturated beads (in the absence of target protein) for 30 minutes at 37°C in SBIT-Ca ”4 buffer.
  • the beads were pelleted and the supernatant, containing molecules that did not bind to the beads, was incubated with VEGF -labeled beads for an additional 30 minutes at 37°C.
  • the input RNA was kept fixed at 25 picomoles, and the protein target was fixed at 0.5 pg.
  • the target was varied between VEGFi 2i and VEGFno and a solution capture method was implemented. Negative selections were implemented as described above, and positive selection involved incubation with biotinylated target without the presence of beads for 30 minutes at 37°C. For capture, 2 pL of streptavidin beads were washed with 0.5 mL of SB I T-Ca -- and the positive selection was used to resuspend the bead pellet. After a five minute incubation, the beads were washed according to Table 4 prior to elution and precipitation as described above.
  • RNA from each round was first hybridized with reverse complement oligonucleotide composed of 2’OMe RNA labeled with Dyligh ⁇ ⁇ 650 (Dy650 ⁇ N30S.R.OMe, sequence identical to N30S.R (SEQ ID NO: 10)).
  • the library was combined with 1 5-fold molar excess of Dy650- N30S.R.QMe, heated at 90°C for 3 minutes and allowed to cool at room temperature for 15 minutes, after which it was incubated with unlabeled“Negative” beads and beads labelled with VEGF121 in SB IT-Ca " ” buffer supplemented with 0.1% BSA and 1 mg/niL ssDNA final.
  • Example 1C Selection, purification and characterization of clones
  • Aptamer 4.2 (r7-01; SEQ ID NO: 7) was chemically synthesized with 2’-fluoro-G and 2’-0-methyl (2’OMe) A/C/IJ modified phosphoramidites along with a 3’ inverted deoxythymidine and a 5’ C6 disulfide linker, which was conjugated to Dylight ® 650 maleimide (Table 5).
  • the aptamer was purified by reversed phase high-performance liquid chromatography (HPLC) and assayed for activity in the flow cytometry assay described above.
  • HPLC reversed phase high-performance liquid chromatography
  • Example 2 Binding affinity and confirmation of pan-variant specificity with surface plasmon resonance.
  • Aptamer 7 has been demonstrated previously to bind directly to the HBD of VEGF-A at an epitope overlapping the binding site of the VEGF 165 co-receptor neuropilin-1 (Ruckman et al., 1998). Briefly, in the SPR studies, 1.0-2.5 pg/mL of glycan biotinylated VEGF ⁇ s, VEGF , and VEGF U o were diluted in HEPES running buffer (10 mM HEPES, 137.5 rnM NaCl, 5.7 IDM KC1, 1 mM MgCl 2, 1 mM CaCl ?.
  • Regeneration conditions of chips were optimized based on the protein immobilized and were effected with either 50 mM NaOH for 30 seconds or 2M guanidine HCi for 2 x 40 seconds.
  • aptamers were subsequently ran in an 11 -point, 2-fold dose response with Aptamer 26 (SEQ ID NO: 7) starting at a top concentration of 1.0 mM, and Aptamer 7 starting at 500 nM.
  • inG and inA are 2’OMe modified RNA
  • fC and fU are 2’F modified RNA
  • C6NH 2 is a hexylainine linker
  • idT is an inverted deoxythymidine residue.
  • aptamers identified in the selection described herein from previously described aptamers to VEGF-A, such as Aptamer 7, which recognize an epitope of VEGF-A contained within the HBD, and thus do not bind the non-HBD bearing variants VEGFno and VEGF .
  • the SPR data in Example 2 demonstrates that Aptamer 26 binds to the RBD of VEGF- A.
  • the mechanism of action of Aptamer 26 was interrogated by testing the ability of Aptamer 26 to directly inhibit KDR binding to either VEGFies or VEGFm as compared to a clone of an anti- VEGF antibody (Ferrara, NYCe, et al.
  • aptamers were heated at 90°C for 3 minutes and allowed to cool to room temperature for a minimum of 5 minutes
  • Aptamers and anti-VEGF antibody were serial diluted in a polypropylene plate and 5 pL was transferred to a white low volume 384-well Optiplate (Perkin Elmer).
  • a solution of VEGFies (Aero BioSystems) or VEGFm (Aero BioSystems) that was giycan biotinylated was prepared and added to the assay plate containing aptamers or anti- VEGF antibody to yield a fi nal assay concentration of 2 nM.
  • FIG. 4A and FIG. 4B demonstrate that Aptamer 26 (SEQ ID NO: 7) and the anti-VEGF mAh directly blocked the interaction of VEGF-A (VEGF 1 ⁇ 25 or VEGFm) with KDR.
  • VEGF-A VEGF 1 ⁇ 25 or VEGFm
  • Aptamer 7 shows no activity in this assay against VEGF 65 , demonstrating that it does not directly inhibit the interaction between VEGF-A and its receptor.
  • Aptamer 7 also showed no inhibition of the interaction between this variant of VEGF-A and KDR.
  • Aptamer 26 blocked the interaction of VEGF 1 ⁇ 25 with KDR with an IC 50 of 1.8 ⁇ 1.3 nM and the interaction of VEGFm with KDR with an IC 50 of 19 ⁇ 19 nM, consistent with the affinity of Aptamer 26 for the respective VEGF-A variants.
  • the anti-VEGF mAb also inhibited the interaction of VEGFies and VEGF 121 with an IC50 of 1.0 ⁇ 0.25 nM and 0.75 ⁇ 0.12 nM, respectively, consistent with its affinity for these VEGF-A variants.
  • FIG. 4A and FIG. 4B demonstrates that these aptamers bind to an epitope consisting of or overlapping with the receptor binding face contained within the RBD of VEGF-A, and thus directly block the interaction of VEGF-A with its cognate receptor.
  • the absence of inhibition of the interaction of VEGF 165 with KDR by Aptamer 7 is consistent with the literature which demonstrates that this aptamer engages VEGF-A by binding to an epitope within the HBD and does not directly block binding of VEGF-A to its receptor, KDR (Ruckman, 1998; Lee, 2005; Ng, 2006).
  • the lack of inhibition of VEGF activity by Aptamer 7 is consistent with the SPR binding data described in Example 2 and the published literature (Ruckman, 1998). Table 8. Anti-VEGF-A inAb
  • HEK293 cells engineered to stably overexpress KDR were plated overnight in collagen coated 96 well plates at 50k cells/well. Aptamers were heated to 90°C for 3 minutes and cooled to room temperature for a minimum of 5 minutes.
  • VEGF (Biolegend) and VEGF, 65 (R.&D Systems) were prepared at 20 nM in DMEM + 0.8% FBS, a 20X stock for the reaction 15 pL of VEGF was added to 15 pL titrated aptamer in a polypropylene plate and diluted to 300 pL with IS buffer (10 niM Iris pH 7.5; 100 mM NaCl; 5.7 mM KC1: 1 mM MgCh; 1 mM CaCh). The aptamer/VEGF mixture was incubated at 37°C for 30 minutes, after which 100 pL was added for 5 minutes at 37°C to the cells in an incubator with 5% CO2.
  • Treatments were aspirated from cells, and cells were lysed with 100 pL cold lysis buffer (20 mM Tris-HCl, pH 7.5, 150 niM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5 mM sodium
  • IC50 values for Aptamer 26 were 1.5 ⁇ 0.6 nM and 20 ⁇ 5.2 nM, for inhibition of phosphorylation of KDR by VEGF
  • Aptamer 7 only partially inhibited phosphorylation of KDR by VEGF165 (maximum inhibition of -80%), with an IC50 of 2.2 nM (FIG. 5A).
  • VEGF 165 mediates formation of complexes containing VEGFRD2 and neuropil in ⁇ 1 that enhance VEGF 165 ⁇ receptor binding.
  • Journal of Cellular Biochemistry 85.2 (2002): 357-368 This result supports the conclusion that binding of Aptamer 26 to the receptor binding face within the RBD of VEGF- A variants confers inhibition of VEGF- A signaling. Further, these results differentiate the mechanism of action of Aptamer 26 from previously described HBD binding aptamers such as Aptamer 7.
  • VEGF is thought to play an important role in inducing angiogenesis in both normal tissues and diseased pathologies (Ferrara, Orlandoe. "Vascular endothelial growth factor: basic science and clinical progress " Endocrine Reviews 25.4 (2004): 581-611.) Therefore, the ability of candidate VEGF antagonists to inhibit VEGF -induced angiogenesis may be evaluated in an angiogenesis assay. In vitro models have been established to study VEGF-stimulated
  • angiogenesis To determine if Aptamer 26 can inhibit VEGF -A induced angiogenesis, Aptamer 26 was tested for its ability to inhibit angiogenesis via binding to either VEGF ⁇ 5 or VEGF .
  • fibroblasts and endothelial ceils leads to the formation of tubes after stimulation with either VEGF gs or VEGF m .
  • ATCC primary human dermal fibroblasts
  • ATCC primary human dermal fibroblasts
  • GFP-infected HUVEC cells were then plated at 10k cells/well with 100 pL HUVEC growth media (without VEGF supplement, PromoCeil) and incubated at room temperature for 1 hour.
  • the plate was then placed in the IncuCyte ®' Zoom (Essen Bioscience) set to the default angiogenesis analysis definition with imaging programmed every 4 hours. After 24 hours of incubation (Day 1), old media was removed and replaced with 150 pL/well of IncuCyte’ Angiogenesis Prime Kit Optimized Assay Medium (Essen Bioscience). After 24 hours (Day 2), media was changed with fresh media with or without treatment. Treatment included 400 pM VEGF 165 or 800 pM
  • VEGF Hi premixed with or without a dilution of aptamer were carried out for 6 days with media changes and subsequent treatments performed on Days 4 and 6.
  • Network length (mm/mm 3 ) of the tubes formed from the GFP-HUVEC cells was measured as a primary' indication of angiogenesis. Percent inhibition was calculated by subtracting assay media background from each value and normalizing to VEGF-A only controls. The values were fit using a four-parameter non-linear fit in GraphPad Prism Version 7.0. IC 50 values for VEGF 165 and VEGFm were calculated at a time point that reflected an EC 90 -EC 100 of the VEGF-A alone induction of tube formation. Representative dose response curves of Aptamer 26, shown in FIG. 6A and FIG.
  • Example 6 Long-acting pan-variant specific anti- VEGF-A aptamer for treatment of retinal diseases by intravitreal (IVT) administration.
  • pan-variant specific anti -VEGF-A inhibitors that can be dosed by IVT administration and maintain a therapeutic effect for 12 weeks or greater.
  • a patient is diagnosed with wet age-related macular degeneration, diabetic retinopathy, diabetic macular edema, myopic choroidal neovascularization, or macular edema following retinal vein occlusion (RVO).
  • the patient is treated with a therapeutically effective dose of a PEGyiated-anti -VEGF-A aptamer by intravitreal (IVT) injection.
  • the aptamer is Aptamer 26 or a more potent derivative of Aptamer 26 and inhibits VEGF-A by binding the receptor binding domain of VEGF-A and is thus pan-variant specific for inhibition of VEGF-A induced angiogenesis.
  • the aptamer maintains a concentration in the vitreous above the IC90 of the aptamer, that is, the aptamer maintains a concentration in the vitreous that would allow for inhibition of at least 90% of the VEGF-A activity in the vitreous.
  • IC90 of an aptamer can be assumed to be 10-fold higher than the IC50.
  • a PEGylated anti-VEGF-A aptamer such as Aptamer 26 or a more potent derivative
  • a peak aptamer concentration of approximately 16 mM in the vitreous would be expected.
  • a PEGylated anti- VEGF-A aptamer with a vitreous half-life of 10 days and an affinity for VEGF-A of 0.2 nM, would provide a therapeutic level of VEGF-A inhibition for approximately 18-19 weeks. Thus, the patient would need to be treated only two to three times a year to maintain a therapeutic level of VEGF-A inhibition (FIFE 8A).
  • a PEGylated anti-VEGF-A aptamer with a vitreous half-life of 10 days and an affinity for VEGF-A of 2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 14 weeks (FIG. 8A). Thus, the patient would need to be treated only three times a year to maintain a therapeutic level of VEGF-A inhibition.
  • the vitreous half-life of the aptamer is greater than 10 days.
  • a PEGylated anti-VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 0.2 nM would provide a therapeutic level of VEGF-A inhibition for
  • FIG. 8A A PEGylated anti-VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 21 weeks (FIG. 8A).
  • FIG. 8A A PEGylated anti-VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 21 weeks.
  • the patient would need to be treated only two to three times a year to maintain a therapeutic level of ⁇ EGF-A inhibition.
  • a PEGylated anti -VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 20 nM would provide a therapeutic level of VEGF-A inhibition for approximately 14 weeks (FIG. 8A).
  • the patient would need to be treated only three times a year to maintain a therapeutic level of VEGF-A inhibition.
  • PEG moieties are utilized that allow for higher concentration aptamer formulations.
  • a PEGylated anti-VEGF-A aptamer such as Aptamer 26 or a more potent derivative, is presented as an isotonic, neutral pH formulation at a concentration of 50 mg/rnL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 100 pL volume for a maximum dosage of 5 mg/eye.
  • a peak aptamer concentration of approximately 82 mM in the vitreous would be expected.
  • a PEGylated anti-VEGF-A aptamer with a vitreous half-life of 10 days and an affinity for VEGF- A of 0.2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 22 weeks.
  • the patient would need to be treated only two to three times a year to maintain a therapeutic level of VEGF-A inhibition (FIG. 8B).
  • a PEGylated anti- VEGF-A aptamer with a vitreous half-life of 10 days and an affinity for VEGF-A of 2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 17 to 18 weeks (FIG. 8B).
  • the patient would need to be treated only two to three times a year to maintain a therapeutic level of VEGF- A inhibition.
  • a PEGylated anti -VEGF-A aptamer with a vitreous half-life of 10 days and an affinity for VEGF-A of 20 nM would provide a therapeutic level of VEGF-A inhibition for approximately 12 to 13 weeks (FIG. 8B).
  • the patient would need to be treated only four to five times a year to maintain a therapeutic level of VEGF-A inhibition
  • the vitreous half-life of the aptamer is greater than 10 days.
  • a PEGylated anti -VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 0.2 nM would provide a therapeutic level of VEGF-A inhibition for
  • FIG. 8B A PEGylated anti -VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 26 w ⁇ eeks (FIG. 8B).
  • FIG. 8B A PEGylated anti -VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 26 w ⁇ eeks.
  • FIG. 8B A PEGylated anti -VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 26 w ⁇ eeks
  • a PEGylated anti- VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 20 nM would provide a therapeutic level of VEGF-A inhibition for approximately 19 weeks (FIG. 8B).
  • the patient would need to be treated only two to three times a year to maintain a therapeutic level of VEGF-A inhibition.
  • the patient is treated at a dosing interval to maintain a drug concentration in the vitreous above its IC90, and not more than once every 12 weeks.
  • the patient is assessed for mean change in visual acuity from baseline, improvement of >5 or more letters in visual acuity testing, central foveai thickness and change in central foveai thickness from baseline.
  • Treatment of the patient with the pan-variant specific anti -VEGF-A aptamer results in an improvement in visual acuity from pre treatment baseline, a gain of 5 or more letters in visual acuity testing, and a marked reduction in central foveal thickness.
  • Example 7 Aptamer engineering for systemic safety hi adult IVT administration.
  • a patient is diagnosed with macular edema following RVO.
  • the patient is treated with a therapeutically effective dose of a PEGylated-anti-VEGF-A, such as Aptamer 26, by intravitreal (IVT) injection.
  • the aptamer inhibits VEGF-A by binding to the receptor binding face of VEGF-A and is thus pan-variant specific for inhibition of VEGF-A induced angiogenesis.
  • Systemic and tissue suppression of VEGF-A activity is contraindicated when a patient has a risk of arterial thromboembolic events (ATEs; USPI Lucentis 03/2018).
  • the IC 90 and IC10 of an aptamer can be assumed to be 10-fold higher and 10-fold lower than the IC50 respectively.
  • an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 30 mg/mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 100 pL volume for a maximum dosage of 3 mg/eye.
  • a peak aptamer concentration of 60mM in the vitreous would be expected.
  • the vitreous concentration of aptamer at day 30 would be 7.5 mM.
  • the safety profile is achieved by lowering the injected dose, ensuring a lower maximum systemic and tissue exposure while diminishing the duration of effect in the vitreous.
  • an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 6 nig/mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 50 mI_. volume for a dosage of 0.3 mg/eye. Assuming an
  • oligonucleotide molecular weight of 12.5 kDa and an adult human vitreous volume of 4 mL a peak aptamer concentration of 6 mM in the vitreous would be expected. Assuming a vitreous half-life of 10 days and a desired therapeutic window of 30-day target suppression, the vitreous concentration of aptamer at day 30 would be 750 nM. Based on serum and tissue exposure of 0.5 nM following a 0.3 mg IVT injection of Aptamer 9 (see Table 6) in adult humans
  • aptamers with an IC50 ranging from 5 to 75 nM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the IC10 in serum and tissues (FIG. 9B).
  • an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 60 mg/rnL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at 100 pL volume for a maximum dosage of 6 mg/eye.
  • a peak aptamer concentration of 120 mM in the vitreous would be expected.
  • the vitreous concentration of aptamer at day 30 would be 30 mM.
  • the maximum serum and tissue exposure of a 6 mg/eye dose with an improved PEG moiety would be 2.5 nM.
  • aptamers with an IC50 ranging from 25 nM to 3 mM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the IC10 in serum and tissues (FIG, 9C).
  • the PEG moiety is attached to the aptamer by a linker that is
  • an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 30 mg/mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 100 mE volume for a maximum dosage of 3 mg/eye.
  • a peak aptamer concentration of 60 mM in the vitreous would be expected.
  • the vitreous concentration of aptamer at day 30 would be 7 5 mM. Due to the metabolic instability of the active compound, a 3 mg/eye dose would yield a maximum serum and tissue exposure of 0.5 nM.
  • aptamers with an IC 50 ranging from 5 to 750 nM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the IC o in serum and tissues (FIG. 9D).
  • the aptamer possesses differential potency for VEGF-A variants (e.g., VEGFies and VEGF ⁇ ).
  • VEGF-A variants e.g., VEGFies and VEGF ⁇ .
  • systemic and tissue target suppression is defined by the aptamer’ s IC 10 against the highest potency VEGF-A variant, while maintenance of therapeutic effect in the vitreous is defined by the aptamer’ s IC 90 against the lowest potency VEGF-A variant.
  • the patient is treated once every 4 w ? eeks or once every 8 weeks or once every ' 16 weeks. After three months of treatment and every month thereafter, the patient is assessed for mean change in visual acuity from baseline, improvement of 5 or more letters in visual acuity testing, central foveal thickness and change in central foveal thickness from baseline.
  • Treatment of the patient with the pan-variant specific anti -VEGF-A aptamer results in an improvement in visual acuity from pre-treatment baseline, a gain of 5 or more letters in visual acuity testing, and a marked reduction in central foveal thickness.
  • a patient is diagnosed with zone I stage 3+ (i.e., stage 3 with plus disease) retinopathy of prematurity (ROP; Mintz-Hittner, 2011).
  • the patient is treated with a therapeutically effective dose of a PEGylated-anti -VEGF-A aptamer by IVT injection.
  • the aptamer inhibits VEGF-A by binding the receptor binding face of VEGF-A and is thus pan variant specific for inhibition of VEGF-A induced angiogenesis.
  • Systemic and tissue suppression of VEGF-A activity is contraindicated for infants and can potentiate the onset of adverse neurodevelopmental outcomes (Lien, 2016).
  • an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 30 mg/mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 25 pL volume for a maximum dosage of 0.75 mg/eye.
  • oligonucleotide molecular weight 12.5 kDa and an infant human vitreous volume of l mL
  • a peak aptamer concentration of 60 mM in the vitreous would be expected.
  • the vitreous concentration of aptamer at day 30 would be 940 nM.
  • rabbits (-2.5-4 kg) are assumed to be a relevant predictor of exposure in human infants. Based on serum and tissue exposure of 4 5 nM following a 1.5 mg IVT injection of a PEGylated aptamer in rabbits, the maximum serum and tissue exposure of a 0.75 mg/eye dose in infants is estimated to be 2.5 nM. Following from this reasoning, aptamers with an IC50 ranging from 25 to 95 nM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the IC10 in serum and tissues (FIG. 10A).
  • the safety profile is achieved by lowering the injected dose, ensuring a lower maximum systemic and tissue exposure while diminishing the duration of effect in the vitreous.
  • an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 10 ng/'mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 25 pL volume for a dosage of 0.25 mg/eye. Assuming an
  • oligonucleotide molecular weight of 12.5 kDa and an infant human vitreous volume of 1 ml a peak aptamer concentration of 20 mM in the vitreous would be expected.
  • the vitreous concentration of aptamer at day 30 would be 310 nM.
  • rabbits (-2.5-4 kg) are assumed to be a relevant predictor of exposure in human infants.
  • aptamers with an IC 50 ranging from 7.5 to 3 1 nM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the IC o in serum and tissues (FIG. 10B).
  • an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 60 mg/mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 25 pL volume for a maximum dosage of 1.5 mg/eye.
  • a peak aptamer concentration of 120 pM in the vitreous would be expected.
  • the vitreous concentration of aptamer at day 30 would be 15 pM.
  • rabbits -2 5-4 kg are assumed to be a relevant predictor of exposure in human infants. Based on serum and tissue exposure of 4.5 nM!
  • aptamers with an IC50 ranging from 7.5 nM to 1.5 mM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the ICio in serum and tissues (FIG, IOC).
  • the PEG moiety is attached to the aptamer by a linker that is
  • an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 30 mg/mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 25 mE volume for a maximum dosage of 0.75 mg/eye.
  • a peak aptamer concentration of 60 mM in the vitreous would be expected.
  • the vitreous concentration of aptamer at day 30 would be 950 nM. Due to the metabolic instability of the active compound a 0.75 mg/eye dose would yield a maximum serum and tissue exposure of 0.5 nM.
  • aptamers with an IC 50 ranging from 5 to 95 nM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the IC i0 in serum and tissues (FIG. 10D).
  • the aptamer possesses differential potency for VEGF-A variants (e.g., VEGFies and VEGFm).
  • VEGF-A variants e.g., VEGFies and VEGFm.
  • systemic and tissue target suppression is defined by the aptamer’ s IC 10 against the highest potency VEGF-A variant, while maintenance of therapeutic effect in the vitreous is defined by the aptamer’ s IC 90 against the lowest potency VEGF-A variant.
  • the patient is treated once every 4 weeks or once every 8 weeks.
  • Treatment of the patient with the pan-variant-specific anti- VEGF-A aptamer results in an absence of recurrence of stage 3+ ROP in one or both eyes in zone I at 54 weeks’ postmenstrual age.
  • These clinical outcomes are significant improvements when compared to an untreated patient and to clinical outcomes when compared to a patient who has been treated with conventional laser therapy.
  • the patient’s risk of adverse neurodevelopmental outcomes is substantially reduced when compared to a patient who has been treated with an anti- VEGF-A antibody fragment therapy due to the reduced exposure of the patient to systemic and tissue inhibition of VEGF-A.
  • G-quartets are a unique, often potassium- dependent nucleic acid structure in which four guanine bases associate through Hoogsteen hydrogen bonding to form a planar guanine tetrad structure, and two or more of the guanine tetrads stack on top of each oilier to form a G-quartet.
  • aptamers Vap7, VEaP121, or 3R02 w ? ere capable of inhibiting VEGF- A activity were tested for their ability to inhibit KDR phosphorylation induced by either VEGF-Aies or VEGF-Am as compared to a clone of an anti-VEGF antibody (Table 8).
  • the KDR phosphorylation assay was performed as described in Example 4, except that the concentration of VEGF-A 155 or VEGF-Am used to induce KDR phosphorylation was 1 nM, and an ELISA was used to detect phosphorylation of KDR.
  • aptamers Prior to use, aptamers were renatured as described by Nonaka et al.
  • TS buffer pH 7.5 10 mM Tris-HCl; 100 mM NaCl; 5.7 mM KC1; 1 mM MgCC; 1 mM CaCI ;
  • Aptamers were tested at a final concentration of 500 nM, and anti-VEGF mAh at a final concentration of 160 nM.
  • TS buffer pH 7.5 10 mM Tris-HCl; 100 mM NaCl; 5.7 mM KC1; 1 mM MgCC; 1 mM CaCI ;
  • Aptamers Vap7, VEaP121 and 3R02 showed no inhibition of VEGF-A induced phosphorylation of KDR.

Abstract

The application discloses methods and compositions for inhibiting functions associated with vascular endothelial growth factor-A (VEGF-A). The methods and compositions may involve the use of pan-variant specific aptamers for binding to VEGF-A, and preventing or reducing association of VEGF-A with Flt-1, KDR, or Nrp-1. The methods and compositions may include one or more aptamers that bind to receptor binding face of VEGF-A. The methods and compositions may include one or more aptamers that bind to a receptor binding domain of VEGF-A. The application further provides anti-VEGF-A aptamers for the treatment of ocular diseases or disorders.

Description

COMPOSITIONS AND METHODS FOR INHIBITING VASCULAR ENDOTHELIAL
GROWTH FACTOR
BACKGROUND OF THE INVENTION
[0001] Visual impairment is a national and global health concern that has a negative impact on physical and mental health. The number of people with visual impairment and blindness is increasing due to an overall aging population. Visual impairment and blindness can be caused by any one of a large number of eye diseases and disorders affecting people of all ages.
[0002] Vascular endothelial growth factor- A (VEGF-A) is thought to be the most significant regulator of angiogenesis in the VEGF family. VEGF-A promotes growth of vascular endothelial cells which leads to the formation of capillary -like structures and may be necessary for the survival of newly formed blood vessels. VEGF-A is thought to play a role in various ocular diseases and disorders. Previous attempts at developing aptamers that inhibit VEGF-A have proven difficult because such aptamers have been unable to target multiple isoforms and variants of VEGF-A. Therefore, there is an un-rnet need for pan-variant specifi c aptamers that demonstrate high specificity and potency towards multiple isoforms and variants of VEGF-A. These needs may be met by the aptamers provided in the present disclosure.
SUMMARY OF THE INVENTION
[0003] In one aspect, an aptamer is provided comprising a nucleic acid sequence that selectively binds to and inhibits at least one of VEGF -A and VEGF-Ano, wherein less than 50% of pyrimidines present in said nucleic acid sequence comprise a C-5 modified pyrimidine. In some cases, less than 25% of pyrimidines present in said nucleic acid sequence comprise a C-5 modified pyrimidine. In some cases, less than 10% of pyrimidines present in said nucleic acid sequence comprise a C-5 modified pyrimidine. In some cases, the nucleic acid sequence does not comprise any C-5 modified pyrimidines. In some cases, the C-5 modified pyrimidine comprises a C-5 modified cytosine or a C-5 modified uridine. In some cases, the C-5 modified pyrimidine comprises a C-5 hydrophobic modification.
[0004] In another aspect, an aptamer is provided comprising a nucleic acid sequence that selectively binds to and inhibits at least one of VEGF -A and VEGF-Ano, wherein less than 100% of uridines present in said nucleic acid sequence comprise a C-5 modified uridine. In some cases, less than 50% of uridines present in said nucleic acid sequence comprise a C-5 modified uridine. In some cases, less than 25% of uridines present in said nucleic acid sequence comprise a C-5 modified uridine. In some cases, less than 10% of uridines present in said nucleic acid sequence comprise a C-5 modified uridine. In some cases, no uridines present in said nucleic acid sequence comprise a C-5 modified uridine. In some cases, the C-5 modified uridine comprises a C-5 hydrophobic modification. In some cases, the C-5 modified uridine is selected from the group consisting of: 5-(N-benzylcarboxyamide)-2'-deoxyuridine (BndU), 5-(N- benzyicarboxyamide)-2'-Q-methyiuridine, 5-(N-benzylcarboxyamide)-2'-fluorouridine, 5-QNJ- phenethylcarboxyamide)-2'-deoxyuiidine (PEdU), 5-(N-thiophenylmethylcarboxyamide)-2'- deoxyuridine (ThdU), 5-(N-isobutylcarboxyamide)-2'-deoxyuridine (IBud!J), 5-(N- tyrosylcarboxyamide)-2'-deoxyuridine (TyrdU), 5-(N-3,4-methylenedioxybenzylcarboxyamide)- 2'-deoxyuridine (MBndU), 5-(N-4-fluorobenzylcarboxyamide)-2'-deoxyuridine (FBndU), 5-(N- 3-phenylpropylcarboxyamide)-2'-deoxyuridine (PPdU), 5-(N-imidizolylethylcarboxyamide)-2'- deoxyuridine (ImdU), 5-(N-isobutylcarboxyamide)-2'-0-methyluridine, 5-(N- isobutyIcarboxyamide)-2'-fluorouridine, 5-(N-tryptaminocarboxyamide)-2'-deoxyuridine (TrpdU), 5-(N-R-threoninylcarboxyamide)-2'-deoxyuridine (ThrdU), 5-(N- tryptaminocarboxyamide)-2'-0-methyluridine, 5-CN-tryptaminocarboxyamide)-2'-fluorouiidine, 5-(N-[l-(3-trimethylamonium)propyl]carboxyamide)-2'-deoxyuridine chloride, 5-(N- naphthylmethylcarboxyamide)-2'-deoxyuridine (NapdU), 5-(N-naphthylmethylcarboxyamide)- 2'-0-methyluridine, 5-(N-naphthylmethylcarboxyamide)-2'-fluorouridine, 5-(N-[l-(2,3- dihydroxypropyl)]carboxyamide)-2'-deoxyuridine), 5-(N-2-naphthylme†hylcarboxyamide)-2'- deoxyuridine (2NapdU), 5-(N-2-naphthylmethylcarboxyamide)-2'-0-methyluridine, 5-(N-2- naphthylmethyiearboxyamide)-2'-fluorouridine, 5-(N-l-naphthylethylcarboxyamide)-2'~ deoxyuridine (NEdU), 5-(N- 1 -naphthyl ethylcarboxyamide)-2'-0-methyJuri dine, 5-(N- 1 - naphthylethylearboxyamide)-2'-fluorouridine, 5-(N-2-naphthylethylcarboxyamide)-2'- deoxyuridine (2NEdU), 5-(N-2-naphthylethylcarboxyamide)-2'-0-methyluridine, 5-(N-2- naphthyl ethyl carboxyamide)-2'-fluorouridine, 5-(N-3-benzofuranylethylcarboxyamide)-2'- deoxyuridine (BFdU), 5-(N-3-benzofuranylethylcarboxyamide)-2'-0-methyluridine, 5-(N-3- benzofuranylethylcarboxyamide)-2i-fluorouridine, 5-(N-3-benzothiophenylethylcarboxyamide)- 2'-deoxyuridine (BTdU), 5-(N-3-benzothiophenylethylcarboxyamide)-2'-0-methyluridine, and 5-(N-3-benzothiophenylethyiearboxyamide)-2'-fluorouridine. In some eases, the C-5 modified uridine is 5-(N-naphthylmethylcarboxyamide)-2'-deoxyuridine (NapdU).
[0005] In some cases, any aptamer of the preceding selectively binds to a receptor binding face or receptor binding domain of VEGF-Am or VEGF-Ano. In some cases, the receptor binding domain comprises at least one of residues 1 -109 of SEQ ID NO: 1. In some cases, the receptor binding domain comprises at least one of residues Phel7, Ile43, Ile46, Glu64, Gln79, Ile83, Lys84, Pro85, Arg82, His86, Asp63, and Glu67 of SEQ ID NO: 1 In some cases, any aptamer of the preceding inhibits VEGF-Am, VEGF-Ano, or both, with an IC50 of less than about 50 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation
AiphaLISA* assay, or an in vitro model of VEGF-A-induced angiogenesis. In some cases, any aptamer of the preceding inhibits VEGF-Am, VEGF-Ano, or both, with an IC50 of less than about 25 nM as measured by a VEGF-A:KDR competition binding assay, a KDR
phosphorylation AlphaLISA® assay, or an in vitro model of VEGF-A-induced angiogenesis. In some cases, any aptamer of the preceding inhibits VEGF-Am, VEGF-Ano, or both, with an IC50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation AlphaLISA8, assay, or an in vitro model of VEGF-A-induced angiogenesis. In some cases, any aptamer of the preceding inhibits VEGF-Am, VEGF-Ano, or both, with an IC50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation Alpha! . ISA " assay, or an in vitro model of VEGF-A-induced angiogenesis. In some cases, any aptamer of the preceding inhibits VEGF-Am, VEGF-Ano, or both, with an IC50 of less than about 1 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation AlphaLISA® assay, or an in vitro model of VEGF-A-induced angiogenesis. In some cases, any aptamer of the preceding binds to VEGF-A , VEGF-Ano, or both, with a Kd of less than about 50 nM as measured by surface plasmon resonance assay. In some cases, any aptamer of the preceding binds to VEGF-Am, VEGF-Ano, or both, with a Kd of less than about 25 nM as measured by surface plasmon resonance assay. In some cases, any aptamer of the preceding binds to VEGF~A , VEGF-Apo, or both, with a Kd of less than about 10 nM as measured by surface plasmon resonance assay. In some cases, any aptamer of the preceding binds to VEGF-Am, VEGF-Ano, or both, with a Kd of less than about 5 nM as measured by surface plasmon resonance assay. In some cases, any aptamer of the preceding binds to VEGF- Am, VEGF-Ano, or both, with a K of less than about 1 nM as measured by surface plasmon resonance assay. In some cases, any aptamer of the preceding selectively binds to and inhibits at least one ofVEGF-A165, VEGF-Ai89, and VEGF-A2o6. In some cases, any aptamer of the preceding inhibits or reduces an interaction of VEGF-A with KDR. In some cases, any aptamer of the preceding inhibits or reduces VEGF-A-induced KDR phosphorylation. In some cases, any aptamer of the preceding comprises RNA or sugar-modified RNA. In some cases, any aptamer of the preceding comprises DNA or sugar-modified DNA. In some cases, at least 50% of said nucleic acid sequence of any aptamer of the preceding comprises sugar-modified nucleotides. In some cases, 100% of said nucleic acid sequence of any aptamer of the preceding comprises sugar-modified nucleotides. In some cases, the sugar-modified nucleotides comprise a 2’F- modified nucleotide, a 2’OMe-modified nucleotide, or both. In some cases, the sugar-modified nucleotides are selected form the group consisting of: 2’F-G, 2’OMe-G, 2’OMe-U, 2’OMe-A, 2’OMe-C, and any combination thereof. In some cases, any aptamer of the preceding further comprises a 3’ terminal inverted deoxythymidine. In some cases, any aptamer of the preceding comprises a nuclease-stabilized nucleic acid backbone. In some cases, the nucleic acid sequence of any aptamer of the preceding comprises from about 30 to about 90 nucleotides, wherein said nucleotides are unmodified nucleotides, modified nucleotides, or a combination of modified nucleotides and unmodified nucleotides. In some cases, any aptamer of the preceding is conjugated to a polyethylene glycol (PEG) molecule. In some cases, the PEG molecule has a molecular weight selected from the group consisting of: less than about 5 kDa, less than about 10 kDa, less than about 20 kDa, less than about 40 kDa, less than about 60 kDa, and less than about 80 kDa.
[0006] In another aspect, an aptamer of any of the preceding is provided for use in treating an ocular disease or disorder in a subject in need thereof. In some cases, one or more symptoms of said ocular disease or disorder are treated.
[0007] In another aspect, a method is provided for treating an ocular disease or disorder in a subject in need thereof, comprising administering to said subject an aptamer of any of the preceding, thereby treating said ocular disease or disorder. In some cases, the ocular disease or disorder is selected from the group consisting of: diabetic retinopathy, retinopathy of
prematurity, central retinal vein occlusion, macular edema, choroidal neovascularization, neovascular age-related macular degeneration, myopic choroidal neovascularization, punctate inner choroidopathy, ocular histoplasmosis syndrome, familial exudative vitreoretinopathy, and retinoblastoma. In some cases, the ocular disease or disorder exhibits elevated levels of VEGF- A
[0008] In yet another aspect, any aptamer of the preceding is provided for use in a formulation of a medicament for treatment of an ocular disease or disorder.
[0009] In yet another aspect, any aptamer of the preceding is provided for use for treatment of an ocular disease or disorder.
[0010] In yet another aspect, a method is provided for modulating vascular endothelial growth factor-A (VEGF-A) in a biological system, said method comprising: administering to said biological system any aptamer of the preceding, thereby modulating VEGF-A in said biological system. In some cases, the biological system comprises a biological tissue or biological cells. In some cases, the biological system is a subject. In some cases, the subject is a human. In some cases, the modulating comprises inhibiting a function associated with VEGF-A. In some cases, the modulating comprises preventing or reducing an association of VEGF-A with one or more of Flt-1, KDR, or Nrp-1.
INCORPORATION BY REFERENCE
[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The novel features of the invention are set forth with particularity in the appended claims A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0013] FIG. 1A depicts a non-limiting example of a primary sequence of an aptamer library suitable for screening for aptamers that target VEGF-A.
[0014] FIG. IB depicts a non-limiting example of a secondary7 structure of the aptamer library depicted in FIG. 1A, with a reverse primer hybridized thereto. {0015] FIG. 1C depicts a non-limiting example of chemical structures of 2’-modified nucleotides that may be used in the VEGF-A aptamer selection process.
[0016] FIG. 2A depicts a non-limiting example of flow cytometry' data demonstrating the interaction of various anti -VEGF-A aptamer selection rounds with unlabeled“negative” beads.
[0017] FIG. 2B depicts a non-limiting example of flow cytometry data demonstrating the interaction of various anti -VEGF-A aptamer selection rounds with VEGFm-functionalized beads.
[0018] FIG. 2C depicts a non-limiting example of flow cytometry7 data demonstrating the dose- dependent interaction of various anti-VEGF-A aptamer selection rounds with VEGF no- functionalized beads.
[0019] FIG. 2D depicts a non-limiting example of flow cytometry data demonstrating the dose- dependent interaction of various anti-VEGF-A aptamer selection rounds with VEGFm- functionalized beads.
[0020] FIG, 2E depicts a non-limiting example of flow cytometry data demonstrating the interaction of various anti-VEGF-A aptamer selection rounds with VEGF -functionalized beads in the presence of a non-bead bound VEGFm decoy.
[0021] FIG. 3 depicts a non-limiting example of flow cytometry data demonstrating the dose- dependent interaction of an anti-VEGF-A aptamer with VEGF!65- and VEGFm-functionalized beads.
[0022] FIG, 4A demonstrates a non-limiting example of the inhibition of VEGF 155 binding to KDR by an anti-VEGF-A aptamer of the disclosure. Compounds were tested in a dose- dependent fashion to determine an IC50 against VEGF 105.
[0023] FIG. 4B demonstrates a non-limiting example of the inhibition of VEGFm binding to KDR by an anti-VEGF-A aptamer of the disclosure. Compounds were tested in a dose- dependent fashion to determine an IC50 against VEGF 121.
[0024] FIG. 5A demonstrates a non-limiting example of the inhibition of V EGF r.s-sti mill ated KDR phosphorylation by an anti-VEGF-A aptamer of the disclosure. Compounds were tested in a dose-dependent fashion to determine an IC50 against VEGF 155.
[0025] FIG, 5B demonstrates a non-limiting example of the inhibition of VEGFm-stimulated KDR phosphorylation by an anti-VEGF-A aptamer of the disclosure. Compounds were tested in a dose-dependent fashion to determine an IC50 against VEGF 121. [0026] FIG. 6A demonstrates a non-limiting example of the inhibition of VEGFies-stimulated angiogenesis by an anti-VEGF-A aptamer of the disclosure. Anti-VEGF-A aptamer was tested in a dose-dependent fashion to determine an IC50 against VEGF165.
[0027] FIG. 6B demonstrates a non-limiting example of the inhibition of VEGFi2i-stimulated angiogenesis by an anti-VEGF-A aptamer of the disclosure. Anti-VEGF-A aptamer was tested in a dose-dependent fashion to determine an IC50 against VEGF^ .
[0028] FIG. 7 depicts representative images of the inhibition of VEGF165- or VEGF m- stimuiated angiogenesis with an anti-VEGF-A aptamer of the disclosure.
[0029] FIG. 8A depicts a non-limiting example of duration of action modeling of a 1 nig/eye dose pan-variant specific anti-VEGF-A aptamer in the vitreous for the treatment of wet age- related macular degeneration, diabetic retinopathy, diabetic macular edema, or macular edema following retinal vein occlusion
[0030] FIG. SB depicts a non-limiting example of duration of action modeling of a 5 mg/eye dose pan-variant specific anti-VEGF-A aptamer in the vitreous for the treatment of wet age- related macular degeneration, diabetic retinopathy, diabetic macular edema, or macular edema following retinal vein occlusion.
[0031] FIG. 9A depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of a high dose of pegylated anti-VEGF-A aptamer of the disclosure delivered by IVT administration for the treatment of macular edema following retinal vein occlusion.
[0032] FIG. 9B depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of a low dose of pegylated anti-VEGF-A aptamer of the disclosure delivered by IVT administration for the treatment of macular edema following retinal vein occlusion.
[0033] FIG. 9C depicts a non-limiting exampl e of potency modeling in the vi treous or in the systemic and tissue compartments of an anti-VEGF-A aptamer of the disclosure pegylated with an optimized PEG moiety and delivered by IVT administration for the treatment of macular edema following retinal vein occlusion.
[0034] FIG. 9D depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of an anti-VEGF-A aptamer of the disclosure that has been engineered for metabolic instability and delivered by IVT administration for the treatment of macular edema following retinal vein occlusion.
[0035] FIG. I0A depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of a high dose of a pegylated anti -VEGF-A aptamer of the disclosure delivered by IVT administration for the treatment of retinopathy of prematurity
[0036] FIG. 10B depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of a low' dose of a pegylated anti-VEGF-A aptamer of the disclosure delivered by IVT administration for the treatment of retinopathy of prematurity.
[0037] FIG. IOC depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of an anti-VEGF-A aptamer of the disclosure pegylated with an optimized PEG moiety and delivered by IVT administration for the treatment of retinopathy of prematurity.
[0038] FIG. 10D depicts a non-limiting example of potency modeling in the vitreous or in the systemic and tissue compartments of an anti-VEGF-A aptamer of the disclosure that has been engineered for metabolic instability and delivered by IVT administration for the treatment of retinopathy of prematurity.
[0039] FIG. 11 depicts a non-limiting example of a circular dichroism (CD) spectra of Aptamer 3R02 according to embodiments of the disclosure.
[0040] FIG. 12 depicts non-limiting examples of inhibition of VEGF-A165 or VEGF-A - stimulated KDR phosphorylation by Aptamers Vap7, VEaP121 or 3R02, as compared to an anti- VEGF mAh according to embodiments of the disclosure. For each condition, the left hand bar represents stimulation with VEGF-Aies and the right hand bar represents stimulation with VEGF-A121. Data is presented as the mean ± standard deviation.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The disclosure herein provides aptamer compositions that selectively bind to and inhibit vascular endothelial growth factor-A (VEGF-A) and methods of using said aptamer
compositions. In various aspects, an aptamer composition of the disclosure may comprise an anti -VEGF-A aptamer that binds to one or more isoforms or variants of VEGF-A. In various aspects, an aptamer composition of the disclosure may comprise a pan-variant specific anti- VEGF-A aptamer that binds to each of VEGF-A no, VEGF-A , VEGF-Aies, VEGF-Ai89, and VEGF-A206· In some cases, the anti-VEGF-A aptamers may hind to the receptor binding face of VEGF-A, or a portion thereof. In some eases, the anti-VEGF-A aptamers may bind to the receptor binding domain (RBD) of VEGF-A, or a portion thereof. In some cases, an aptamer of the disclosure does not bind to the heparin-binding domain (HBD) of VEGF-A. Without wishing to be hound by theory, anti-VEGF-A aptamers of the disclosure may prevent or reduce binding of VEGF-A to a VEGF receptor (VEGFR). For example, an anti-VEGF-A aptamer of the disclosure may prevent or reduce binding of VEGF-A to VEGFR1 (also known as Fms- reiated tyrosine kinase 1 (1· It i )), VEGFR2 (also known as Kinase insert domain receptor (KDR) or Flk-1), Neuropilin-1 (Nrp-1), or any combination thereof. In some cases, an anti-VEGF-A aptamer of the disclosure may inhibit a function associated with VEGF-A (e.g, engaging a VEGF receptor, a signaling pathway downstream of VEGF-A, or both).
[0042] The disclosure herein further provides methods for inhibiting VEGF-A (and/or a downstream signaling pathway of VEGF-A). In some cases, the methods include administering an anti-VEGF-A aptamer (or a composition comprising said aptamer) to a biological system (e.g, biological ceils, biological tissue, a subject, and the like). The disclosure further provides methods for treating ocular diseases or disorders including administering an anti-VEGF-A aptamer (or a pharmaceutical composition comprising said aptamer) to a subject having, suspected of having, or at risk of developing, an ocular disease or disorder. In some cases, the ocular disease or disorder may be diabetic retinopathy. In some cases, the ocular disease or disorder may be retinopathy of prematurity'. In some cases, the ocular disease or disorder may be central retinal vein occlusion. In some cases, the ocular disease or disorder may be macular edema. In some cases, the ocular disease or disorder may be choroidal neovascularization. In some cases, the ocular disease or disorder may be neovascular (or wet) age-related macular degeneration. In some cases, the ocular disease or disorder may be myopic choroidal neovascularization. In some cases, the ocular disease or disorder may be punctate inner choroidopathy. In some cases, the ocular disease or disorder may be presumed ocular histoplasmosis syndrome. In some cases, the ocular disease or disorder may be familial exudative vitreoretinopathy. In some cases, the ocular disease or disorder may be
retinoblastoma. In some cases, a subject having, suspected of having, or at risk of developing, an ocular disease or disorder may exhibit elevated levels of one or more variants or isoforms of VEGF-A. For example, a subject having, suspected of having, or at risk of developing, an ocular disease or disorder may exhibit elevated levels of one or more of VEGF-A206, VEGF-A gy, VEGF-A!65, VEGF~Am, and VEGF-AU0.
[0043] In some aspects of the disclosure, the methods and compositions may involve the inhibition of a function associated with VEGF-A. In some cases, the methods and compositions may involve preventing or reducing VEGF-A binding to or interaction with one or more VEGF receptors. For example, the methods and compositions may involve preventing or reducing VEGF-A binding to or interaction with Fit- 1 , KDR, Nrp-1, or any combination thereof. In some cases, the methods and compositions may involve preventing or reducing downstream signaling associated with Flt-1, KDR, Nrp-1, or any combination thereof. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of ocular diseases or disorders. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of diabetic retinopathy. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of retinopathy of prematurity. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of central retinal vein occlusion. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of macular edema. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of choroidal neovascularization. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of neovascular (or wet) age-related macular degeneration. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of myopic choroidal neovascularization. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of punctate inner choroidopathy. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of presumed ocular histoplasmosis syndrome. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of familial exudative vitreoretinopathy. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of retinoblastoma. In some cases, the methods and compositions may involve the inhibition of a function associated with VEGF-A for the treatment of an ocular disease or disorder exhibiting elevated levels of one or more isoforms or variants of VEGF-A [0044] In various aspects, the compositions may include one or more aptamers that selectively bind to and inhibit a function associated with VEGF-A. In some cases, the compositions may include one or more aptamers that bind to the receptor binding face of VEGF-A. In some cases, the compositions may include one or more aptamers that bind to the receptor binding domain of VEGF-A. In some cases, the compositions may include one or more aptamers that bind to a region of VEGF-A other than the heparin binding domain of VEGF-A. Put another way, the compositions may include one or more aptamers that do not bind to the heparin binding domain of VEGF-A. In some cases, the compositions may include one or more aptamers that bind to one or more variants or isoforms of VEGF-A. For example, the compositions may include one or more aptamers that bind to one or more of VEGF-A206, VEGF-Aig9, VEGF-A ½5, VEGF-Am, and VEGF-A io. In some cases, the compositions may include one or more pan-variant specific anti -VEGF-A aptamers. In particular cases, the compositions may include pan-variant specific aptamers that bind to each of VEGF-Am,, VEGF-Am, VEGF-A165, VEGF-A189, and VEGF- A206· Generally, a pan-variant specific apta ner disclosed herein may bind to a structural feature of VEGF-A that is shared amongst VEGF-AU0, VEGF-Am, VEGF-A165, VEGF-A189, and VEGF-A206. In some cases, the structural feature is the receptor binding face or the receptor binding domain.
[0045] In some cases, the compositions may include one or more aptamers that prevent or reduce binding of VEGF-A to Fit- 1 , KDR, Nrp-1, or any combination thereof. In some cases, the compositions may include one or more aptamers that prevent or reduce downstream signaling pathways associated with Flt-1, KDR, Nrp-1 , or any combination thereof. In some cases, the aptamers may be RNA aptamers, DNA aptamers, modified RNA aptamers, or modified DNA aptamers.
[0046] In some cases, the aptamers do not contain non-naturally occurring hydrophobic modifications. In some aspects, less than 100% of the pyrimidines (e.g., C, T, or U) present in a nucleic acid sequence of an aptarner herein comprise a C-5 modified pyrimidine. For example, less than 100%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% of the pyrimidines present in a nucleic acid sequence of an aptamer herein comprise a C-5 modified pyrimidine. In particular aspects, none of the pyrimidines present in a nucleic acid sequence of an aptamer herein comprise a C-5 modified pyrimidine. In another particular aspect, none of the bases in an aptamer sequence herein comprise a C-5 modification. In some aspects, less than 100% of the uridines present in a nucleic acid sequence of an aptamer herein comprise a C-5 modified uridine. For example, less than 100%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% of the uridines present in a nucleic acid sequence of an aptamer herein comprise a C-5 modified uridine. In particular aspects, none of the uridines present in a nucleic acid sequence of an aptamer herein comprise a C-5 modified uridine. In some cases, the C-5 modified pyrimidine or C-5 modified uridine is selected from the group consisting of: 5-(N-benzylcarboxyamide)-2'-deoxyuridine (BndU), 5-(N- benzylcarboxyamide)-2'-0-methyluridine, 5-(N-benzylcarboxyamide)-2'-fluorouridine, 5-(N- phenethylcarboxyamide)-2'-deoxyuridine (PEdU), 5-(N-thiophenylmethylcarboxyamide)-2'- deoxyuridine (ThdU), 5-(N-isobutylcarboxyamide)-2'-deoxyuridine (iBudU), 5-(N- tyrosylcarboxyamide)-2'-deoxyuridine (TyrdU), 5-(N-3,4-methylenedioxybenzylcarboxyamide)- 2'-deoxyuridine (MBndU), 5-(N-4-fluorobenzylcarboxyamide)-2'-deoxyuridine (FBndU), 5-(N~ 3-phenylpropylcarboxyamide)-2'-deoxyuridine (PPd!J), 5-(N-imidizolylethylcarboxyamide)-2'- deoxyuridine (ImdU), 5-(N-isobutylcarboxyamide)-2'-0-methyluridine, 5-(N- isobutylcarboxyamide)-2'-fluorouridine, 5-(N-tryptaminocarboxyamide)-2'-deoxyuridine (TrpdU), 5-(N-R-threoninylcarboxyamide)-2'-deoxyuridine (ThrdU), 5-(N- tryptaminocarboxyamide)-2'-0-methyluridine, 5-(N-tryptaminocarboxyamide)-2'-fluorouridine, 5-(N-[l-(3-trimethylamonium)propyl]carboxyamide)-2'-deoxyuridine chloride, 5-(N- naphthylmethylcarboxyamide)-2'-deoxyuridine (NapdU), 5-(N-naphthylmethylcarboxyamide)- 2'-0-methyluridine, 5-(N-naphthylmethylcarboxyamide)-2'-fluorouridine, 5-(N-[l-(2,3- dihydroxypropyl)]carboxyamide)-2'-deoxyuridine), 5-(N-2-naphthylmethylcarboxyamide)-2'- deoxyuridine (2NapdU), 5-(N-2-naphthylmethylcarboxyamide)-2'-0-methyluridine, 5-(N-2- naphthy!methylcarboxyamide)-2'-fiuorouridine, 5-(N-l-naphthylethylcarboxyamide)-2'- deoxyuridine (NEdU), 5-(N-l-naphthylethylcarboxyamide)-2'-0-methyluridine, 5-(N-l - naphthylethy!carboxyamide)-2'-fluorouridine, 5-(N-2-naphthylethylcarboxyamide)-2'- deoxyuridine (2NEdU), 5-(N-2-naphthylethylcarboxyamide)-2'-0-methyluridine, 5-(N-2- naphthylethylcarboxyamide)-2'-fluorouridine, 5-(N-3-benzofuranylethylcarboxyamide)-2'- deoxyuridine (BFdU), 5-(N-3-benzofuranylethylcarboxyamide)-2'-0-methyluridine, 5-(N-3- benzofuranylethylcarboxyamide)-2'-fluorouridine, 5-(N-3-benzothiophenylethylcarboxyamide)- 2'-deoxyuridine (BTdU), 5-(N-3-benzothiophenylethylcarboxyamide)-2'-0-methyluridine, and 5-(N-3-benzothiophenylethylcarboxyamide)-2'-fluorouridine. In particular aspects, none of the aptamers provided herein comprise NapdU. In other particular cases, the aptamers are not SOMAmers.
[0047] In general,“sequence identity” refers to an exact nucleotide-to-nucleotide or amino acid- to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their“percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Aitschui, Proc. Natl. Acad. Sci USA , 87:2264-2268 (1990) and as discussed in Aitschui, et al, J Mol. Biol. , 215:403-410 (1990); Karlin And Aitschui,
Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993); and Aitschui et al, Nucleic Acids Res., 25:3389-3402 (1997). The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are approximately 50% to 100% and integer values therebetween. In general, this disclosure encompasses sequences with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any sequence provided herein. [0048] In general,“modification identity” refers to two polynucleotides with identical patterns of modifications on a nucleotide-to-nucleotide level. Techniques for determining modification identity may include determining the modifications of a polynucleotide and comparing these modifications to modifications of a second polynucleotide. The percent modification identity of two sequences is the number of exact modification matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Ranges of desired degrees of modification identity are generally approximately 50% to 100%, and integer values
therebetween. In general, this disclosure encompasses sequences with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% modification identity with any sequence provided herein.
[0049] The term“aptamer” as used herein refers to an oligonucleotide and/or nucleic acid analogues that can hind to a specific target molecule Aptamers can include RNA, DNA, modified RNA, modified DNA, any nucleic acid analogue, and/or combinations thereof.
Aptamers can be single-stranded oligonucleotides. In some cases, aptamers may comprise more than one nucleic acid strand (e.g., two or more nucleic acid strands). Aptamers may bind to a target (e.g., a protein) with high affinity and specificity through non-Watson-Crick base pairing interactions. Generally, the aptamers described herein are non-naturally occurring
oligonucleotides {e.g, synthetically produced) that are i solated and used for the treatment of a disorder or a disease. Aptamers can bind to essentially any target molecule including, without limitation, proteins, oligonucleotides, carbohydrates, lipids, small molecules, and even bacterial cells. Aptamers may be monomeric (composed of a single unit) or multimeric (composed of multiple units). Multimeric aptamers can be homomeric (composed of multiple identical units) or heteromeric (composed of multiple non-identical units) Aptamers herein may be described by their primary structures, meaning the linear nucleotide sequence of the aptamer. Aptamer sequences herein are generally described from the 5’ end to the 3’ end, unless otherwise stated. Additionally or alternatively, aptamers herein may be described by their secondary structures which may refer to the combination of single-stranded regions and base-pairing interactions within the aptamer. Whereas many naturally occurring oligonucleotides, such as mRNA, encode information in their linear base sequences, aptamers generally do not encode information in their linear base sequences. Further, aptamers can be distinguished from naturally occurring oligonucleotides in that binding of aptamers to target molecules is dependent upon secondary' and tertiary structures of the aptamer. Aptamers may be suitable as therapeutic agents and may be preferable to other therapeutic agents because: 1) aptamers may be fast and economical to produce because aptamers can be developed entirely by in vitro processes; 2) aptamers may have low toxicity and may lack an immunogenic response; 3) aptamers may have high specificity and affinity for their targets, 4) aptamers may have good solubility; 5) aptamers may have tunable pharmacokinetic properties; 6) aptamers may be amenable to site-specific conjugation of PEG and other carriers; and 7) aptamers may be stable at ambient temperatures.
[0050] The term“VEGF-A”, as used herein includes any variant or isoform of VEGF-A.
Therefore, unless otherwise specified,“VEGF-A” may mean one or more of VEGF-Ano, VEGF-
Figure imgf000017_0001
[0051] The term“pan-variant specific aptamer” as used herein refers to an aptamer that selectively binds to at least VEGF-Ano, VEGF-Am, VEGF-Ai65, VEGF-A i89, and VEGF-A206· A pan-variant specific aptamer may, but not necessarily, bind to one or more additional VEGF-A isoforms or variants. Generally, a pan-variant specific aptamer binds to a structural feature of VEGF-A that is common amongst VEGF-Ano, VEGF-Am, VEGF-A!65, VEGF-A189, and VEGF-A206.
[0052] The terms“about” and“approximately” as used herein, generally refer to a range that is 15% greater than or less than a stated numeri cal value within the context of the particular usage. For example,“about 10” or“approximately 10” would include a range from 8.5 to 1 1.5.
[0053] As used herein, the term“or” is used nonexclusively to encompass“or” and“and.” For example,“A or B” includes“A but not B,”“B but not A,” and“A and B”, unless otherwise indicated.
Vascular endothelial growth factor- A (VEGF-A)
[0054] This disclosure generally provides compositions that bind to vascular endothelial growth factor-A (VEGF-A), and methods of using such compositions to modulate VEGF-A signaling pathways. VEGF-A is thought to be the most significant regulator of angiogenesis in the VEGF family. VEGF-A promotes growth of vascular endothelial cells which leads to the formation of capillary-like structures and may be necessary for the survival of newly formed blood vessels. Vascular endothelial cells are thought to be major effectors of VEGF signaling. Retinal pigment epithelial (RPE) cells may also express VEGF receptors and have been shown to proliferate and migrate upon exposure to VEGF In addition, VEGF is thought to play roles beyond the vascular system. For example, VEGF may play roles in normal physiological functions, including, but not limited to, bone formation, hematopoiesis, wound healing, and development. In various aspects, the compositions provided herein include aptamers that bind to VEGF -A, thereby inhibiting or reducing angiogenesis, e.g., by inhibiting or preventing growth of vascular endothelial cells, retinal pigment epithelial cells, or both. In some cases, the anti- VEGF -A aptamers provided herein may prevent or reduce binding or association of VEGF -A with a VEGF receptor (e.g., Fit- 1 , KDR, Nrp-1) expressed on vascular endothelial cells, retinal pigment epithelial cells, or both.
[0055] The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factor (PIGF). The aptamers disclosed herein primarily bind to variants and isoforms of VEGF-A. In some cases, such aptamers may also bind to one or more of VEGF- B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PIGF. Transcription of VEGF mRNA may be upregulated under hypoxic conditions. Furthermore, various growth factors and cytokines have been shown to upregulate VEGF mRNA expression, including, without limitation, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-cx), transforming growth factor- beta (TGF-b), keratin ocyte growth factor (KGF), insulin-like growth factor- 1 (IGF-1), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), interleukin 1 -alpha (IL-l-cx), interleukin-6 (EL-6), and interleukin-8 (118). VEGF-A is thought to play a role in various ocular diseases and disorders such as, but not limited to, diabetic retinopathy, retinopathy of
prematurity, central retinal vein occlusion, macular edema, choroidal neovascularization, neovascular (or wet) age-related macular degeneration, myopic choroidal neovascularization, punctate inner choroidopathy, presumed ocular histoplasmosis syndrome, familial exudative vitreoretinopathy, and retinoblastoma.
[0056] In some eases, the aptamers provided herein may be used to treat an ocular disease or disorder involving one or more factors that upregulate VEGF-A expression and/or activity, including, but not limited to, hypoxic conditions; a growth factor such as EGF, TGF-a, TGF-b, KGF, IGF-1, FGF, or PDGF; and a cytokine such as IL-i-a, IL6, and 118. In some cases, the aptamers provided herein may be used to treat an ocular disease or disorder selected from the group consisting of: diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, macular edema, choroidal neovascularization, neovascular (or wet) age-related macular degeneration, myopic choroidal neovascularization, punctate inner choroidopathy, presumed ocular histoplasmosis syndrome, familial exudative vitreoretinopathy, and retinoblastoma.
[0057] The gene for human VEGF-A contains eight exons and encodes at least 16 isoforms. The most common isoforms generated by alternative splicing mechanisms are VEGF-A121, VEGF- A165, VEGF-A189, and VEGF-A206. Of these, VEGF-A165, VEGF-A189, and VEGF-A206 each contain a C -terminal heparin binding domain (HBD). In contrast, VEGF-Am lacks a heparin- binding domain. Furthermore, plasmin activation may result in proteolytic cleavage of VEGF- Ai65, VEGF-A189, and VEGF-A20&, resulting in the release of the soluble VEGF-Ano variant, which also lacks a heparin-binding domain.
[0058] In various aspects, the aptamers provided herein may bind to and inhibit a function associated with one or more VEGF-A isoforms or variants. For example, the aptamers provided herein may bind to and inhibit a function associated with one or more of VEGF-Ano, VEGF- Am, VEGF-A 165, VEGF-Ai89, and VEGF-A206. In some cases, the aptamers provided herein may be pan-variant specific aptamers. In some cases, a pan-variant specific aptamer may bind to each of VEGF-Ano, VEGF-Am, VEGF-Ai65, VEGF-A1S9, and VEGF-A206. In some cases, the aptamers provided herein may bind to a structural feature that is common to each of VEGF-Ano, VEGF-Am, VEGF-A165, VEGF-A189, and VEGF-A206· For example, the aptamers provided herein may bind to the receptor binding face, or a portion thereof, of each of VEGF-Ano, VEGF- Am, VEGF-A 165, VEGF-Ai89, and VEGF-A2o6· hi another example, the aptamers provided herein may bind to the receptor binding domain, or a portion thereof, of each of VEGF-Ano, VEGF-Am, VEGF-A165, VEGF-A 139, and VEGF-A206. In some cases, the aptamers provided herein may bind to a structural feature of VEGF-A other than the heparin binding domain found in VEGF-Ai65, VEGF-AI89, and VEGF-A206.
[0059] VEGF-A is known to interact with the receptor tyrosine kinases VEGFIll (also known as Fit- 1 ), VEGFR2 (also known as KDR or Flk-1), and Neuropilin-1 (Nrp-1). Nrp- i is thought to be a co-receptor for KDR. VEGF receptors have been shown to be expressed by endothelial cells, macrophages, hematopoietic cells, and smooth muscle cells. KDR is a class IV receptor tyrosine kinase that binds 2: 1 to VEGF-A dimers. Fit- 1 is a receptor tyrosine kinase that binds to VEGF-A with a 3-10 fold higher affinity than KDR, and has also been shown to bind to VEGF-B and P1GF Flt-1 expression may be upregulated by hypoxia, and its affinity for VEGF-A has been proposed as a negative regulator of signaling by KDR by acting as a decoy receptor. An alternative splicing variant of Fit- 1 results in a soluble variant of the receptor (sFlt-1) which has been suggested to act as an anti-angiogenic sink for VEGF-A. Association of VEGF-Ai65 with KDR may be enhanced by the interaction of the heparin binding domain with co-receptor Nrp-1, which may enhance downstream signaling of KDR. Nrp-1 also has strong affinity for Fit- 1 , which may prevent Nrp-1 association with VEGF-A165 and may be a secondary' regulatory mechanism for VEGF-A induced angiogenesis.
[0060] In various aspects, aptarners provided herein may bind to one or more isoforms or variants of VEGF-A, and may prevent or reduce binding or association of VEGF-A with a VEGF receptor. For example, aptarners provided herein may prevent or reduce binding of one or more isoforms or variants of VEGF-A with Fit- 1 , KDR, Nrp-1, or any combination thereof. In some cases, aptarners provided herein may prevent or reduce binding of one or more of VEGF-Ano, VEGF-Am, VEGF-A165, VEGF-A189, and VEGF-A206 to one or more of Fit- 1, KDR, and Nrp-1. In particular cases, aptarners provided herein may prevent or reduce binding of one or more isoforms or variants of VEGF-A to KDR. In some aspects, the aptarners are pan-variant specific aptarners that bind to each of VEGF-Ano, VEGF-Am, VEGF-A165, VEGF-A!89, and VEGF- A2O6, and reduce or prevent binding or association thereof with one or more of Fit- 1, KDR, and Nrp-1.
[0061] In one instance, an amino acid sequence of human VEGF-A206 may comprise the following sequence:
APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGG CCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSV RGKGKGQKRKRKKSRYKSWS VYVGARCCLMPW SLPGPHPCGPC SERRKHLFVQDPQT CKCSCKNTDSRCKARQLELNERTCRCDKPRR (SEQ ID NO: 1).
[0062] In one instance, an amino acid sequence of human VEGF-A 8 may comprise the fol 1 owing sequence :
APMAEGGGQNHHEVVKF DVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGG
Cr/NDEGLEC TEESNITMGIMRiKPHQGQHiGEMSFLQHNKCECRPKKDRARQEKKSV RGKGKGQKRKRKKSRYKSWSVPCGPCSERRKHLFVQDPQTGKCSCKNTDSRCKARQL ELNERTCRCDKPRR (SEQ ID NO: 2)
[0063] In one instance, an amino acid sequence of human VEGF-Aies may comprise the following sequence: APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGG CCNDEGLECVPTEFiSNITMQiMRlKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCG PCSiiRRKHLFVQDPQTCKCSCKNTDSRGKARQLE-LNERTGRGDKPRR (SEQ ID NO: 3) [0064] In one instance, an amino acid sequence of human VEGF-Au; may comprise the following sequence:
APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGG GCNDEGLECVPTEFiSNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKCDK PRR (SEQ ID NO: 4)
[0065] In one instance, an amino acid sequence of human VEGF-Auo may comprise the following sequence:
APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGG
CCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDR (SEQ ID NO: 5)
Aptamers
[0066] In some cases, the methods and compositions described herein include the use of one or more aptamers for the treatment of an ocular disease or disorder. In some cases, the methods and compositions described herein include the use of one or more aptamers for inhibiting an activity associated with VEGF-A.
[0067] Aptamers as described herein may include any number of modifications that can affect the function or affinity of the aptamer. For example, aptamers may be unmodified or they may contain modified nucleotides to improve stability, nuclease resistance or delivery characteristics. Examples of such modifications may include chemical substitutions at the sugar and/or phosphate and/or base positions, for example, at the T position of ribose, the 5 position of pyrimidines, and the 8 position of purines, various :2'-modified pyrimidines and purines and modifications with 2'-amino (2'-NH2), 2 -fluoro (2!-F), and/or 2'-0-methyl (2'-OMe) substituents. In some cases, aptamers described herein comprise a 2’-OMe and/or a 2’F modification to increase in vivo stability. In some eases, the aptamers described herein contain modified nucleotides to improve the affinity and specificity of the aptamers for a target. Examples of modified nucleotides include those modified with guanidine, indole, amine, phenol,
hydroxymethyl, or boronic acid. In other cases, pyrimidine nucleotide triphosphate analogs or CE-phosphoramidites may be modified at the 5 position to generate, for example, 5- benzylaminocarbonyl-2’-deoxyuridine (BndU); 5-[N-(phenyl-3-propyl)carboxamide]-2'- deoxyuridine (PPdU); 5-(N-thiophenylmethylcarboxyamide)-2'-deoxyuridine (ThdU); 5-(N-4- fluorobenzylcarboxyamide)-2'-deoxyuridine (FBndU); 5-(N-(l -naphthylmethyl)carboxamide)-2'- deoxyuridine (NapdU); 5 -(N-2-naphthylmethylcarboxyamide)-2'-deoxyuridine (2NapdU); 5-(N- l-naphthylethylcarboxyamide)-2'-deoxyuridine (NEdU); 5-(N-2-naphthylethylcarboxyamide)-2'- deoxyuridine (2NEdU); 5-(N-tryptaminocarboxyamide)-2'-deoxyuridine (TrpdU); 5- isobutylaminocarbonyl-2’-deoxyuridine (IbdU); 5-(N-tyrosylcarboxyamide)-2'-deoxyuridine (TyrdU); 5-(N-isobutylaminocarbonyl-2’-deoxyuridine (iBudU); 5-(N-benzylcarboxyamide)-2'- O-rnethy!uridine, 5~(N-benzylcarboxyamide)~2'-fluorouridine, 5-(N-phenethylcarboxyamide)-2'- deoxyuridine (PEdU), 5-(N-3,4-methylenedioxybenzylcarboxyamide)-2'-deoxyuridine
(MBndLI), 5-(N-imidizolylethylcarboxyamide)-2'-deoxyuridine (ImdU), 5-(N- isobutylcarboxyamide)-2'-0-methyluridine, 5-(N-isobutylcarboxyamide)-2'-fluorouridine, 5-(N— R-threoninylcarboxyamide)-2'-deoxyuridine (ThrdU), 5-(N-tryptaminocarboxyamide)-2'-0- methyluridine, 5-(N-tryptaminocarboxyamide)-2'-fluorouridine, 5-(N-[ I -(3- trimethylamonium)propyl]carboxyamide)-2'-deoxyuridine chloride, 5-(N- naphthylmethylcarboxyamide)-2'-0-methyluridine, 5-(N-naphthylmethylcarboxyamide)-2'- fluorouridine, 5-(N-[l -(2,3-dihydroxypropyl)]carboxyamide)-2'-deoxyuridine), 5-(N-2- naphthylmethylcarboxyamide)-2'-0-methyluridine, 5-(N-2-naphthylmethylcarboxyamide)-2'- fluorouridine, 5-(N- 1 -naphthyl ethyl carboxyamide)-2'-0-methyluri dine, 5-(N- 1 - naphthylethylcarboxyamide)-2'-fluorouridine, 5-(N-2-naphthyiethyicarboxyamide)-2'-G- n ethyiuridine, 5-(N-2-naphthylethylcarboxyamide)-2'-fluorouridine, 5-(N-3- benzofuranylethylcarboxyamide)-2'-deoxyuridine (BFdU), 5-(N-3- benzofuranylethylcarboxyamide)-2'-0-methyluridine, 5-(N-3-benzofuranylethylcarboxyamide)- 2'-fluorouridine, 5-(N-3-benzothiophenylethylcarboxyamide)-2'-deoxyuridine (BTdlJ), 5-(N-3- benzothiophenylethylcarboxyamide)-2'-0-methyluridine, 5-(N-3- benzothiophenylethylcarboxyamide)-2!-fluorouridine; 5-[N-(l-morphoiino-2- ethyl)carboxamide]-2'-deoxyuridine (MOEdu); R-tetrahydrofuranylmethyl-2'-deoxyuridine (RTMdU); 3-methoxybenzyl-2'-deoxyuridine (3MBndU); 4-methoxybenzyl-2'-deoxyuridine (4MB ndU); 3 ,4-dimethoxybenzyl-2'-deoxyuridine (3 ,4DMBndU); S-tetrahydrofuranylmethyl-2'- deoxyuridine (STMdlJ); 3,4-methylenedioxyphenyl-2-ethyl-2'-deoxyuridine (MPEdU); 4- pyridinylmethyl-2'-deoxyuridine (PyrdU); or l-benzimidazol-2-ethyl-2'-deoxyuridine (BidU); 5- (amino-l-propenyl)-2'-deoxyuridine; 5-(indole-3-acetamido-l-propenyl)-2'-deoxyuridine; or 5- (4-pivaloylbenzamido-l-propenyl)-2'-deoxyuridine.
[0068] Modifications of the aptamers contemplated in this disclosure include, without limitation, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and functionality to the nucleic acid aptamer bases or to the nucleic acid aptamer as a whole. Modifications to generate
oligonucleotide populations that are resistant to nucleases can also include one or more substitute internucleotide linkages, altered sugars, altered bases, or combinations thereof. Such
modifications include, but are not limited to, mposition sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate, phosphorodithioate, or alkyl phosphate modifications, methylations, and unusual base-pairing combinations such as the isobases isocytidine and isoguanosine.
Modifications can also include 3’ and 5' modifications such as capping, e.g., addition of a 3'-3'- dT cap to increase exonuclease resistance, or conjugation of a PEG to the 5’ or 3’ end to increase exonuclease and endonuclease resistance
[0069] Aptamers of the disclosure may generally comprise nucleotides having ribose in the b-D- ribofuranose configuration. In some cases, 100% of the nucleotides present in the aptamer have ribose in the b-D-ribofuranose configuration. In some cases, at least 50% of the nucleotides present in the aptamer have ribose in the b-D-ribofuranose configuration. In some cases, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the nucleotides present in the aptamer have ribose in the b-D- ribofuranose configuration.
[0070] The length of the aptamer can be variable. In some cases, the length of the aptamer is less than 100 nucl eotides. In some cases, the length of the aptamer is greater than 10 nucleotides. In some cases, the length of the aptamer is between 10 and 90 nucleotides. The aptamer can be, without limitation, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, or about 90 nucleotides in length.
[0071] In some instances, a polyethylene glycol (PEG) polymer chain is covalently bound to the aptamer, referred to herein as PEGylation. Without wishing to be bound by theory, PEGylation may increase the half-life and stability of the aptamer in physiological conditions. In some cases, the PEG polymer is covalently bound to the 5' end of the aptamer. In some cases, the PEG polymer is covalently hound to the 3' end of the aptamer. In some cases, the PEG polymer is covalently bound to both the 5’ end and the 3' end of the aptamer. In some cases, the PEG polymer is covalently bound to a specific site on a nueleobase within the aptamer, including the 5-position of a pyrimidine or 8-position of a purine. In some cases, the PEG polymer is covalently bound to an abasic site within the aptamer.
[0072] In some cases, an aptamer described herein may be conjugated to a PEG having the general formula, H-CQ-CH -CEBjn-OH. In some cases, an aptamer described herein may be conjugated to a methoxy-PEG (mPEG) of the general formula, CH30-(CH2-CH2-0)n-H. In some cases, the aptamer is conjugated to a linear chain PEG or mPEG. The linear chain PEG or mPEG may have an average molecular weight of up to about 30 kD. Multiple linear chain PEGs or mPEGs can be linked to a common reactive group to form multi-arm or branched PEGs or mPEGs. For example, more than one PEG or mPEG can be linked together through an amino acid linker (e.g., lysine) or another linker, such as glycerine. In some cases, the aptamer is conjugated to a branched PEG or branched mPEG. Branched PEGs or mPEGs may be referred to by their total mass (e.g., two linked 2QkD mPEGs have a total molecular weight of 40kD).
Branched PEGs or mPEGs may have more than two aims. Multi -arm branched PEGs or mPEGs may he referred to by their total mass (e.g, four linked 10 kD mPEGs have a total molecular weight of 40 kD). In some cases, an aptamer of the present disclosure is conjugated to a PEG polymer having a total molecular weight from about 5 kD to about 200 kD, for example, about 5 kD, about 10 kD, about 20 kD, about 30 kD, about 40 kD, about 50 kD, about 60 kD, about 70 kD, about 80 kD, about 90 kD, about 100 kD, about 1 10 kD, about 120 kD, about 130 kD, about 140 kD, about 150 kD, about 160 kD, about 170 kD, about 180 kD, about 190 kD, or about 200 kD. In one non-limiting example, the aptamer is conjugated to a PEG having a total molecular weight of about 40 kD.
[0073] In some cases, the reagent that may be used to generate PEGylated aptamers is a branched PEG N-Hydroxysuccinimide (mPEG-NHS) having the general formula:
Figure imgf000025_0001
with a 20 kD, 40 kD or 60 kD total molecular weight (e.g., where each mPEG is about lOkD, 20 kD or about 30 kD). As described above, the branched PEGs can be linked through any appropriate reagent, such as an amino acid (e.g, lysine or glycine residues).
[0074] In one non-limiting example, the reagent used to generate PEGylated aptamers is [N2- (monomethoxy 20K polyethylene glycol carbamoyl)-N°-(monomethoxy 20K polyethylene glycol carbamoyl)] -lysine N-hydroxysuccinimide having the formula:
Figure imgf000025_0002
[0075] In yet another non-limiting example, the reagent used to generate PEGylated aptamers has the formula:
Figure imgf000025_0003
[0076] where X is N-hydroxysuccinimide and the PEG arms are of approximately equivalent molecular weight. Such PEG architecture may provide a compound with reduced viscosity compared to a similar aptamer conjugated to a two-armed or single-arm linear PEG. [0077] In some examples, the reagent used to generate PEGyiated aptamers has the formula:
Figure imgf000026_0001
where X is N-hydroxysuccinimide and the PEG arms are of different molecular weights, for example, a 40 kD PEG of this architecture may be composed of 2 amis of 5 kD and 4 arms of 7.5 kD. Such PEG architecture may provide a compound with reduced viscosity compared to a similar aptamer conjugated to a two-armed PEG or a single-ami linear PEG.
[0078] In some cases, the reagent that may be used to generate PEGyiated aptamers is a non- branched mPEG-Succinimidyl Propionate (mPEG-SPA), having the general formula:
Figure imgf000026_0002
where mPEG is about 20 kD or about 30 kD. In one example, the reactive ester may be
Figure imgf000026_0003
CH2-CO2-NHS.
[0079] In some instances, the reagent that may be used to generate PEGyiated aptamers may include a branched PEG linked through glycerol, such as the SUNBRIGHT® series from NOF Corporation, Japan. Non-limiting examples of these reagents include:
Figure imgf000026_0004
400GS2);
Figure imgf000027_0001
and
Figure imgf000027_0002
[0080] In another example, the reagents may include a non-branched mPEG Succinimidyl alpha-methylbutanoate (mPEG-SMB) having the general formula:
Figure imgf000027_0003
where mPEG is between 10 and 30 kD. In one example, the reactive ester may be -O-CH2.CH2. CH(CH3)-C02-NHS.
[0081] In other instances, the PEG reagents may include nitrophenyl carbonate-linked PEGs, having the general formula:
Figure imgf000027_0004
[0082] Compounds including nitrophenyl carbonate can be conjugated to primary amine containing linkers.
[0083] In some cases, the reagents used to generate PEGy!ated aptamers may include PEG with thiol -reactive groups that can be used with a thiol -modi tied linker. One non-limiting example may include reagents having the following general structure:
Figure imgf000028_0001
where mPEG is about 10 kD, about 20 kD or about 30 kD.
[0084] Another non-limiting example may include reagents having the following general structure:
Figure imgf000028_0002
where each mPEG is about 10 kD, about 20 kD, or about 30 kD and the total molecular weight is about 20 kD, about 40 kD, or about 60 kD, respectively. Branched PEGs with thiol reactive groups that can be used with a thiol-modified linker, as described above, may include reagents in which the branched PEG has a total molecular weight of about 40 kD or about 60 kD (e.g., where each mPEG is about 20 kD or about 30 kD).
[0085] In some cases, the reagents used to generated PEGylated aptamers may include reagents having the following structure:
Figure imgf000028_0003
[0086] In some cases, the reaction to conjugate the PEG to the aptamer is carried out between about pH 6 and about pH 10, or between about pH 7 and pH 9 or about pH 8. [0087] In some cases, the reagents used to generate PEGylated aptamers may include reagents having the following structure:
Figure imgf000029_0001
[0088] In some cases, the reagents used to generate PEGylated aptamers may include reagents having the following structure:
Figure imgf000029_0002
[0089] In some cases, the aptamer is associated with a single PEG molecule. In other cases, the aptamer is associated with two or more PEG molecules.
[0090] In some cases, the aptamers described herein may be bound or conjugated to one or more molecules having desired biological properties. Any number of molecules can be bound or conjugated to aptamers, non-limiting examples including antibodies, peptides, proteins, carbohydrates, enzymes, polymers, drugs, small molecules, gold nanoparticles, radiolabels, fluorescent labels, dyes, haptens (e.g., biotin), other aptamers, or nucleic acids (e.g, siRNA). In some cases, aptamers may be conjugated to molecules that increase the stability, the solubility or the bioavailability of the aptamer. Non-limiting examples include polyethylene glycol (PEG) polymers, carbohydrates and fatty acids. In some cases, molecules that improve the transport or deliver}- of the aptamer may be used, such as cell penetrating peptides. Non-limiting examples of cell penetrating peptides can include peptides derived from Tat, penetratin, polyarginine peptide Args sequence, Transportan, VP22 protein from Herpes Simplex Virus (HSV), antimicrobial peptides such as Buforin I and SynB, polyproline sweet arrow' peptide molecules, Pep-1 and MPG. In some embodiments, the aptamer is conjugated to a lipophilic compound such as cholesterol, dialkyl glycerol, diacyl glycerol, or a non-immunogenic, high molecular weight compound or polymer such as polyethylene glycol (PEG) or other water-soluble pharmaceutically acceptable polymers including, but not limited to, polyaminoamines (PAMAM) and polysaccharides such as dextran, or polyoxazolines (POZ).
[0091] The molecule to be conjugated can be covalently bonded or can be associated through non-covalent interactions with the aptamer of interest. In one example, the molecule to be conjugated is covalently attached to the aptamer. The covalent attachment may occur at a variety of positions on the aptamer, for example, to the exocyclic amino group on the base, the 5- position of a pyrimidine nucleotide, the 8-position of a purine nucleotide, the hydroxyl group of the phosphate, or a hydroxyl group or other group at the 5! or 3' terminus. In one example, the covalent attachment is to the 5' or 3’ hydroxyl group of the aptamer.
[0092] In some cases, the aptamer can be attached to another molecule directly or with the use of a spacer or linker. For example, a lipophilic compound or a non-immunogenic, high molecular weight compound can be attached to the aptamer using a linker or a spacer. Various linkers and attachment chemistries are known in the art. In a non-limiting example, 6- (trifluoroacetamido)hexanol (2-cyanoethyl-N,N-diisopropyl)phosphoramidite can be used to add a hexylamino linker to the 5! end of the synthesized aptamer. This linker, as with the other amino linkers provided herein, once the group protecting the amine has been removed, can be reacted with PEG-NHS esters to produce covalently linked PEG-aptamers. Other non-limiting examples of linker phosphoramidites may include: TFA-amino C4 CED phosphoramidite having the structure:
Figure imgf000030_0001
5'-amino modifier C3 TFA having the structure:
Figure imgf000031_0001
MMT amino modifier C6 CED phosphoramidite having the structure:
Figure imgf000031_0002
5'-amino modifier 5 having the structure:
Figure imgf000031_0003
MMT : 4-Monomethoxy trity 1
S'-amino modifier C 12 having the structure:
Figure imgf000031_0004
MMT : 4-Monomethoxytrityl 5' thiol-modifier C6 having the structure:
Figure imgf000032_0001
5' thiol-modifier C6 having the structure:
Figure imgf000032_0002
and 5' thiol -modifier C6 having the structure:
Figure imgf000032_0003
[0093] The 5'-thiol modified linker may be used, for example, with PEG-maleimides, PEG- vinylsulfone, PEG-iodoacetamide and PEG-orthopyridyl-disulfide. In one example, the aptamer may be bonded to the 5'-thiol through a maleimide or vinyl sulfone functionality.
[0094] In some cases, the aptamer formulated according to the present disclosure may also be modified by encapsulation within or displayed on the surface of a liposome. In other cases, the aptamer formulated according to the present disclosure may also be modified by encapsulation within or displayed on the surface of a micelle. Liposomes and micelles may be comprised of any lipids, and in some cases the lipids may be phospholipids, including phosphatidylcholine. Liposomes and micelles may also contain or be comprised in part or in total of other polymers and amphipathic molecules including PEG conjugates of poly lactic acid (PLA), poly DL-lactic- co-glycolic acid (PLGA), or poly caprolactone (PCL) [0095] In some cases, the aptamers described herein may be designed to inhibit a function associated with VEGF-A. In some cases, the aptamers described herein may be designed to bind the receptor binding face of VEGF-A, or a portion thereof. In some cases, the aptamers described herein may be designed to bind the receptor binding domain of VEGF-A, or a portion thereof. The receptor binding domain of VEGF-A may include any one or more of residues 1- 109 as described in SEQ ID NOs: 1-5. In some cases, the aptamers described herein may bind to a structural feature of VEGF-A other than the heparin binding domain of VEGF-A. The heparin binding domain of VEGF-A may include any one or more of residues 111-165 as described in SEQ ID NOs: 1-3. In some cases, the aptamers described herein may block or reduce binding of one or more isoforms or variants of VEGF-A to one or more of Flk-1, KDR, and Nrp-1.
[0096] In some instances, an aptamer is isolated or purified. “Isolated” (used interchangeably with“substantially pure” or“purified”) as used herein means an aptamer that is synthesized chemically, or has been separated from other aptamers.
[0097] In some cases, an aptamer of the disclosure may comprise one of the following sequences described in Table 1 or Table 2.
Table 1. VEGF-A Aptamer Sequence
Figure imgf000033_0001
Table 2. VEGF-A Aptamer Sequences
Figure imgf000033_0002
[0098] In some aspects, an aptamer of the disclosure may have a nucleic acid sequence comprising any one of SEQ ID NOs: 6 or 7, or may have a nucleic acid sequence that shares at least 50% sequence identity to any one of SEQ ID NOs: 6 or 7. In some aspects, an aptamer of the disclosure may have a nucleic acid sequence consisting of any one of SEQ ID NOs: 6 or 7, or may have a nucleic acid sequence that shares at least 50% sequence identity to a sequence that consists of any one of SEQ ID NOs: 6 or 7. In some cases, the nucleic acid sequence may comprise one or more modified nucleotides. In some cases, at least 50% of said nucleic acid sequence may comprise the one or more modified nucleotides. In some cases, the one or more modified nucleotides may comprise a 2'F-modified nucleotide, a 2'QMe-modified nucleotide, or a combination thereof. In some cases, the one or more modified nucleotides may be selected from the group consisting of: 2'F-G, 2'OMe-G, 2'QMe-U, 2'OMe-A, 2'OMe-C, an inverted deoxythymidine at the 3' terminus, and any combination thereof. In some cases, the aptamer may have a nucleic acid sequence comprising any one of SEQ ID NOs: 6 or 7, wherein the nucleic acid sequence comprises modified nucleotides as described in Table 2. In some cases, the aptamer is selected from the group consisting of: Aptamer 4.2 as described in Table 2, and Aptamer 26 as described in Table 2. In some cases, the aptamer may be conjugated to a polyethylene glycol (PEG) molecule. In some cases, the PEG molecule may have a molecular weight of 80 kDa or less (e.g., 40kX)a).
[0099] In some cases, an aptamer of the disclosure may have at least 50%, 55%, 60%, 65%,
70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%>, or 99% sequence identity with any aptamer described herein. For example, an anti-VEGF-A aptamer of the disclosure may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any aptamer described in Table 1 or Table 2. In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%o sequence identity with any one of SEQ ID NOs: 6 or 7
[00100] In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 50% sequence identity with any one of SEQ ID NOs: 6 or 7. In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 55% sequence identity with any one of SEQ ID NOs: 6 or 7. In some eases, an anti-VEGF-A aptamer of the disclosure may have at least 60% sequence identity with any one of S EQ ID NOs: 6 or 7. In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 65% sequence identity with any one of SEQ ID NOs: 6 or 7. In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 70% sequence identity with any one of SEQ ID NOs: 6 or 7, In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 75% sequence identity with any one of SEQ ID NOs: 6 or 7. In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 80% sequence identity with any one of SEQ ID NOs: 6 or 7. In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 85% sequence identity with any one of SEQ ID NOs: 6 or 7. In some cases, an anti-VEGF- A aptamer of the disclosure may have at least 90% sequence identity with any one of SEQ ID NOs: 6 or 7. In some cases, an anti-VEGF-A aptamer of the disclosure may have at least 95% sequence identity with any one of SEQ ID NOs: 6 or 7,
[00101] In some cases, an aptamer of the disclosure may have a primary nucleotide sequence that shares at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 contiguous nucleotides with a nucleotide sequence described in Table 1 or Table 2 In some cases, an aptamer of the disclosure may have a primary nucleotide sequence that shares at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 contiguous nucleotides with any one of SEQ ID NOs: 6 or 7.
[00102] In such cases wiiere specific nucleotide modifications have been recited, it should be understood that any number and type of nucleotide modifications may be substituted. For example, 2’OMe-G may be substituted for 2’F-G. Non-limiting examples of nucleotide modifi cations have been provided herein. In some instances, all of the nucleotides of an aptamer are modified. In some instances, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleotides of an aptamer of the disclosure may be modified. In some aspects, an aptamer of the disclosure has the primary nucleotide sequence of any one of SEQ ID NOS: 6 or 7 and has a modified nucleotide sequence as described in Table 2.
[00103] In some cases, an aptamer of the disclosure may have a modified nucleotide sequence.
In some cases, an aptamer of the disclosure may have a modified nucleotide sequence as described in Table 2 In some cases, an aptamer of the disclosure may have a primary nucleotide sequence according to any aptamer described in Table 2, and a modified nucleotide sequence that is different than that described in Table 2. In such cases, an aptam er of the disclosure may have a modified nucleotide sequence that shares at least 10% modification identity with any modified nucleotide sequence described in Table 2 For example, an aptamer of the disclosure may have a modified nucleotide sequence that shares at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% modification identity with any modified nucleotide sequence described in Table 2.
[00104] In some cases, an aptamer of the disclosure may have a primary nucleotide sequence of any one of SEQ ID NOS: 6 or 7, and a modified nucleotide sequence in which at least 10% of the C nucleotides are modified (e.g., 2'OMe-C). For example, an aptamer of the disclosure may have a modified nucleotide sequence in which at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 99%, or 100% of the C nucleotides are modified (e.g., 2’OMe-C). In some cases, an aptamer of the disclosure may have a modified nucleotide sequence wherein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 99%, or 100% of the C nucleotides (C) are modified according to Table 2.
[00105] In some cases, an aptamer of the disclosure may have a primary nucleotide sequence of any one of SEQ ID NOS: 6 or 7, and a modified nucleotide sequence in which at least 10% of the A nucleotides are modified (e.g, 2'OMe-A). For example, an aptamer of the disclosure may have a modified nucleotide sequence in which at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the A nucleotides are modified (e.g., 2’OMe-A). In some cases, an aptamer of the disclosure may have a modified nucleotide sequence wherein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the A nucleotides are modified according to Table 2.
[00106] In some cases, an aptamer of the disclosure may have a primary nucleotide sequence of any one of SEQ ID NOS: 6 or 7, and a modified nucleotide sequence in which at least 10% of the U nucleotides are modified (e.g., 2'OMe-U). For example, an aptamer of the disclosure may have a modified nucleotide sequence in which at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 99%, or 100% of the U nucleotides are modified (e.g., 2'OMe-U). In some cases, an aptamer of the disclosure may have a modified nucleotide sequence wherein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 99%, or 100% of the U nucleotides are modified according to Table 2.
[00107] In some cases, an aptamer of the disclosure may have a primary nucleotide sequence of any one of SEQ ID NOS: 6 or 7, and a modified nucleotide sequence in which at least 10% of the G nucleotides are modified (e.g., 2'F-G, 2'OMe-G). For example, an aptamer of the disclosure may have a modified nucleotide sequence in which at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 99%, or 100% of the G nucleotides are modified (e.g., 2'F-G, 2'OMe- G). In some cases, an aptamer of the disclosure may have a modified nucleotide sequence wherein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the G nucleotides are modified according to Table 2.
Anti- VEGF-A Compositions
[00108] In some aspects, the disclosure provides anti -VEGF-A compositions that inhibit a function associated with VEGF-A. The anti -VEGF-A compositions may include one or more anti -VEGF-A aptamers that bind to specific regions of VEGF-A with high specificity and high affinity. In some cases, the anti-VEGF-A compositions may include one or more anti -VEGF-A aptamers that bind to a region of VEGF-A that includes the receptor binding face of VEGF-A.
In some cases, the anti-VEGF-A compositions may include one or more anti-VEGF-A aptamers that bind to a region of VEGF-A that includes the receptor binding domain of VEGF-A, or a portion thereof. The receptor binding domain of VEGF-A may include any one or more of residues 1-109 as described in SEQ ID NOs: 1-5. In some cases, the anti-VEGF-A
compositions may include one or more anti-VEGF-A aptamers that prevent or reduce binding of one or more isoforms or variants of VEGF-A with Fit- 1 , KDR, Nrp-1, or any combination thereof.
Anti-VEGF-A Aptamers
[00109] In some aspects, anti-VEGF-A aptamers of the disclosure may block or reduce the interaction of VEGF-A with Fit- 1 , may block or reduce the interaction of VEGF-A with KDR, may block or reduce the interaction of VEGF-A with Nrp-1, or any combination thereof.
[00110] In some aspects, anti-VEGF-A aptamers of the disclosure bind to structural features that are common to VEGF-AU0, VEGF-Am, VEGF-A165, VEGF-A189, and VEGF-A2o6. In some cases, anti-VEGF-A aptamers of the disclosure bind to regions of VEGF-A other than the heparin binding domain present in VEGF-Ai65, VEGF-A i89, and VEGF-A2o6· The heparin binding domain may include residues 111-165 as described by SEQ ID NOs: 1-3. Without wishing to be bound by theory, the cationic heparin binding domain of VEGF-A is thought to be the dominant epitope for aptamer recognition due to the anionic nature of the oligonucleotide sugar phosphate backbone. Therefore, selection of aptamers to regions of VEGF-A other than the heparin binding domain have proven difficult. For example, pegaptanib (brand name Macugen8) is an oligonucleotide inhibitor of VEGF-A that binds to the heparin binding domain. Because VEGF-Am and VEGF-Ano lack the heparin binding domain, pegaptanib does not bind to or inhibit VEGF-Am and VEGF-Auo, thereby providing inferior VEGF-A suppression as compared to an inhibitor that binds to the receptor binding domain of VEGF-A. Similarly, additional aptamer inhibitors of VEGF-A have been described which bind to the heparin binding domain of VEGF-A. For example, a DNA aptamer specific for VEGF-Ates, but not VEGF-Am has been described (Hasegawa, Hijiri, Koji Sode, and Kazunori Ikebukuro. "Selection of DNA aptamers against VEGF165 using a protein competitor and the aptamer blotting method." Biotechnology letters 30.5 (2008): 829-834., Kaur, Harleen, and Lin-Yue Lanry Yung. "Probing high affinity sequences of DNA aptamer against VEGF165." PLoS One 7.2 (2012): e31196.). Additionally, a 2’ -O-Methyl aptamer has been described that binds to VEGF-Aies, but not VEGF-Am (Burmeister, Paula E., et al. "Direct in vitro selection of a 2'-0-methyJ aptamer to VEGF." Chemistry & biology 12.1 (2005): 25-33.). Similarly, DNA aptamers with an expanded 6-base nucleotide alphabet have been described that recognize VEGF-Aies, but not VEGF-Am (Kimoto, Michiko, et al. "Generation of high-affinity DNA aptamers using an expanded genetic alphabet " Nature biotechnology 31.5 (2013): 453.) Furthermore, aptamer selections against other proteins that contain heparin binding domains tend to generate aptamers to those epitopes. For example, RNA aptamers that bind to the heparin binding domain have been described for thrombin (Jeter, Martha L., et al. "RNA aptamer to thrombin binds anionUbinding exosite P 2 and alters protease inhibition by heparin□ binding serpins." FEBS letters568.1-3 (2004): 10-14.; Long, Stephen B., et al. "Crystal structure of an RNA aptamer bound to thrombin." Rna (2008)), basic fibroblast growth factor (Jellinek, D., et al. "High-affinity RNA ligands to basic fibroblast growth factor inhibit receptor binding." Proceedings of the National Academy of Sciences 90.23 (1993): 11227-11231.), interleukin-8 (Sung, Ho Jin, et al. "Inhibition of human neutrophil activity by an RNA aptamer bound to interleukin-8." Biomaterials 35.1 (2014): 578-589.), and Plasmodium falciparum erythrocyte membrane protein 1 (Barfod, Anders, Tina Persson, and Johan Lindh. "In vitro selection of RNA aptamers against a conserved region of the Plasmodium falciparum erythrocyte membrane protein 1." Parasitology research 105.6 (2009): 1557-1566.).
[00111] In some cases, anti -VEGF-A aptamers of the disclosure may bind to a region ofVEGF- A that includes the receptor-binding face of any isoform or variant of VEGF-A, or portions thereof. The receptor-binding face of VEGF-A may include strands b2, b5, and b6, and loops bΐ to b2 of one monomer, and the N-terminal a helix and loop b3 to b4 of a second monomer. The receptor-binding face of VEGF-A may be as described by Muller et al.“The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding." Structure 5.10 (1997): 1325-1338. In some cases, anti-VEGF-A aptamers of the disclosure may bind to one or more amino acid residues of any isoform or variant of VEGF -A, including, without limitation, Phe17, He43, 1!e46, Glu64, Gln79, Ile83, Lys84, Pro85, Arg82, His86, Asp63, and Glu67 as described by SEQ O) NOs: 1-5. In some cases, anti -VEGF - A aptamers that bind to the receptor-binding face of VEGF- A, or a portion thereof, may prevent or reduce the association of VEGF- A with one or more of Fit- 1, KDR, or Nrp-1. In some cases, anti-VEGF-A aptamers that bind to the receptor-binding face of VEGF- A, or a portion thereof, may interact with recombinant bead-bound VEGF -A es, VEGF -Am, or VEGF-Ano as measured by flow cytometry or may interact with recombinant surface-bound VEGF-AI65, VEGF- A , or VEGF- A io as measured by surface piasmon resonance (see Examples 1 and 2, respectively). In some cases, anti-VEGF-A aptamers that bind to the receptor-binding face of VEGF -A, or a portion thereof, may inhibit or reduce the interaction of VEGF-A165, VEGF -Am, or VEGF-Ano with KDR as measured by a reduction in FRET signal (see Example 3). In some cases, anti- VEGF-A aptamers that bind to the receptor-binding face of VEGF- A, or a portion thereof, may inhibit or reduce VEGF-A105, VEGF -Am, or VEGF-Ano induced trans autophosphorylation of the intracellular domain of KDR as measured by phospho-KDR AlphaLISA® ( see Example 4 ).
In some cases, anti-VEGF-A aptamers that bind to the receptor-binding face of VEGF- A, or a portion thereof, may inhibit or reduce VEGF-Ai65, VEGF-Am. or VEGF-Ano induced gene expression of tissue factor in HUVEC ceils as measured by qPCR. In some cases, anti-VEGF-A aptamers that bind to the receptor-binding face of VEGF -A, or a portion thereof, may inhibit or reduce VEGF-Aies, VEGF-Am, or VEGF-Ano induced tube formation of GFP-HUVECs in co- culture with human dermal fibroblasts cells as measured by change in network length or network area (see Example 5). In some cases, anti-VEGF-A aptamers that bind to the receptor-binding face of VEGF -A, or a portion thereof, may inhibit or reduce vascular leakage in a mouse, rat, rabbit, or primate eye following exogenous VEGF-A365, VEGF-Am, or VEGF-Ano challenge as measured by fluorescein angiography and Evans-blue albumin staining.
[00112] In some cases, anti-VEGF-A aptamers of the disclosure may bind to a region of VEGF - A that includes the receptor binding domain of any isoform or variant of VEGF- A, or portions thereof. The receptor binding domain of VEGF -A may include one or more of residues 1-109, as described in SEQ ID NOs: 1-5. In some cases, anti-VEGF-A aptamers of the disclosure may bind to one or more amino acid residues of any isoform or variant of VEGF-A, including, without limitation, Phel7, Tyr21, Tyr25, Ile43, Ue46, Ile83, Asp63, Glu64, Pro85, and His86, as described by SEQ ID NQs: 1-5 In some cases, anti-VEGF-A aptamers of the disclosure may bind to a region within the receptor binding domain of VEGF-A which results in global conformational changes in VEGF-A such that it no longer binds to and activates signaling via KDR. In some cases, anti-VEGF-A aptamers that bind to the receptor binding domain of VEGF- A, or a portion thereof, may prevent or reduce the association of VEGF-A with one or more of Flt-1, KDR, and Nrp-1. In some cases, anti-VEGF-A aptamers that bind to the receptor binding domain of VEGF-A, or a portion thereof, may interact with recombinant bead-bound VEGF- Ai65, VEGF-A 121. or VEGF-Ano as measured by flow cytometr7 or may interact with
recombinant surface-bound VEGF-A 65, VEGF-A , or VEGF-Ano as measured by surface plasmon resonance (see Examples 1 and 2, respectively). In some cases, anti-VEGF-A aptamers that bind to the receptor-binding domain of VEGF-A, or a portion thereof, may inhibit or reduce the interaction of VEGF-A165, VEGF-Am, or VEGF-Ano with KDR as measured by a reduction in FRET signal (see Example 3). In some cases, anti-VEGF-A aptamers that bind to the receptor-binding domain of VEGF-A, or a portion thereof, may inhibit or reduce VEGF-A165, VEGF-Ai2i, or VEGF-Ano induced trans autophosphorylation of the intracellular domain of KDR as measured by phospho-KDR AlphaLISA® (see Example 4). In some cases, anti-VEGF- A aptamers that bind to the receptor binding domain of VEGF-A, or a portion thereof, may inhibit or reduce VEGF-A es, VEGF-A , or VEGF-Ano induced gene expression of tissue factor in HUVEC cells as measured by qPCR. In some cases, anti-VEGF-A aptamers that bind to the receptor binding domain of VEGF-A, or a portion thereof, may inhibit or reduce VEGF- Ai65, VEGF-A 121, or VEGF-Ano induced tube formation of GFP-HUVECs in co-culture with human dermal fibroblasts cells as measured by change in network length or network area (see Example 5). In some cases, anti-VEGF-A aptamers that bind to the receptor binding domain of VEGF-A, or a portion thereof, may inhibit or reduce vascular leakage in a mouse, rat, rabbit, or primate eye following exogenous VEGF-Ai65, VEGF-Ani. or VEGF-Ano challenge as measured by fluorescein angiography and Evans-blue albumin staining.
Binding Affinity
[00113] The dissociation constant (¾) can be used to describe the affinity of an aptamer for a target (or to describe how tightly the aptamer binds to the target) or to describe the affinity of an aptamer for a specific epitope of a target. The dissociation constant may be defined as the molar concentration at which half of the binding sites of a target are occupied by the aptamer. Thus, the smaller the Kd, the tighter the binding of the aptamer to its target. In some cases, an anti-VEGF- A aptamer of the disclosure may have a ¾ for one or more isoforms or variants of VEGF-A of less than about 1000 nM, for example, less than about 500 nM, less than about 100 nM, less than about 50 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM, as measured by a surface plasmon resonance assay (see Example 2). In some cases, an anti -VEGF-A aptamer may have a dissociation constant (¾) for one or more isoforms or variants of VEGF-A of less than about 50 nM, as measured by a surface plasmon resonance assay (see Example 2). In some cases, an anti -VEGF-A aptamer may have a dissociation constant (¾) for one or more isoforms or variants of VEGF-A of less than about 25 nM, as measured by a surface plasmon resonance assay (see Example 2). In some cases, an anti- VEGF-A aptamer may have a dissociation constant (¾) for one or more isoforms or variants VEGF-A of less than about 10 nM, as measured by a surface plasmon resonance assay (see Example 2). In some cases, an anti -VEGF-A aptamer may have a dissociation constant (¾) for one or more isoforms or variants of VEGF-A of less than about 5 nM, as measured by a surface plasmon resonance assay (see Example 2). In some cases, an anti -VEGF-A aptamer may have a dissociation constant (¾) for one or more isoforms or variants of VEGF-A of less than about l nM, as measured by a surface plasmon resonance assay (see Example 2). In some cases, an anti- VEGF-A aptamer may have a dissociation constant (¾) for one or more isoforms or variants of VEGF-A of less than about 0.5 nM, as measured by a surface plasmon resonance assay (see Example 2). In some cases, an anti -VEGF-A aptamer may have a dissociation constant (¾) for one or more Isoforms or variants of VEGF-A of less than about 0 1 nM, as measured by a surface plasmon resonance assay (see Example 2). In some cases, the aptamer may be a pan-variant specific aptamer that binds to each ofVEGF-Ano, VEGF-A , VEGF-Aies, VEGF-A 89, and VEGF-A206 with a KQ of less than about 1000 nM, for example, less than about 500 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM, as measured by a surface plasmon resonance assay (see Example 2). In some cases, the aptamer may bind to any region of VEGF-A described herein, or a portion thereof, with a K of less than about 1000 nM, for example, less than about 500 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM, as measured by a surface plasmon resonance assay (see Example 2). In some eases, the aptamer may bind to the receptor-binding face or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 1000 nM, for example, less than about 500 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM, as measured by a surface plasmon assay (see Example 2). In some cases, the anti-VEGF-A aptamer may bind to the receptor-binding face or the receptor binding domain of VEGF-A, or portions thereof with a I from about 0.5 nM to about 25 nM, as measured by a surface plasmon resonance assay (see Example 2).
[00114] In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor-binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 50 nM as measured by a surface plasmon assay (see Example 2), and may have an IC50 of less than about 50 nM as measured by a VEGF-A :KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay ( see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some eases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor biniding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 50 nM as measured by a surface plasmon resonance assay ( see Example 2), and may have an IC50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay ( see Example 3), a KDR phosphorylation AlphaLISA8, assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K of less than about 50 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA8 assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 50 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF-A- induced angiogenesis (see Example 5). In some eases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 50 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a ¾ of less than about 50 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF -A -induced angiogenesis (see Example 5).
[00115] In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 10 nM as measured by a surface pl asmon resonance assay (see Example 2), and may have an IC50 of less than about 50 nM as measured by a VEGF- A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 10 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay ( see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see, Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K of less than about 10 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF- A, such as the receptor binding face of VEGF- A, or the receptor binding domain of VEGF- A, or portions thereof, with a Kd of less than about 10 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF- A- induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF- A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 10 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.5 nM as measured by a VEGF-A: KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K4 of less than about 10 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5).
[00116] In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 50 nM as measured by a VEGF- A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 10 nM as measured by a VEGF-A: KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K of less than about 5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an ICso of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesi s (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a ¾ of less than about 5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA^ assay (see Example 4), or an in vitro model of VEGF-A- induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a ¾ of less than about 5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.5 nM: as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA'8' assay (see Example 4), or an i vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a KQ of less than about 5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.1 nM as measured by a VEGF~A:KDR competition binding assay (see Example 3), a KDR phosphorylation Alpha) . ISA " assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
[00117] In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a ¾ of less than about 1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 50 nM as measured by a VEGF- A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA1® assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA'4' assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K of less than about 1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF-A- induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a K of less than about 1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA4’’ assay (see Example 4), or an in vitro model of VEGF -A -induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a KQ of less than about 1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.1 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA®' assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). [00118] In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a ¾ of less than about 0.5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 50 nM as measured by a VEGF- A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Ka of less than about 0.5 nM as measured by a surface plasmon resonance assay ( see Example 2), and may have an IC50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA®' assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a ICj of less than about 0.5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a ¾ of less than about 0.5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 1 nM as measured by a VEGF- A: KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF-A- induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a KQ of less than about 0.5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.5 nM as measured by a VEGF~A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA®' assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis ( see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 0.5 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.1 nM as measured by a VEGF-A :KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5).
[00119] In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 0.1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 50 nM as measured by a VEGF- A: KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 0.1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF- A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 0.1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF -A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 0.1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 1 nM as measured by a VEGF- A: KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA® assay (see Example 4), or an in vitro model of VEGF-A- induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 0.1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.5 nM as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA* assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5). In some cases, the aptamers disclosed herein may bind to a region of VEGF-A, such as the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A, or portions thereof, with a Kd of less than about 0.1 nM as measured by a surface plasmon resonance assay (see Example 2), and may have an IC50 of less than about 0.1 nM: as measured by a VEGF-A:KDR competition binding assay (see Example 3), a KDR phosphorylation AlphaLISA'8' assay (see Example 4), or an in vitro model of VEGF-A-induced angiogenesis (see Example 5).
[00120] In some aspects, the aptamers disclosed herein may have an improved half-life as compared to other therapeutics, including antibodies. In some cases, the aptamers may have an improved half-life in a biological fluid or solution as compared to an antibody. In some cases, the aptamers may have an improved half-life in vivo as compared to an antibody. In one example, the aptamers may have an improved half-life when injected into the eye (intraocular half-life) as compared to an antibody. In some cases, the aptamers may have an improved intraocular half-life when injected into the eye of a human. In some cases, the aptamers may demonstrate improved stability over antibodies under physiological conditions.
[00121] In some cases, the aptamers described herein may have an intraocular half-life of at least 7 days in a human. In some cases, the aptamers described herein may have an intraocular half-life of at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 20 days or greater in a human.
[00122] In some cases, the aptamers described herein may have an intraocular half-life of at least 1 day in a non-human animal ( e.g rodent/rabbit/monkey/chimpanzee/pig). In some cases, the aptamers described herein may have an intraocular half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days or greater in a non-human animal such as a rodent, rabbit or monkey.
[00123] In some aspects, the aptamers described herein may have a shorter half-life as compared to other therapeutics. For example, an unmodified or unconjugated aptamer may have a lower half-life as compared to a modified or conjugated aptamer, however, the low molecular weight of the unmodified or unconjugated forms may allow for orders of magnitude greater initial concentrations, thereby achieving greater duration/efficacy. In some examples, the aptamer may have an intraocular half-life of less than about 7 days in a human. In some examples, the aptamers described herein may have an intraocular half-life of less than about 6 days, less than about 5 days or even less than about 4 days in a human
[00124] The aptamers disclosed herein may demonstrate high specificity for VEGF-A versus other members of the VEGF family, including VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and P1GF. In some eases, the aptamer may be selected such that the aptamer has high affinity for VEGF-A, but with little to no affinity for VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or P1GF. In some cases, the aptamers of the disclosure may bind to VEGF-A with a specificity of at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 1,000-fold, at least 5, 000-fold, at least 10,000-fold, at least 50,000-fold, or at least 100,000-fold, or greater than 100,000-fold than the aptamers bind to VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or P1GF at relative serum concentrations.
[00125] The activity of a therapeutic agent can be characterized by the half maximal inhibitory concentration (IC50). The IC50 may be calculated as the concentration of therapeutic agent in nM at which half of the maximum inhibitory effect of the therapeutic agent is achieved. The IC50 may be dependent upon the assay utilized to calculate the value. In some examples, the IC50 of an aptamer described herein may be less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM or less than 0.01 nM as measured by a VEGF-A :KDR competition binding assay {see Example 3). In some examples, the IC50 of an aptamer described herein may be less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM or less than 0.01 nM as measured by a KDR phosphorylation AlphaLISA® assay {see Example 4). In some examples, the IC50 of an aptamer described herein may be less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM or less than 0.01 nM as measured by an in vitro model of VEGF-A- induced angiogenesis (see Example 5)
[00126] Aptamers generally have high stability at ambient temperatures for extended periods of time. The aptamers described herein may demonstrate greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or greater than 99.9% activity in solution under physiological conditions at 30 days or later
[00127] In some cases, a composition of the disclosure comprises anti-VEGF-A aptamers, wherein essentially 100%o of the anti-VEGF-A aptamers comprise nucleotides having ribose in the b-D-ribofuranose configuration. In other examples, a composition of the disclosure may comprise anti-VEGF-A aptamers, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90% of the anti-VEGF-A aptamers have ribose in the b-D- ribofuranose configuration.
Indications
[00128] In some aspects, the methods and compositions provided herein may be suitable for the treatment of ocular diseases or disorders. In some aspects, the methods and compositions provided herein may be suitable for the prevention of ocular diseases or disorders. In some aspects, the methods and compositions provided herein may be suitable to slow or halt the progression of ocular diseases or disorders. In some cases, the ocular disease or disorder is diabetic retinopathy. In some cases, the ocular disease or disorder is retinopathy of prematurity. In some cases, the ocular disease or disorder is central retinal vein occlusion. In some cases, the ocular disease or disorder is macular edema. In some cases, the ocular disease or disorder is choroidal neovascularization. In some cases, the ocular disease or disorder is neovascular (or wet) age-related macular degeneration. In some cases, the ocular disease or disorder is myopic choroidal neovascularization. In some cases, the ocular disease or disorder is punctate inner choroidopathy. In some cases, the ocular disease or disorder is presumed ocular histoplasmosis syndrome. In some cases, the ocular disease or disorder is familial exudative vitreoretinopathy. In some cases, the ocular disease or disorder is retinoblastoma.
[00129] Additional examples of ocular diseases or disorders that may be amendable to treatment by the methods and compositions provided herein may include, without limitation, pterygium, inflammatory conjunctivitis, including allergic and giant papillary conjunctivitis, infectious conjunctivitis, vernal keratoconjunctivitis, Stevens- Johnson disease, corneal herpetic keratitis, rhegmatogenous retinal detachment, pseudo-exfoliation syndrome, endophthalmitis, sc!eritis, corneal ulcers, dry eye syndrome, glaucoma, ischemic retinal disease, corneal transplant rejection, complications related to intraocular surgery such intraocular lens implantation and inflammation associated with cataract surgery, Behcet's disease, Stargardt disease, immune complex vasculitis, Fuch's disease, Vogt-Koyanagi-Harada disease, subretinal fibrosis, keratitis, vitreo-retinal inflammation, ocular parasitic infestation/migration, retinitis pigmentosa, cytomegalovirus retinitis and choroidal inflammation, ectropion, lagophthalmos,
blepharochalasis, ptosis, xanthelasma of the eyelid, parasitic infestation of the eyelid, dermatitis of the eyelid, dacryoadenitis, epiphora, dysthyroid exophthalmos, conjunctivitis, scleritis, adenovirus keratitis, corneal ulcer, corneal abrasion, snow blindness, arc eye, Thygeson’s superficial punctate keratopathy, corneal neovascularization, Fuchs’ dystrophy, keratoconus, keratoconjunctivitis sicca, iritis, sympathetic ophthalmia, cataracts, chorioretinal inflammation, focal chorioretinal inflammation, focal chorioretinitis, focal choroiditis, focal retinitis, focal retinochoroiditis, disseminated chorioretinal inflammation, disseminated chorioretinitis, disseminated choroiditis, disseminated retinitis, disseminated retinochoroiditis, exudative retinopathy, posterior cyclitis, pars p!anitis, Harada’s disease, chorioretinal scars, macula scars of posterior pole, solar retinopathy, choroidal degeneration, choroidal atrophy, choroidal sclerosis, angioid streaks, hereditary choroidal dystrophy, choroideremia, choroidal dystrophy (central arealor), gyrate atrophy (choroid), omithinaemia, choroidal haemorrhage and rupture, choroidal haemorrhage (not otherwise specified), choroidal haemorrhage (expulsive), choroidal detachment, retinoschisis, retinal artery occlusion, retinal vein occlusion, hypertensive retinopathy, diabetic retinopathy, retinopathy, retinopathy of prematurity, macular degeneration, Bull’s Eye maculopathy, epiretinal membrane, peripheral retinal degeneration, hereditary retinal dystrophy, retinitis pigmentosa, retinal haemorrhage, separation of retinal layers, central serous retinopathy, retinal detachment, macular edema, glaucoma - optic neuropathy, glaucoma suspect - ocular hypertension, primary open-angle glaucoma, primary angle-closure glaucoma, floaters, Leber’s hereditary' optic neuropathy, optic disc drusen, strabismus, ophthalmoparesis, progressive external ophthaloplegia, esotropia, exotropia, disorders of refraction and
accommodation, hypermetropia, myopia, astigmastism, anisometropia, presbyopia, internal ophthalmoplegia, amblyopia, Leber’s congenital amaurosis, scotoma, anopsia, color blindness, achromatopsia, maskun, nyctalopia, blindness, River blindness, micropthalmia, coloboma, red eye, Argyll Robertson pupil, keratomycosis, xerophthalmia, aniridia, sickle cell retinopathy, ocular neovascularization, retinal neovascularization, subretinal neovascularization; rubeosis iritis inflammatory diseases, chronic posterior and pan uveitis, neoplasms, retinoblastoma, pseudoglioma, neovascular glaucoma; neovascularization resulting following a combined vitrectomy-2 and lensectomy, vascular diseases, retinal ischemia, choroidal vascular
insufficiency, choroidal thrombosis, neovascularization of the optic nerve, diabetic macular edema, cystoid macular edema, proliferative vitreoretinopathy, and neovascularization due to penetration of the eye or ocular injury.
[00130] In some aspects, the methods and compositions provided herein are suitable for the treatment of diseases that cause one or more ocular symptoms. Non-limiting examples of symptoms which may be amenable to treatment with the methods disclosed herein include, but are not limited to choroidal or vitreal neovascularization, vascular leakage, reduced reading speed, reduced color vision, macular edema, increased retinal thickening, increase in central retinal volume and/or, macular sensitivity, loss of retinal cells, increase in area of retinal atrophy, reduced best corrected visual acuity such as measured by Snellen or ETDRS scales, reduced Best Corrected Visual Acuity under low luminance conditions, impaired night vision, impaired light sensitivity, impaired dark adaptation, impaired contrast sensitivity, worsened patient reported outcomes, and any combination thereof.
[00131] In some cases, the methods and compositions provided herein may alleviate or reduce a symptom of a disease. In some cases, treatment with an aptamer provided herein may result in a reduction in the severity of any of the symptoms described herein. In some cases, treatment with an aptamer described herein may slow, halt or reverse the progression of any of the symptoms described herein. In some cases, treatment with an aptamer described herein may prevent the development of any of the symptoms described herein. In some cases, treatment with an aptamer described herein may slow, halt or reverse the progression of a disease, as measured by the number and severity of symptoms experienced. Examples of symptoms and relevant endpoints where the aptamer may have a therapeutic effect include choroidal or retinal neovascularization, vascular leakage, reduced reading speed, reduced color vision, macular edema, increased retinal thickening, increase in central retinal volume and/or, macular sensitivity, loss of retinal cells, increase in area of retinal atrophy, reduced best corrected visual acuity such as measured by Snellen or ETDRS scales, reduced Best Corrected Visual Acuity under low luminance conditions, impaired night vision, impaired light sensitivity, impaired dark adaptation, impaired contrast sensitivity, and worsening patient reported outcomes. In some instances, treatment with an aptamer described herein may have beneficial effects as measured by clinical endpoints including reading speed, choroidal or retinal neovascularization or vascular leakage as measured by fluorescein angiography, retinal thickness as measured by Optical Coherence Tomography or other techniques, central retinal volume, number and density of retinal cells, area of retinal atrophy as measured by Fundus Photography or Fundus Autofluoresence or other techniques, best corrected visual acuity such as measured by Snellen or ETDRS scales. Best Corrected Visual Acuity under low luminance conditions, light sensitivity, dark adaptation, contrast sensitivity, and patient reported outcomes as measured by such tools as the National Eye Institute Visual Function Questionnaire and Health Related Quality of Life Questionnaires.
[00132] In some cases, the methods and compositions provided herein may alleviate or reduce a symptom of a neovascular eye disease. In some cases, treatment with an aptamer provided herein may result in a reduction in the severity of any symptoms associated with a neovascular eye disease. In some cases, treatment with an aptamer described herein may slow, halt or reverse the progression of any symptom associated with a neovascular eye disease. In some cases, treatment with an aptamer described herein may prevent the development of any symptom associated with a neovascular eye disease. In some cases, treatment with an aptamer described herein may slow, halt or reverse the progression of a neovascular eye disease, as measured by the number and severity of symptoms experienced. Non-limiting examples of symptoms associated with neovascular eye diseases where the aptamer may have a therapeutic effect include choroidal or retinal neovascularization, vascular leakage within the eye, macular edema, central retinal thickness and visual acuity.
[00133] The terms“subject” and“patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, rodents (e.g., mice, rats, rabbits, etc.) simians, humans, research animals (e.g., beagles, etc.), farm animals (e.g., pigs, horses, cows, llamas, alpacas, etc.), sport animals, and pets. In some cases, the methods described herein may be used on tissues or cells derived from a subject and the progeny of such tissues or cells. For example, aptamers described herein may be used to affect some function in tissues or cells of a subject. The tissues or cells may be obtained from a subject in vivo. In some cases, the tissues or cells are cultured in vitro and contacted with a composition provided herein (e.g., an aptamer). [00134] In some aspects, the methods and compositions provided herein are used to treat a subject in need thereof. In some cases, the subject has, is suspected of having, or is at risk of developing, an ocular disease or disorder. In some cases, the subject is a human. In some cases, the human is a patient at a hospital or a clinic. In some cases, the subject is a non-human animal, for example, a non-human primate, a livestock animal, a domestic pet, or a laboratory animal. For example, a non-human animal can be an ape (e.g, a chimpanzee, a baboon, a gorilla, or an orangutan), an old world monkey (e.g., a rhesus monkey), a new' world monkey, a dog, a cat, a bison, a camel, a cow, a deer, a pig, a donkey, a horse, a mule, a lama, a sheep, a goat, a buffalo, a reindeer, a yak, a mouse, a rat, a rabbit, or any other non-human animal
[00135] In cases where the subject is a human, the subject may be of any age. In some cases, the subject has an age-related ocular disease or disorder (e.g., age-related macular degeneration). In some cases, the subject is about 50 years or older. In some cases, the subject is about 55 years or older. In some cases, the subject is about 60 years or older. In some cases, the subject is about 65 years or older. In some cases, the subject is about 70 years or older. In some cases, the subject is about 75 years or older. In some cases, the subject is about 80 years or older. In some cases, the subject is about 85 years or older. In some cases, the subject is about 90 years or older. In some cases, the subject is about 95 years or older. In some cases, the subject is about 100 years or older. In some cases, the subject is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or greater than 100 years old. In some cases, the subject is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater than 20 years old.
[00136] In some aspects, the methods and compositions provided herein may be used to treat a subject having, suspected of having, or at risk of developing ocular symptoms as described herein. In some aspects, the methods and compositions provided herein may be used to treat a subject having, suspected of having, or at risk of developing an ocular disease as provided herein. In some cases, the methods and compositions provided herein may be used to treat a subject having, suspected of having, or at risk of developing an ocular disease or disorder. In some cases, the ocular disease or disorder is diabetic retinopathy. In some cases, the ocular disease or disorder is retinopathy of prematurity. In some cases, the ocular disease or disorder is central retinal vein occlusion. In some cases, the ocular disease or disorder is macular edema. In some cases, the ocular disease or disorder is choroidal neovascularization. In some cases, the ocular disease or disorder is neovascular (or wet) age-related macular degeneration. In some cases, the ocular disease or disorder is myopic choroidal neovascularization. In some cases, the ocular disease or disorder is punctate inner choroidopathy. In some cases, the ocular disease or disorder is presumed ocular histoplasmosis syndrome. In some cases, the ocular disease or disorder is familial exudative vitreoretinopathy. In some cases, the ocular disease or disorder is retinoblastoma. In some cases, the methods and compositions provided herein may be used to treat a subject having, suspected of having, or at risk of developing an ocular disease or disorder exhibiting elevated levels of one or more isoforms or variants of VEGF-A.
Pharmaceutical compositions or medicaments
[00137] Disclosed herein are pharmaceutical compositions or medicaments, used
interchangeably, for use in a method of therapy, or for use in a method of medical treatment. Such use may be for the treatment of ocular diseases or disorders. In some cases, the
pharmaceutical compositions can be used for the treatment of an ocular disease or disorder. In some cases, the pharmaceutical compositions comprise one or more anti-VEGF-A aptamers for the treatment of an ocular disease or disorder. In some cases, the ocular disease or disorder is diabetic retinopathy. In some cases, the ocular disease or disorder is retinopathy of prematurity. In some cases, the ocular disease or disorder is central retinal vein occlusion. In some cases, the ocular disease or disorder is macular edema. In some cases, the ocular disease or disorder is choroidal neovascularization. In some cases, the ocular disease or disorder is neovascular (or wet) age-related macular degeneration. In some cases, the ocular disease or disorder is myopic choroidal neovascularization. In some cases, the ocular disease or disorder is punctate inner choroidopathy. In some cases, the ocular disease or disorder is presumed ocular histoplasmosis syndrome. In some cases, the ocular disease or disorder is familial exudative vitreoretinopathy. In some cases, the ocular disease or disorder is retinoblastoma. In some cases, the
pharmaceutical compositions can be used for the treatment of an ocular disease or disorder that exhibits elevated levels of one or more isoforms or variants of VEGF-A.
[00138] In some cases, the one or more anti-VEGF-A aptamers may bind to one or more isoforms or variants of VEGF-A. In some cases, the one or more anti-VEGF-A aptamers are pan-variant specific aptamers that bind to each of VEGF-Ano, VEGF-Am, VEGF-Aies, VEGF- Ai89, and VEGF-A2o6· In some cases, the one or more anti-VEGF-A aptamers may bind to the receptor binding face of VEGF-A, or a portion thereof. The receptor binding face of VEGF-A may include strands b2, b5, and bό and loop bΐ and b2 of a first monomer, and the N -terminal a helix and loop b3 to b4 of the second monomer (see Muller, Yves A., et al . "The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding " Structure 5.10 (1997): 1325-1338 ). In some cases, anti- VEGF-A aptamers that bind to the receptor binding face of VEGF-A may bind to one or more of residues Phel7, Ile43, Ile46, Glu64, Gln79, Ile83, Lys84, Pro85, Arg82, His86, Asp63, Glu67, as described in SEQ ID NOs: 1-5. In some cases, the one or more anti -VEGF -A aptamers may bind to the receptor binding domain of VEGF-A, or a portion thereof. The receptor binding domain of VEGF-A may include any one or more of residues 1-109, as described in SEQ ID NOs: 1-5. In some cases, the one or more anti-VEGF-A aptamers may prevent or reduce the binding of one or more isoforms or variants of VEGF-A with Flt-1, KDR, or Nrp-1. In some cases, the compositions may include, e.g., an effective amount of the aptamer, alone or in combination, with one or more vehicles (e.g., pharmaceutically acceptable compositions or e.g., pharmaceutically acceptable carriers).
Formulations
[00139] Compositions as described herein may comprise a liquid formulation, a solid
formulation or a combination thereof. Non -limiting examples of formulations may include a tablet, a capsule, a gel, a paste, a liquid solution and a cream. The compositions of the present disclosure may further comprise any number of excipients. Excipients may include any and all solvents, coatings, flavorings, colorings, lubricants, disintegrants, preservatives, sweeteners, binders, diluents, and vehicles (or carriers). Generally, the excipient is compatible with the therapeutic compositions of the present disclosure. The pharmaceutical composition may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as, for example, sodium acetate, and
triethanolamine oleate.
Dosage and Routes of Administration
[00140] Therapeutic doses of formulations of the disclosure can be administered to a subject in need thereof. In some cases, a formulation is administered to the eye of a subject for the treatment of an ocular disease as described herein. Administration to the eye can be; a) local ocular delivery; or b) systemic. A topical formulation can be applied directly to the eye (e.g., eye drops, contact lens loaded with the formulation) or to the eyelid (e.g, cream, lotion, gel). In some cases, topical administration can be to a site remote from the eye, for example, to the skin of an extremity. This form of administration may be suitable for targets that are not produced directly by the eye. In some cases, a formulation of the disclosure is administered by local ocular delivery'. Non-limiting examples of local ocular delivery' include intravitreai (IVT), intracamarel, subconjunctival, subtenon, retrobulbar, posterior juxtascleral, and peribulbar. In some cases, a formulation of the disclosure is delivered by intravitreai administration (IVT). Local ocular delivery may generally involve injection of a liquid formulation. In other cases, a formulation of the disclosure is administered systemically. Systemic administration can involve oral administration. In some cases, systemic administration can be intravenous administration, subcutaneous administration, infusion, implantation, and the like.
[00141] Other formulations suitable for delivery of the pharmaceutical compositions described herein may include a sustained release gel or polymer formulations by surgical implantation of a biodegradable microsize polymer system, e.g., microdevice, microparticle, or sponge, or other slow release transscleral devices, implanted during the treatment of an ophthalmic disease, or by an ocular delivery device, e.g., polymer contact lens sustained delivery device. In some cases, the formulation is a polymer gel, a self-assembling gel, a durable implant, an eluting implant, a biodegradable matrix or biodegradable polymers. In some cases, the formulation may be administered by iontophoresis using electric current to drive the composition from the surface to the posterior of the eye. In some cases, the formulation may be administered by a surgically implanted port with an intravitreai reservoir, an extra-vitreal reservoir or a combination thereof. Examples of implantable ocular devices can include, without limitation, the Durasert™ technology developed by Bausch & Lomb, the ODTx device developed by On Demand
Therapeutics, the Port Delivery System developed by ForSight VISION4 and the Replenish MicroPump IM System developed by Replenish, Inc. In some cases, nanotechnologies can be used to deliver the pharmaceutical compositions including nanospheres, nanoparticles, nanocapsules, liposomes, nanomicelles and dendrimers.
[00142] A composition of the disclosure can be administered once or more than once each day.
In some cases, the composition is administered as a single dose (i.e., one-time use). In this example, the single dose may be curative. In other cases, the composition may be administered serially (e.g., taken every day without a break for the duration of the treatment regimen). In some cases, the treatment regime can be less than a week, a week, two weeks, three weeks, a month, or greater than a month. In some cases, the composition is administered over a period of at least 12 weeks, at least 16 weeks, at least 20 weeks, or at least 24 weeks. In other cases, the composition is administered for a day, at least two consecutive days, at least three consecutive days, at least four consecutive days, at least five consecutive days, at least six consecutive days, at least seven consecutive days, at least eight consecutive days, at least nine consecutive days, at least ten consecutive days, or at least greater than ten consecutive days. In some cases, a therapeutically effective amount can be administered one time per week, two times per week, three times per week, four times per week, five times per week, six times per week, seven times per week, eight times per w'eek, nine times per week, 10 times per week, 11 times per week, 12 times per week,
13 times per week, 14 times per week, 15 times per w'eek, 16 times per week, 17 times per week,
18 times per week, 19 times per week, 20 times per week, 25 times per week, 30 times per week,
35 times per w'eek, 40 times per week, or greater than 40 times per week. In some cases, a therapeutically effective amount can be administered one time per day, two times per day, three times per day, four times per day, five times per day, six times per day, seven times per day, eight times per day, nine times per day, 10 times per day, or greater than 10 times per day. In some cases, the composition is administered at least twice a day. In further eases, the
composition is administered at least every hour, at least every two hours, at least every three hours, at least ever}' four hours, at least every five hours, at least every six hours, at least every' seven hours, at least every eight hours, at least every' nine hours, at least every' 10 hours, at least every' 11 hours, at least every' 12 hours, at least every' 13 hours, at least every 14 hours, at least every' 15 hours, at least every 16 hours, at least every 17 hours, at least every' 18 hours, at least every 19 hours, at least every' 20 hours, at least every 21 hours, at least every' 22 hours, at least every 23 hours, or at least every day
[00143] Aptamers as described herein may be particularly advantageous over antibodies as they may sustain therapeutic intravitrea! concentrations of drug for longer peri ods of time, thus requiring less frequent administration. The aptamers described herein may have a longer intraocular half-life, and/or sustain therapeutic intravitreal concentrations of drug for longer periods of time than an anti-VEGF-A antibody therapy and can be dosed less frequently. In some cases, the aptamers of the disclosure are dosed at least once every' 4 weeks (q4w), once every 5 weeks (qSw), once every 6 weeks (q6w), once every 7 weeks (q7w), once every 8 weeks (q8w), once ever}' 9 weeks (q9w), once every 10 weeks (qlOw), once every 11 weeks (ql Iw) once every 12 weeks (ql2w), once every 13 weeks (ql3w), once every 14 weeks (ql4w), once every 15 weeks (q15w), once every 16 weeks (ql6w), once every 17 weeks (ql7w), once every 18 weeks (ql 8w), once every 19 weeks (ql9w), once every 20 weeks (q20w), once every 21 weeks (q21w), once every 22 weeks (q22w), once every 23 weeks (q23w), once every 24 weeks (q24w), or greater than once every 24 weeks.
[00144] In some aspects, a therapeutically effective amount of the aptamer may be administered. A“therapeutically effective amount” or“therapeutically effective dose” are used interchangeably herein and refer to an amount of a therapeutic agent (e.g, an aptamer) that provokes a therapeutic or desired response in a subject. The therapeutically effective amount of the composition may he dependent on the route of administration. In the case of systemic administration, a therapeutically effective amount may be about 10 mg/kg to about 100 mg/kg. In some cases, a therapeutically effective amount may be about 10 pg/kg to about 1000 pg/kg for systemic administration. For i travitreal administration, a therapeutically effective amount can be about 0.01 mg to about 150 mg in about 25 pi to about 100 mΐ volume per eye.
Methods of Treatment
[00145] Disclosed herein are methods for the treatment of ocular diseases or disorders. In some cases, the ocular disease or disorder may be diabetic retinopathy. In some cases, the ocular disease or disorder may be retinopathy of prematurity. In some cases, the ocular disease or disorder may be central retinal vein occlusion. In some cases, the ocular disease or disorder may be macular edema. In some cases, the ocular disease or disorder may be choroidal
neovascularization. In some cases, the ocular disease or disorder may be neovascular (or wet) age-related macular degeneration. In some cases, the ocular disease or disorder may be myopic choroidal neovascularization. In some cases, the ocular disease or disorder may be punctate inner choroidopathy. In some cases, the ocular disease or disorder may be presumed ocular histoplasmosis syndrome. In some cases, the ocular disease or disorder may be familial exudative vitreoretinopathy. In some cases, the ocular disease or disorder may be
retinoblastoma. In some cases, the ocular disease or disorder may exhibit elevated levels of one or more isoforms or variants of VEGF-A. [00146] In some cases, the method involves administering a therapeutically effective amount of a composition to a subject to treat an ocular disease. In some cases, the composition includes one or more aptamers as described herein. The aptamers may bind to and inhibit a function associated with one or more isoforms or variants of VEGF-A as described herein. The methods can be performed at a hospital or a clinic, for example, the pharmaceutical compositions can be administered by a health-care professional. In other cases, the pharmaceutical compositions can be self-administered by the subject. Treatment may commence with the diagnosis of a subject with an ocular disease. In the event that further treatments are necessary, follow-up appointments may be scheduled for the administration of subsequent doses of the composition, for example, administration every 8, 12, 16, 20, or 24 weeks.
[00147] Further disclosed herein are methods of using an anti-VEGF-A composition of the disclosure to inhibit a function associated with VEGF-A. For example, the methods may involve administering a composition of the disclosure, including one or more anti-VEGF-A aptamers, to a biological system (e.g, biological cells, biological tissue, a subject) to inhibit a function associated with VEGF-A. In some cases, the anti-VEGF-A aptamers may bind to the receptor binding face of VEGF-A, or portions thereof. In some cases, the anti-VEGF-A aptamers may bind to the receptor binding domain of VEGF-A. In some cases, the methods may be used to prevent or reduce binding of VEGF-A to Fit- 1 , KDR, Nrp-1, or any combination thereof. In some cases, the methods may be used to inhibit downstream signaling pathways associated with VEGF-A.
Methods of Generating Aptamers
The SELEX™ Method
[00148] The aptamers described herein can be generated by any method suitable for generating aptamers. In some cases, the aptamers described herein are generated by a process known as Systematic Evolution of Ligands by Exponential Enrichment" ("SELEX1M"). The SELEX f process is described in, e.g, U.S. patent application Ser. No. 07/536,428, filed Jun. 1 1, 1990, now abandoned, U.S. Pat. No. 5,475,096 entitled "Nucleic Acid Ligands", and U.S. Pat. No. 5,270,163 (see, also WO 91/19813) entitled "Nucleic Acid Ligands", each of which are herein incorporated by reference. By performing iterative cycles of selection and amplification, SELEX™ may be used to obtain aptamers with any desired level of target binding affinity. [00149] The SELEX1M method generally relies as a starting point upon a large library or pool of single stranded oligonucleotides comprising randomized sequences. The oligonucleotides can be modified or unmodified DNA, RNA, or DNA/RNA hybrids. In some examples, the pool comprises 100% random or partially random oligonucleotides. In other examples, the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and/or conserved sequence incorporated within randomized sequence. In other examples, the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and/or conserved sequence at its 5' and/or 3' end which may comprise a sequence shared by all the molecules of the oligonucleotide pool. Fixed sequences are sequences common to oligonucleotides in the pool which are incorporated for a preselected purpose such as, CpG motifs, hybridization sites for PCR primers, promoter sequences for RNA polymerases (e.g., T3, T4, T7, and SP6), sequences to form stems to present the randomized region of the library' within a defined terminal stem structure, restriction sites, or homopolymeric sequences, such as poly A or poly T tracts, catalytic cores, sites for selective binding to affinity columns, and other sequences to facilitate cloning and/or sequencing of an oligonucleotide of interest. Conserved sequences are sequences, other than the previously described fixed sequences, shared by a number of aptamers that bind to the same target.
[00150] The oligonucleotides of the pool can include a randomized sequence portion as well as fixed sequences necessary for efficient amplification. Typically the oligonucleotides of the starting pool contain fixed 5' and 3' terminal sequences which flank an internal region of 30-50 random nucleotides. The randomized nucleotides can be produced in a number of ways including chemical synthesis and size selection from randomly cleaved cellular nucleic acids. Sequence variation in test nucleic acids can also be introduced or increased by mutagenesis before or during the selection/amplification iterations.
[00151] The random sequence portion of the oligonucleotide can be of any length and can comprise ribonucleotides and/or deoxyribonucleotides and can include modified or non-natural nucleotides or nucleotide analogs. Typical syntheses carried out on automated DNA synthesis equipment yield 10 4-l 0io individual molecules, a number sufficient for most SELEX‘M experiments. Sufficiently large regions of random sequence in the sequence design increases the likelihood that each synthesized molecule is likely to represent a unique sequence. [00152] The starting library of oligonucleotides may be generated by automated chemical synthesis on a DNA synthesizer. To synthesize randomized sequences, mixtures of all four nucleotides are added at each nucleotide addition step during the synthesis process, allowing for random incorporation of nucleotides. As stated above, in some cases, random oligonucleotides comprise entirely random sequences; however, in other cases, random oligonucleotides can comprise stretches of nonrandom or partially random sequences. Partially random sequences can be created by adding the four nucleotides in different molar ratios at each addition step.
[00153] The starting library of oligonucleotides may be RNA, DNA, substituted RNA or DNA or combinations thereof. In those instances where an RNA library is to be used as the starting library it is typically generated by synthesizing a DNA library', optionally PCR amplifying, then transcribing the DNA library' in vitro using T7 RNA polymerase or modified T7 RNA
polymerases, and purifying the transcribed library. The nucleic acid library is then mixed with the target under conditions favorable for binding and subjected to step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. More specifically, starting with a mixture containing the starting pool of nucleic acids, the SELEX1M method includes steps of: (a) contacting the mixture with the target under conditions favorable for binding; (b) partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; (c) dissociating the nucleic acid-target complexes; (d) amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids; and (e) reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule. In those instances where RNA aptamers are being selected, the SELEXiM method further comprises the steps of: (i) reverse transcribing the nucleic acids dissociated from the nucleic acid-target complexes before amplification in step (d); and (ii) transcribing the amplified nucleic acids from step (d) before restarting the process.
[00154] Within a nucleic acid mixture containing a large number of possible sequences and structures, there is a wide range of binding affinities for a given target. Those which have the higher affinity (lower dissociation constants) for the target are most likely to bind to the target. After partitioning, dissociation and amplification, a second nucleic acid mixture is generated, enriched for the higher binding affinity candidates. Additional rounds of selection progressively favor the best ligands until the resulting nucleic acid mixture is predominantly composed of only one or a few sequences. These can then be cloned, sequenced and individually tested as ligands or aptamers for 1) target binding affinity; and 2) ability to effect target function.
[00155] Cycles of selection and amplification are repeated until a desired goal is achieved. In the most general case, selection/amplification is continued until no significant improvement in binding strength is achieved on repetition of the cycle. The method is typically used to sample approximately iO14 different nucleic acid species but may be used to sample as many as about l(3lS different nucleic acid species. Generally, nucleic acid aptamer molecules are selected in a 3 to 20 cycle procedure.
[00156] In some cases, the aptamers of the disclosure are generated using the SELEX1M method as described above. In other cases, the aptamers of the disclosure are generated using any modification or variant of the SELEX method.
Methods of generating pan-variant specific anti-VEGF-A aptamers
[00157] Further provided herein are methods for generating and screening pan-variant specific anti-VEGF-A aptamers. In some cases, the pan-variant specific aptamers bind to the receptor binding face of VEGF-A, or the receptor binding domain of VEGF-A. Generally, the methods provided herein bias the selection process towards aptamers that selectively bind to the receptor binding face or receptor binding domain of VEGF-A. In some cases, such aptamers do not bind to the heparin binding domain of VEGF-A. In various aspects, the methods may involve incubating an aptamer library with an isoform or variant of VEGF-A which contains a receptor binding domain but does not contain a heparin binding domain. In some cases, the isoform or variant of VEGF-A is VEGF-Am or VEGF-Ano. In various aspects, the methods involve immobilizing the VEGF-A variant on a solid support in a manner that does not preclude access of the library to the receptor binding face or receptor binding domain of VEGF-A. In various aspects, the methods involve performing the selection in the absence of Ca^
[00158] In various aspects, methods for screening pan-variant specific anti-VEGF-A aptamers are provided. In some cases, the methods may involve measuring the interaction of a candidate aptamers with recombinant bead-bound VEGF-Ai6 , VEGF-Am. or VEGF-Ano by flow cytometry' {see Example 1). In such cases, interaction with each isoform or variant would indicate binding to the receptor binding domain, while only binding to VEGF-Ai65 would indicate recognition of the heparin binding domain. In some cases, the methods may involve measuring the ability of the candidate aptamer to inhibit or reduce the interaction of VEGF~A!65, VEGF-Am, or VEGF-Ano with KDR by a reduction in FRET signal {see Example 3). In such cases, efficacy against each variant would indicate binding to the receptor binding domain, while only inhibiting VEGF-Aies would indicate binding to the heparin binding domain. In some cases, the methods may involve measuring the ability of the candidate aptamer to inhibit or reduce VEGF-Aies, VEGF-Am, or VEGF-Ano induced trans autophosphorylation of the intracellular domain of KDR by phospho-KDR AlphaLISA^ {see Example 4). In such cases, efficacy against each variant would indicate binding to the receptor binding domain, while only inhibiting VEGF165 would indicate binding to the heparin binding domain. In some cases, the methods may involve measuring the ability of the candidate aptamer to inhibit or reduce VEGF- Ai65, VEGF-Am, or VEGF-Ano induced gene expression of tissue factor in HUVEC cells as measured by qPCR. In such cases, efficacy against each variant would indicate binding to the receptor binding domain, while only inhibiting VEGFies would indicate binding to the heparin binding domain. In some cases, the methods may involve measuring the ability of the candidate aptamer to inhibit or reduce VEGF-Aies, VEGF-Am, or VEGF-Ano induced tube formation of GFP-HUVECs in co-culture with human dermal fibroblasts ceils by change in network length or network area {see Example 5). In such cases, efficacy against each variant would indicate binding to the receptor binding domain, while only inhibiting VEGF-Ai65 would indicate binding to the heparin binding domain. In some cases, the methods may involve measuring the ability of the candidate aptamer to inhibit or reduce vascular leakage in a mouse, rat, rabbit, or primate eye following exogenous VEGF-Aies, VEGF-Am. or VEGF-Ano challenge by fluorescein angiography and Evans-blue albumin staining. In such cases, efficacy against each VEGF isoform or variant would indicate binding to the receptor binding domain, while only inhibiting VEGF-Ai65 would indicate binding to the heparin binding domain.
EXAMPLES
[00159] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
Example 1A. Selection of anti-VEGF-A aptamers
[00160] Anti-VEGF-A (VEGF) aptamers targeting the receptor binding domain (RBD) of VEGF-A were identified using an N30 library (N30S) comprised of a 30-nucleotide random region flanked by constant regions containing a built-in stem region as depicted in FIG. 1A. The sequence in italics represents the forward and reverse primer binding sites. The built-in stem region is shown in bold and underlined. FIG. IB depicts a representation of the N30S library with the reverse primer hybridized. For nuclease stability, the library was composed of 2’- fluoro-G (2’F GTP) and T -O-methyl (2’OMe) A/C/TJ. FIG. 1C depicts structures of modified nucleotides used to generate the N30S library for selection against target VEGF. For simplicity, the nucleosides, and not the nucleotide triphosphates are shown. The library sequence
(underlined sequences represent the built-in stem) and the sequence of oligos used to amplify the library are described in Table 3.
Table 3. Library sequence and sequence of oligos used to amplify the library
Figure imgf000067_0001
[00161] The starting library was transcribed from a pool of ~1014 double-stranded DNA
(dsDNA) molecules. The dsDNA library w'as generated by primer extension using Klenow exo (-) DNA polymerase, the pool forward primer (N30S.F; SEQ ID NO: 9) and a synthetic single- stranded DNA (ssDNA) molecule encoding the reverse complement of the library. The dsDNA w'as subsequently converted to 100% backbone modified RNA via transcription using a mixture of 2’F GXP, 2’OMe ATP/CTP/UTP and a modified phage polymerase in buffer optimized to facilitate efficient transcription. Following transcription, RNAs were treated with DNAse to remove the template dsDNA and were purified.
[00162] In order to isolate pan-variant specific aptamer inhibitors of VEGF-A that bind to the RBD and inhibit VEGF-A via directly blocking its interaction with its receptor, KDR (also known as VEGFR-2), and to avoid the identification of anti -VEGF aptamers that bind
exclusively to the heparin binding domain (HBD) of VEGF, aptamer selections were performed using a combination of the 121 and 110 variants of VEGF-A. Both of these variants lack the HBD but possess the RBD and maintain high affinity for KDR.
[00163] Aptamer selection was facilitated by the use of biotin-tagged recombinant human VEGF 121 or VEGF no. To ensure the biotin tag did not interfere with the availability of desired epitopes within the RBD during the selection, biotin was chemically conjugated to the terminal sugars on the glycan. All VEGF-A monomers bear a single N-linked glycosylation site on Asn74, which is located in the center of the RBD, distal to the receptor binding face, making this an ideal site for target immobilization. In brief, VEGF-A glycans were oxidized with 100 mM sodium periodate for 30 minutes, and excess periodate was quenched with 1 mM glycerol. A 10- fold excess of biotin-PEGi2-alkoxyamine and anthranilic acid (10 mM final concentration) were added to the reaction and allowed to incubate for another 2 hours. Reactions were cleaned up by membrane filtration using a 10 kDa cutoff filter.
[00164] Biotinylated VEGF was immobilized on magnetic streptavidin capture beads
(Dynabeads^ My One 1 Strepavidin Cl) at a constant ratio of 1 pL beads per 1 pg protein.
Briefly, beads were washed three times with binding buffer SB 1T-Ca++ (40 mM F1EPES, pH 7.5, 125 mM NaCi, 5 mM KC1, 1 mM MgCl2, 0.05% Tween-20) and were resuspended in 50 pi. of recombinant VEGF in SB 1 T Ca and incubated at room temperature for 30 minutes. The beads were subsequently blocked with excess biotin for 10 minutes. The amount of target protein varied with each round (Table 4). The beads w?ere washed three times with SB 1 T-Ca buffer to remove any unbound protein.
[00165] For the first round of selection, ~1 nanomole of the Round 0 RNA pool (~3x coverage for lxlO14 sequences) was used. Prior to each round, the library' was thermally equilibrated by heating at 90°C for 3 minutes and cooled at room temperature for 15 minutes in the presence of a 1.5-fold molar excess of reverse primer (N30S.R; SEQ ID NO: 10) to allow the library to refold.
Figure imgf000069_0001
Following renaturation, the final volume of the reaction was adjusted to 50 pL in SB I F-i'a supplemented with 1 pg/rnl ssDNA and 0.1% BSA.
[00166] For the first round, the library was added to VEGFno immobilized on beads and incubated at 37°C for 30 minutes on a tube rotary. After 30 minutes, the beads were washed three times using 0.5 ml SB1T-Ca++ buffer to remove unbound aptamers. After washing, VEGFno-bound aptamers were eluted using 200 pL elution buffer (2M Guanidine-HCl in SBIT buffer) two times (total volume 400 pL). The eluted aptamers, in 400 pL of elution buffer, were ethanol precipitated. The recovered library was converted to DNA by reverse transcription using Super Script41 IV reverse transcriptase, and the ssDNA was subsequently amplified by PCR. The resulting dsDNA library was subsequently converted back into modified RNA via transcription as described above. DNased, purified RNA was used for subsequent rounds.
[00167] Following the first round, a negative selection step was included in ail of the subsequent rounds. For the negative selection, the pool was prepared as described before and incubated with biotin-saturated beads (in the absence of target protein) for 30 minutes at 37°C in SBIT-Ca”4 buffer. The beads were pelleted and the supernatant, containing molecules that did not bind to the beads, was incubated with VEGF -labeled beads for an additional 30 minutes at 37°C. For rounds 2-4, the input RNA was kept fixed at 25 picomoles, and the protein target was fixed at 0.5 pg.
[00168] For rounds 5-7, the target was varied between VEGFi2i and VEGFno and a solution capture method was implemented. Negative selections were implemented as described above, and positive selection involved incubation with biotinylated target without the presence of beads for 30 minutes at 37°C. For capture, 2 pL of streptavidin beads were washed with 0.5 mL of SB I T-Ca-- and the positive selection was used to resuspend the bead pellet. After a five minute incubation, the beads were washed according to Table 4 prior to elution and precipitation as described above.
Table 4. Selection Details
Figure imgf000069_0002
Figure imgf000070_0001
Exampie IB. Assessing the progress of selection
[00169] Flow cytometry was used to assess the progress of the selection. For these assays, RNA from each round was first hybridized with reverse complement oligonucleotide composed of 2’OMe RNA labeled with Dyligh†~ 650 (Dy650~N30S.R.OMe, sequence identical to N30S.R (SEQ ID NO: 10)). Briefly, the library was combined with 1 5-fold molar excess of Dy650- N30S.R.QMe, heated at 90°C for 3 minutes and allowed to cool at room temperature for 15 minutes, after which it was incubated with unlabeled“Negative” beads and beads labelled with VEGF121 in SB IT-Ca"” buffer supplemented with 0.1% BSA and 1 mg/niL ssDNA final.
Following incubation for 30 minutes at 37°C, the beads were saturated by the addition of 1 mM free biotin, incubated for 5 minutes, washed three times with SB IT Ca”+, re-suspended in SB IT - Car buffer, and analyzed by flow cytometry. As shown in FIG. 2A and FIG, 2B, an
improvement in fluorescent signal was observed by Round 4. After Round 5, there was little change in the binding signal through Round 7. “121” refers to the signal of VEGF 121 -labelled beads in the absence of labeled RNA. Titrations of Rounds 5-7 prepared as described above also showed dose-dependent responses in median fluorescence for the bead populations
functionalized with VEGF and VEGFno, indicating that the selection strategy was successful in enriching for aptamers that bind to the RBD of VEGF -A (FIG. 2C and FIG. 2D).
Importantly, similar binding experiments performed in the presence of 1 mM non-biotinylated labeled VEGFm demonstrated a significant loss in binding signal (FIG. 2E). Thus, the iterative rounds of selection resulted in the enrichment of an aptamer population capable of specifically binding the RBD of VEGF- A when both, immobilized on beads, and in solution.
Example 1C. Selection, purification and characterization of clones
[00170] The enriched aptamer population from Round 7 was cloned into a TOPO® TA vector, transformed into competent cells, and sequenced by Sanger sequencing. All in silica analyses were performed using Geneious software (Biomatters Inc., Newark NJ, USA). Of the 45 sequences identified, 10 were unique and ail fell into a single family with one sequence representing 32 identical reads. The dominant sequence, Aptamer 4.2 (r7-01; SEQ ID NO: 7) was chemically synthesized with 2’-fluoro-G and 2’-0-methyl (2’OMe) A/C/IJ modified phosphoramidites along with a 3’ inverted deoxythymidine and a 5’ C6 disulfide linker, which was conjugated to Dylight® 650 maleimide (Table 5). The aptamer was purified by reversed phase high-performance liquid chromatography (HPLC) and assayed for activity in the flow cytometry assay described above. A dose response against beads bearing VEGFm or VEGFies demonstrated that Aptamer 4.2 was capable of binding to both isoforms. Therefore, Aptamer 4.2 bound to a common epitope on multiple isoforms of VEGF-A, with VEGF-A recognition independent of the HBD (FIG. 3) . In combination with the bead binding results above for VEGFno, the surface plasmon resonance binding data shown in Example 2, and the receptor competition shown below in Example 3, these data support that aptamers in this family of molecules bind to the VEGF-A RED.
Table 5. Chemical synthesis and testing of Round 7 seqnenees
Figure imgf000071_0001
Example 2. Binding affinity and confirmation of pan-variant specificity with surface plasmon resonance.
[00171] To measure binding affinity of the identified aptamer and confirm its pan-variant specific binding to VEGF-A variants described in Example 1, surface plasmon resonance (SPR) experiments were conducted against VEGFies, VEGFm, and VEGFno on Aptamer 26 (Table 5) as compared to a previously described HBD binding aptamer, Aptamer 7 (Table 6) (Ruckman et a!., 1998). Aptamer 26 was identical in sequence to Aptamer 4.2 but contained a hexyl amine linker in place of the C6 disulfide linker at the 5’ terminus (Table 5). Aptamer 7 has been demonstrated previously to bind directly to the HBD of VEGF-A at an epitope overlapping the binding site of the VEGF165 co-receptor neuropilin-1 (Ruckman et al., 1998). Briefly, in the SPR studies, 1.0-2.5 pg/mL of glycan biotinylated VEGF^s, VEGF , and VEGFUo were diluted in HEPES running buffer (10 mM HEPES, 137.5 rnM NaCl, 5.7 IDM KC1, 1 mM MgCl2, 1 mM CaCl?., and 0.05% Tween20; pH 7.4) and were immobilized to a streptavidin dextran chip (GE Healthcare Life Sciences) for 1-2 minutes. The chip was then blocked with 0.2 mg/mL biotin in running buffer, followed by three cycles of buffer blanks and regeneration with 50 mM NaOH to equilibrate the chip. This resulted in 200-3000 RIU immobilized. After coating and blocking, samples were screened at 100 nM in duplicate injections at a flow rate of 25 pL/niin, with an association time of 3 minutes 20 seconds and a dissociation time of 10 minutes. Regeneration conditions of chips were optimized based on the protein immobilized and were effected with either 50 mM NaOH for 30 seconds or 2M guanidine HCi for 2 x 40 seconds. To determine binding affinities, aptamers were subsequently ran in an 11 -point, 2-fold dose response with Aptamer 26 (SEQ ID NO: 7) starting at a top concentration of 1.0 mM, and Aptamer 7 starting at 500 nM.
Table 6. An anti-VEGF HBD aptamer
Figure imgf000072_0001
where inG and inA are 2’OMe modified RNA; fC and fU are 2’F modified RNA; C6NH2 is a hexylainine linker; and idT is an inverted deoxythymidine residue.
[00172] Data was fit to a 1 : 1 kinetic binding model. Binding affinities were calculated based on these fits and are shown in Table 7. Results of the assay demonstrate that Aptamer 26 bound to all three variants ofVEGF-A tested, whereas, consistent with the literature, Aptamer 7 only bound to VEGF165. The affinity for Aptamer 7 to VEGF es as determined here is consistent with the literature reported value (Ruckman et ah, 1998). This result provides further evidence that the selection effectively identified pan-variant specific anti-VEGF- A aptamers, which recognize an epitope of VEGF-A contained within the RBD. This differentiates the aptamers identified in the selection described herein from previously described aptamers to VEGF-A, such as Aptamer 7, which recognize an epitope of VEGF-A contained within the HBD, and thus do not bind the non-HBD bearing variants VEGFno and VEGF .
Table 7. Binding affinity (nM) of aptamers to VEGF-A variants as determined by SPR
Figure imgf000073_0001
Example 3, Characterization of mechanisms of interaction by VEGF-A:KDR competition binding.
[00173] The SPR data in Example 2 demonstrates that Aptamer 26 binds to the RBD of VEGF- A. To further define the epitope within the RBD of VEGF-A recognized by Aptamer 26, the mechanism of action of Aptamer 26 was interrogated by testing the ability of Aptamer 26 to directly inhibit KDR binding to either VEGFies or VEGFm as compared to a clone of an anti- VEGF antibody (Ferrara, Napoleone, et al. "Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer." Nature reviews Drug discovery 3.5 (2004): 391.) known to bind an epitope contained in the RBD that defines the receptor binding face of VEGF- A (SEQ ID NO: 13 and SEQ ID NO: 14 shown in Table 8), and as compared to Aptamer 7.
[00174] Briefly, the aptamers were heated at 90°C for 3 minutes and allowed to cool to room temperature for a minimum of 5 minutes Aptamers and anti-VEGF antibody were serial diluted in a polypropylene plate and 5 pL was transferred to a white low volume 384-well Optiplate (Perkin Elmer). A solution of VEGFies (Aero BioSystems) or VEGFm (Aero BioSystems) that was giycan biotinylated was prepared and added to the assay plate containing aptamers or anti- VEGF antibody to yield a fi nal assay concentration of 2 nM. A mixture of human recombinant his-tagged KDR (Sino Biological), AlphaLISA^ nickel chelate acceptor beads (Perkin Elmer), and AlphaScreen®' streptavidin donor beads (Perkin Elmer) was then prepared and 10 pL was added to the assay plate. The final concentration of KDR and beads was 5 nM and 5 ug/mL, respectively. The assay plate was sealed and incubated in the dark for approximately 2 hours, and then was read on a Biotek CYTATION " 5 plate reader using the Alpha 384-well optical cub e. The low control was determined using excess anti-VEGF inhibitor and the high control was determined by buffer only
[00175] Percent inhibition for each sample was calculated by the following formula;
% inhibition = 1 -(sample-low control)/(high control-low control)* 100
The values were fit by using a four-parameter non-linear fit in GraphPad Prism Version 7.0.
[00176] The results (FIG. 4A and FIG. 4B) demonstrate that Aptamer 26 (SEQ ID NO: 7) and the anti-VEGF mAh directly blocked the interaction of VEGF-A (VEGF ½5 or VEGFm) with KDR. In contrast, despite its high affinity for VEGFigs as provided in Example 2, Aptamer 7 shows no activity in this assay against VEGF 65, demonstrating that it does not directly inhibit the interaction between VEGF-A and its receptor. As expected, given the lack of interaction between Aptamer 7 and VEGFm. Aptamer 7 also showed no inhibition of the interaction between this variant of VEGF-A and KDR. Aptamer 26 blocked the interaction of VEGF ½5 with KDR with an IC50 of 1.8 ± 1.3 nM and the interaction of VEGFm with KDR with an IC50 of 19 ± 19 nM, consistent with the affinity of Aptamer 26 for the respective VEGF-A variants.
Similarly, the anti-VEGF mAb also inhibited the interaction of VEGFies and VEGF 121 with an IC50 of 1.0 ± 0.25 nM and 0.75 ± 0.12 nM, respectively, consistent with its affinity for these VEGF-A variants.
[00177] The data presented in FIG. 4A and FIG. 4B demonstrates that these aptamers bind to an epitope consisting of or overlapping with the receptor binding face contained within the RBD of VEGF-A, and thus directly block the interaction of VEGF-A with its cognate receptor. The absence of inhibition of the interaction of VEGF165 with KDR by Aptamer 7 is consistent with the literature which demonstrates that this aptamer engages VEGF-A by binding to an epitope within the HBD and does not directly block binding of VEGF-A to its receptor, KDR (Ruckman, 1998; Lee, 2005; Ng, 2006). The lack of inhibition of VEGF activity by Aptamer 7 is consistent with the SPR binding data described in Example 2 and the published literature (Ruckman, 1998). Table 8. Anti-VEGF-A inAb
Figure imgf000075_0001
Example 4, Characterization of inhibition of VEGF-A signal transduction by KDR phosphorylation
Figure imgf000075_0002
[00178] When the RBD of VEGF-A binds to KDR, the receptor dimerizes leading to trans autophosphorylation and activation of VEGF signaling. To determine if Aptamer 26 binding to the RBD of VEGF resulted in inhibition of VEGF-A activity, Aptamer 26 was tested for its ability to inhibit KDR phosphorylation induced by either VEGFies or VEGFm as compared to a clone of an anti- VEGF antibody (Ferrara, Napoleone, et al. "Discovery and development of bevacizumab, an anti -VEGF antibody for treating cancer." Nature reviews Drug discovery 3.5 (2004): 391.) (SEQ ID NO: 13 and SEQ ID NO: 14, shown in Table 8), and as compared to Aptamer 7 for inhibition of VEGF ^-induced KDR phosphorylation.
[00179] Briefly, HEK293 cells engineered to stably overexpress KDR were plated overnight in collagen coated 96 well plates at 50k cells/well. Aptamers were heated to 90°C for 3 minutes and cooled to room temperature for a minimum of 5 minutes. VEGF (Biolegend) and VEGF, 65 (R.&D Systems) were prepared at 20 nM in DMEM + 0.8% FBS, a 20X stock for the reaction 15 pL of VEGF was added to 15 pL titrated aptamer in a polypropylene plate and diluted to 300 pL with IS buffer (10 niM Iris pH 7.5; 100 mM NaCl; 5.7 mM KC1: 1 mM MgCh; 1 mM CaCh). The aptamer/VEGF mixture was incubated at 37°C for 30 minutes, after which 100 pL was added for 5 minutes at 37°C to the cells in an incubator with 5% CO2.
Treatments were aspirated from cells, and cells were lysed with 100 pL cold lysis buffer (20 mM Tris-HCl, pH 7.5, 150 niM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5 mM sodium
orthovanadate (freshly prepared), 1 mM PMSF (freshly prepared), lx protease inhibitor cocktail (freshly prepared)) on ice for 10 minutes. Plates were centrifuged at 4000 x g for 10 minutes before transferring the cell lysis to the AlphaLISA® assay plate.
[00180] To perform the AlphaLISA®' assay, 10 pL of cell lysis was transferred to a white low volume 384 well Optiplate (Perkin Elmer). A mixture of the following components was made in order of which they are listed: 1.25 nM anti-hVEGFR2 polyclonal goat IgG antibody (R&D Systems), 10 pg/ml AlphaLISA® anti-goat IgG acceptor beads (Perkin Elmer), 1.25 nM P- tyrosine biotinylated mouse mAb (Cell Signaling Technology), and 10 pg/ml AlphaScreen® streptavidin donor beads (Perkin Elmer). 10 pL of this reagent mixture was added to the assay- plate that contained 10 pL of cell lysate. The assay plate was sealed and incubated in the dark for approximately 2 hours, then was read on a Biotek CYTATION 5 plate reader using the Alpha 384 well optical cube. Percent inhibition was calculated by subtracting TS buffer background from each value and normalizing to VEGF-only controls. The values were fit by using a four-parameter non-linear fit in GraphPad Prism Version 7.0.
[00181] The results demonstrate that Aptamer 26 (SEQ ID NO: 7) and the anti-VEGF m Ab (SEQ ID NO: 13 and SEQ ID NO: 14) achieved full inhibition of the phosphorylation of KDR by inhibiting its interaction with VEGF165 or VEGF121 (FIG. 5A and FIG. SB, respectively). Consistent with their measured affinities for VEGFies and VEGFm, calculated IC50 values for Aptamer 26 were 1.5 ± 0.6 nM and 20 ± 5.2 nM, for inhibition of phosphorylation of KDR by VEGF|65 or YEGF]2I, respectively; and calculated IC50 values for the anti-VEGF mAb were 1.1 ± 0.2 nM and 2.8 ± 1.7 nM, for inhibition of phosphorylation of KDR by VEGF165 or VEGFm, respectively. Conversely, Aptamer 7 only partially inhibited phosphorylation of KDR by VEGF165 (maximum inhibition of -80%), with an IC50 of 2.2 nM (FIG. 5A).
[00182] The potency and complete inhibition of VEGFi65 or VEGFm induced phosphorylation of KDR by Aptamer 26, and its comparable activity profile to an anti-VEGF mAb, is consistent with the ability of Aptamer 26 to bind to the receptor binding face present within the RBD of VEGF-A and directly block the interaction between VEGF-A and KDR. The partial inhibition of VEGFies induced phosphorylation of KDR by Aptamer 7 is consistent with indirect inhibition of KDR phosphorylation via blocking the interaction of neuropilin-1 with VEGF165 (Soker, Shay, et al. "VEGF 165 mediates formation of complexes containing VEGFRD2 and neuropil in□ 1 that enhance VEGF 165□ receptor binding. " Journal of Cellular Biochemistry 85.2 (2002): 357-368). This result supports the conclusion that binding of Aptamer 26 to the receptor binding face within the RBD of VEGF- A variants confers inhibition of VEGF- A signaling. Further, these results differentiate the mechanism of action of Aptamer 26 from previously described HBD binding aptamers such as Aptamer 7.
Figure imgf000077_0001
[00183] VEGF is thought to play an important role in inducing angiogenesis in both normal tissues and diseased pathologies (Ferrara, Napoleone. "Vascular endothelial growth factor: basic science and clinical progress " Endocrine Reviews 25.4 (2004): 581-611.) Therefore, the ability of candidate VEGF antagonists to inhibit VEGF -induced angiogenesis may be evaluated in an angiogenesis assay. In vitro models have been established to study VEGF-stimulated
angiogenesis. To determine if Aptamer 26 can inhibit VEGF -A induced angiogenesis, Aptamer 26 was tested for its ability to inhibit angiogenesis via binding to either VEGF ^5 or VEGF .
[00184] Co-culture of fibroblasts and endothelial ceils leads to the formation of tubes after stimulation with either VEGF gs or VEGFm. Briefly, at Day 0, primary human dermal fibroblasts (ATCC) were plated at 20k ceils/well in 100 mE fibroblast growth media (ATCC) and incubated at room temperature for 1 hour. GFP-infected HUVEC cells (Lonza, infected in house) were then plated at 10k cells/well with 100 pL HUVEC growth media (without VEGF supplement, PromoCeil) and incubated at room temperature for 1 hour. The plate was then placed in the IncuCyte®' Zoom (Essen Bioscience) set to the default angiogenesis analysis definition with imaging programmed every 4 hours. After 24 hours of incubation (Day 1), old media was removed and replaced with 150 pL/well of IncuCyte’ Angiogenesis Prime Kit Optimized Assay Medium (Essen Bioscience). After 24 hours (Day 2), media was changed with fresh media with or without treatment. Treatment included 400 pM VEGF 165 or 800 pM
VEGF Hi premixed with or without a dilution of aptamer. Assays were carried out for 6 days with media changes and subsequent treatments performed on Days 4 and 6.
[00185] Network length (mm/mm3) of the tubes formed from the GFP-HUVEC cells was measured as a primary' indication of angiogenesis. Percent inhibition was calculated by subtracting assay media background from each value and normalizing to VEGF-A only controls. The values were fit using a four-parameter non-linear fit in GraphPad Prism Version 7.0. IC50 values for VEGF165 and VEGFm were calculated at a time point that reflected an EC90-EC100 of the VEGF-A alone induction of tube formation. Representative dose response curves of Aptamer 26, shown in FIG. 6A and FIG. 6B, indicate that Aptamer 26 had an inhibitory effect against VEGFies with IC50 values of 1.9 ± 0.9 nM and VEGF121 with an IC50 of 18 ± 5.5 nM, respectively, demonstrating the anti -angiogenic activity of Aptamer 26. This is also represented qualitatively in FIG. 7, in which Aptamer 26 is shown to inhibit tube formation when GFP- HUVEC cells (shown) were treated with either VEGFm or VEGF165. Together, along with the data shown in Examples 2, 3 and 4, which demonstrate the pan-variant specific nature of Aptamer 26 against VEGF165, VEGFm, and VEGFno. these data further support the conclusion that binding of Aptamer 26 to the receptor binding face contained within the RBD of VEGF-A provides effective inhibition of VEGF-A activity.
Example 6. Long-acting pan-variant specific anti- VEGF-A aptamer for treatment of retinal diseases by intravitreal (IVT) administration.
[00186] There is an unmet need for pan-variant specific anti -VEGF-A inhibitors that can be dosed by IVT administration and maintain a therapeutic effect for 12 weeks or greater. In this example, a patient is diagnosed with wet age-related macular degeneration, diabetic retinopathy, diabetic macular edema, myopic choroidal neovascularization, or macular edema following retinal vein occlusion (RVO). The patient is treated with a therapeutically effective dose of a PEGyiated-anti -VEGF-A aptamer by intravitreal (IVT) injection. The aptamer is Aptamer 26 or a more potent derivative of Aptamer 26 and inhibits VEGF-A by binding the receptor binding domain of VEGF-A and is thus pan-variant specific for inhibition of VEGF-A induced angiogenesis. To maintain on-mechanism inhibition of VEGF-A, the aptamer maintains a concentration in the vitreous above the IC90 of the aptamer, that is, the aptamer maintains a concentration in the vitreous that would allow for inhibition of at least 90% of the VEGF-A activity in the vitreous. Using a binding isotherm model for a non-cooperative 1 : 1 ligand to target interaction, y=Ax/(B+x), where y is the fraction bound, A is the maximal fraction bound, x is the concentration of ligand, and B is the dissociation constant, the IC90 of an aptamer can be assumed to be 10-fold higher than the IC50. [00187] For example, a PEGylated anti-VEGF-A aptamer, such as Aptamer 26 or a more potent derivative, is presented as an isotonic, neutral pH formulation at a concentration of 10 mg/rnL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 100 mE volume for a maximum dosage of 1 mg/eye. Assuming an oligonucleotide molecular weight of 12.5 kDa and an adult human vitreous volume of approximately 4 rnL, a peak aptamer concentration of approximately 16 mM in the vitreous would be expected. A PEGylated anti- VEGF-A aptamer with a vitreous half-life of 10 days and an affinity for VEGF-A of 0.2 nM, would provide a therapeutic level of VEGF-A inhibition for approximately 18-19 weeks. Thus, the patient would need to be treated only two to three times a year to maintain a therapeutic level of VEGF-A inhibition (FIFE 8A). A PEGylated anti-VEGF-A aptamer with a vitreous half-life of 10 days and an affinity for VEGF-A of 2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 14 weeks (FIG. 8A). Thus, the patient would need to be treated only three times a year to maintain a therapeutic level of VEGF-A inhibition.
[00188] In some cases, the vitreous half-life of the aptamer is greater than 10 days. At a dose of 1 mg/eye, a PEGylated anti-VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 0.2 nM would provide a therapeutic level of VEGF-A inhibition for
approximately 28 weeks. Thus, the patient would need to be treated only two times a year or less to maintain a therapeutic level of VEGF-A inhibition (FIG. 8A). A PEGylated anti-VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 21 weeks (FIG. 8A). Thus, the patient would need to be treated only two to three times a year to maintain a therapeutic level of \EGF-A inhibition. A PEGylated anti -VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 20 nM would provide a therapeutic level of VEGF-A inhibition for approximately 14 weeks (FIG. 8A). Thus, the patient would need to be treated only three times a year to maintain a therapeutic level of VEGF-A inhibition.
[00189] In some cases, alternative PEG moieties are utilized that allow for higher concentration aptamer formulations. For example, a PEGylated anti-VEGF-A aptamer, such as Aptamer 26 or a more potent derivative, is presented as an isotonic, neutral pH formulation at a concentration of 50 mg/rnL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 100 pL volume for a maximum dosage of 5 mg/eye. Assuming an oligonucleotide molecular weight of 12.5 kDa and an adult human vitreous volume of approximately 4 rnL, a peak aptamer concentration of approximately 82 mM in the vitreous would be expected. A PEGylated anti-VEGF-A aptamer with a vitreous half-life of 10 days and an affinity for VEGF- A of 0.2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 22 weeks. Thus, the patient would need to be treated only two to three times a year to maintain a therapeutic level of VEGF-A inhibition (FIG. 8B). A PEGylated anti- VEGF-A aptamer with a vitreous half-life of 10 days and an affinity for VEGF-A of 2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 17 to 18 weeks (FIG. 8B). Thus, the patient would need to be treated only two to three times a year to maintain a therapeutic level of VEGF- A inhibition. A PEGylated anti -VEGF-A aptamer with a vitreous half-life of 10 days and an affinity for VEGF-A of 20 nM would provide a therapeutic level of VEGF-A inhibition for approximately 12 to 13 weeks (FIG. 8B). Thus, the patient would need to be treated only four to five times a year to maintain a therapeutic level of VEGF-A inhibition
[00190] In some cases, the vitreous half-life of the aptamer is greater than 10 days. At a dose of 5 mg/eye, a PEGylated anti -VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 0.2 nM would provide a therapeutic level of VEGF-A inhibition for
approximately 33 weeks. Thus, the patient would need to be treated only two times a year or less to maintain a therapeutic level of VEGF-A inhibition (FIG. 8B). A PEGylated anti -VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 2 nM would provide a therapeutic level of VEGF-A inhibition for approximately 26 w^eeks (FIG. 8B). Thus, the patient would need to be treated only two times a year to maintain a therapeutic level of VEGF- A inhibition. A PEGylated anti- VEGF-A aptamer with a vitreous half-life of 15 days and an affinity for VEGF-A of 20 nM would provide a therapeutic level of VEGF-A inhibition for approximately 19 weeks (FIG. 8B). Thus, the patient would need to be treated only two to three times a year to maintain a therapeutic level of VEGF-A inhibition.
[00191] In all of the above cases, the patient is treated at a dosing interval to maintain a drug concentration in the vitreous above its IC90, and not more than once every 12 weeks. After three months of treatment and every month thereafter, the patient is assessed for mean change in visual acuity from baseline, improvement of >5 or more letters in visual acuity testing, central foveai thickness and change in central foveai thickness from baseline. Treatment of the patient with the pan-variant specific anti -VEGF-A aptamer results in an improvement in visual acuity from pre treatment baseline, a gain of 5 or more letters in visual acuity testing, and a marked reduction in central foveal thickness. These clinical outcomes are significant improvements when compared to an untreated patient and comparable to clinical outcomes when compared to a patient who has been treated with an anti-VEGF-A antibody or antibody fragment therapy once every' 12 weeks or less. It is understood in this example that, any range of dose level, affinity and half-life for a pan-variant specific PEGylated anti-VEGF-A aptamer that provides for a vitreous concentration above the IC90 for 12 weeks or greater following IVT administration would maintain a therapeutic effect over this window of time for the treatment of wet age-related macular degeneration, diabetic retinopathy, diabetic macular edema, myopic choroidal
neovascularization, or macular edema following RVO.
Example 7. Aptamer engineering for systemic safety hi adult IVT administration.
[00192] In this example, a patient is diagnosed with macular edema following RVO. The patient is treated with a therapeutically effective dose of a PEGylated-anti-VEGF-A, such as Aptamer 26, by intravitreal (IVT) injection. The aptamer inhibits VEGF-A by binding to the receptor binding face of VEGF-A and is thus pan-variant specific for inhibition of VEGF-A induced angiogenesis. Systemic and tissue suppression of VEGF-A activity is contraindicated when a patient has a risk of arterial thromboembolic events (ATEs; USPI Lucentis 03/2018). As such, maintenance of therapeutic aptamer concentrations in the vitreous (concentrations above the IC90 of the aptamer) while simultaneously preventing systemic and tissue target suppression (concentrations above the IC o of the aptamer) is necessary for achieving the desired safety profile. Using a binding isotherm model for a non-cooperative 1 :1 ligand to target interaction, y=Ax/(B+x), where y is the fraction bound, A is the maximal fraction bound, x is the
concentration of ligand, and B is the dissociation constant, the IC90 and IC10 of an aptamer can be assumed to be 10-fold higher and 10-fold lower than the IC50 respectively.
[00193] For example, an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 30 mg/mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 100 pL volume for a maximum dosage of 3 mg/eye. Assuming an oligonucleotide molecular weight of 12.5 kDa and an adult human vitreous volume of 4 mL, a peak aptamer concentration of 60mM in the vitreous would be expected. Assuming a vitreous half-life of 10 days and a desired therapeutic window of 30-day target suppression, the vitreous concentration of aptamer at day 30 would be 7.5 mM. Based on serum and tissue exposure of 5 nM following a 3 mg/eye IVT injection of Aptamer 9 (see Table 6) in adult humans (Pegaptanib NDA application 21-756), the maximum serum and tissue exposure here would be the same. Following from this reasoning, aptamers with an IC50 ranging from 50 to 750 tiM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the IC10 in serum and tissues (FIG. 9A)
[00194] In some cases, the safety profile is achieved by lowering the injected dose, ensuring a lower maximum systemic and tissue exposure while diminishing the duration of effect in the vitreous. For example, an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 6 nig/mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 50 mI_. volume for a dosage of 0.3 mg/eye. Assuming an
oligonucleotide molecular weight of 12.5 kDa and an adult human vitreous volume of 4 mL, a peak aptamer concentration of 6 mM in the vitreous would be expected. Assuming a vitreous half-life of 10 days and a desired therapeutic window of 30-day target suppression, the vitreous concentration of aptamer at day 30 would be 750 nM. Based on serum and tissue exposure of 0.5 nM following a 0.3 mg IVT injection of Aptamer 9 (see Table 6) in adult humans
(Pegaptanib NDA application 21-756), the maximum serum and tissue exposure here would be the same. Following from this reasoning, aptamers with an IC50 ranging from 5 to 75 nM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the IC10 in serum and tissues (FIG. 9B).
[00195] In some cases, alternative PEG moieties are utilized that allow for higher concentration aptamer formulations and increased aptamer retention in the vitreous, which thereby increase the duration of effect, and also decrease effective systemic and tissue exposure. For example, an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 60 mg/rnL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at 100 pL volume for a maximum dosage of 6 mg/eye. Assuming an oligonucleotide molecular weight of 12.5 kDa and an adult human vitreous volume of 4 mL, a peak aptamer concentration of 120 mM in the vitreous would be expected. Assuming a vitreous half-life of 15 days and a desired therapeutic window of 30-day target suppression, the vitreous concentration of aptamer at day 30 would be 30 mM. Based on serum and tissue exposure of 5 nM following a 3 mg IVT injection of Aptamer 9 (see Table 6) in adult humans (Pegaptanib NDA application 21-756), the maximum serum and tissue exposure of a 6 mg/eye dose with an improved PEG moiety would be 2.5 nM. Following from this reasoning, aptamers with an IC50 ranging from 25 nM to 3 mM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the IC10 in serum and tissues (FIG, 9C).
[00196] In some cases, the PEG moiety is attached to the aptamer by a linker that is
preferentially labile in serum and tissues, leading to increased clearance and lorver effective systemic and tissue exposure. In some cases, serum and tissue-labile moieties are inserted within the sequence of the aptamer, leadi ng to i ncreased metabolic processing in serum and tissues, resulting in lower effective systemic and tissue exposure. For example, an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 30 mg/mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 100 mE volume for a maximum dosage of 3 mg/eye. Assuming an oligonucleotide molecular weight of 12.5 kDa and an adult human vitreous volume of 4 mL, a peak aptamer concentration of 60 mM in the vitreous would be expected. Assuming a vitreous half-life of 10 days and a desired therapeutic window of 30-day target suppression, the vitreous concentration of aptamer at day 30 would be 7 5 mM. Due to the metabolic instability of the active compound, a 3 mg/eye dose would yield a maximum serum and tissue exposure of 0.5 nM. Following from this reasoning, aptamers with an IC50 ranging from 5 to 750 nM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the IC o in serum and tissues (FIG. 9D).
[00197] In some cases, the aptamer possesses differential potency for VEGF-A variants (e.g., VEGFies and VEGF ·). In such cases, systemic and tissue target suppression is defined by the aptamer’ s IC10 against the highest potency VEGF-A variant, while maintenance of therapeutic effect in the vitreous is defined by the aptamer’ s IC90 against the lowest potency VEGF-A variant.
[00198] In all of the above cases, the patient is treated once every 4 w?eeks or once every 8 weeks or once every' 16 weeks. After three months of treatment and every month thereafter, the patient is assessed for mean change in visual acuity from baseline, improvement of 5 or more letters in visual acuity testing, central foveal thickness and change in central foveal thickness from baseline. Treatment of the patient with the pan-variant specific anti -VEGF-A aptamer results in an improvement in visual acuity from pre-treatment baseline, a gain of 5 or more letters in visual acuity testing, and a marked reduction in central foveal thickness. These clinical outcomes are significant improvements when compared to an untreated patient and comparable to clinical outcomes when compared to a patient who has been treated with an anti-VEGF-A antibody fragment therapy once every 4 weeks. Importantly, the patient’s risk of arterial thrombotic events is substantially reduced when compared to a patient who has been treated with an anti-VEGF-A antibody fragment therapy once ever}' 4 weeks due to the reduced exposure of the patient to systemic and tissue inhibition of VEGF-A.
Example 8, Aptamer engineering for systemic safety in pediatric IVT administration
[00199] In this example, a patient is diagnosed with zone I stage 3+ (i.e., stage 3 with plus disease) retinopathy of prematurity (ROP; Mintz-Hittner, 2011). The patient is treated with a therapeutically effective dose of a PEGylated-anti -VEGF-A aptamer by IVT injection. The aptamer inhibits VEGF-A by binding the receptor binding face of VEGF-A and is thus pan variant specific for inhibition of VEGF-A induced angiogenesis. Systemic and tissue suppression of VEGF-A activity is contraindicated for infants and can potentiate the onset of adverse neurodevelopmental outcomes (Lien, 2016). As such, maintenance of therapeutic aptamer concentrations in the vitreous (concentrations above the IC90 of the aptamer) while simultaneously preventing systemic and tissue target suppression (concentrations above the IC10 of the aptamer) is necessary for achieving the desired safety profile. Using a binding isotherm model for a non-cooperative 1 : 1 ligand to target interaction, y=Ax/(B+x), where y is the fraction bound, A is the maximal fraction bound, x is the concentration of ligand, and B is the dissociation constant, the IC90 and IC10 of an aptamer can be assumed to be 10-fold higher and 10-fold lower than the IC50 respectively.
[00200] For example, an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 30 mg/mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 25 pL volume for a maximum dosage of 0.75 mg/eye. Assuming an oligonucleotide molecular weight of 12.5 kDa and an infant human vitreous volume of l mL, a peak aptamer concentration of 60 mM in the vitreous would be expected. Assuming a vitreous half-life of 5 days and a desired therapeutic window of 30-day target suppression, the vitreous concentration of aptamer at day 30 would be 940 nM. Given similarities in body size and vitreous volume, rabbits (-2.5-4 kg) are assumed to be a relevant predictor of exposure in human infants. Based on serum and tissue exposure of 4 5 nM following a 1.5 mg IVT injection of a PEGylated aptamer in rabbits, the maximum serum and tissue exposure of a 0.75 mg/eye dose in infants is estimated to be 2.5 nM. Following from this reasoning, aptamers with an IC50 ranging from 25 to 95 nM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the IC10 in serum and tissues (FIG. 10A).
[00201] In some cases, the safety profile is achieved by lowering the injected dose, ensuring a lower maximum systemic and tissue exposure while diminishing the duration of effect in the vitreous. For example, an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 10 ng/'mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 25 pL volume for a dosage of 0.25 mg/eye. Assuming an
oligonucleotide molecular weight of 12.5 kDa and an infant human vitreous volume of 1 ml, a peak aptamer concentration of 20 mM in the vitreous would be expected. Assuming a vitreous half-life of 5 days and a desired therapeutic window of 30-day target suppression, the vitreous concentration of aptamer at day 30 would be 310 nM. Given similarities in body size and vitreous volume, rabbits (-2.5-4 kg) are assumed to be a relevant predictor of exposure in human infants. Based on serum and tissue exposure of 4.5 nM following a 1.5 mg IVT injection of a PEGylated aptamer in rabbits, the maximum serum and tissue exposure of a 0.25 mg/eye dose in infants is estimated to be 0.75 nM. Following from this reasoning, aptamers with an IC50 ranging from 7.5 to 3 1 nM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the IC o in serum and tissues (FIG. 10B).
[00202] In some cases, alternative PEG moieties are utilized that allow for higher concentration aptamer formulations, and increased aptamer retention in the vitreous, which thereby increase the duration of effect, and also decreased effective systemic and tissue exposure. For example, an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 60 mg/mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 25 pL volume for a maximum dosage of 1.5 mg/eye. Assuming an oligonucleotide molecular weight of 12.5 kDa and an infant human vitreous volume of 1 mL, a peak aptamer concentration of 120 pM in the vitreous would be expected. Assuming a vitreous half-life of 10 days and a desired therapeutic window of 30-day target suppression, the vitreous concentration of aptamer at day 30 would be 15 pM. Given similarities in body size and vitreous volume, rabbits (-2 5-4 kg) are assumed to be a relevant predictor of exposure in human infants. Based on serum and tissue exposure of 4.5 nM! following a 1 5 mg IVT injection of a PEGylated aptamer in rabbits, the maximum serum and tissue exposure of a 1.5 mg/eye dose in infants is estimated to be 0.75 nM. Following from this reasoning, aptamers with an IC50 ranging from 7.5 nM to 1.5 mM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the ICio in serum and tissues (FIG, IOC).
[00203] In some cases, the PEG moiety is attached to the aptamer by a linker that is
preferentially labile in serum and tissues, leading to increased clearance and low'er effective systemic and tissue exposure. In some cases, serum and tissue-labile moieties are inserted within the sequence of the aptamer, leadi ng to i ncreased metabolic processing in serum and tissues, resulting in lower effective systemic and tissue exposure. For example, an aptamer is presented as an isotonic, neutral pH formulation at a concentration of 30 mg/mL based on oligonucleotide molecular weight and is IVT administered via a 30-gauge needle at a 25 mE volume for a maximum dosage of 0.75 mg/eye. Assuming an oligonucleotide molecular weight of 12.5 kDa and an infant human vitreous volume of 1 rnL, a peak aptamer concentration of 60 mM in the vitreous would be expected. Assuming a vitreous half-life of 5 days and a desired therapeutic window of 30-day target suppression, the vitreous concentration of aptamer at day 30 would be 950 nM. Due to the metabolic instability of the active compound a 0.75 mg/eye dose would yield a maximum serum and tissue exposure of 0.5 nM. Following from this reasoning, aptamers with an IC50 ranging from 5 to 95 nM will fully suppress vitreous VEGF-A activity for at least 30 days while not exceeding the ICi0 in serum and tissues (FIG. 10D).
[00204] In some cases, the aptamer possesses differential potency for VEGF-A variants (e.g., VEGFies and VEGFm). In such cases, systemic and tissue target suppression is defined by the aptamer’ s IC10 against the highest potency VEGF-A variant, while maintenance of therapeutic effect in the vitreous is defined by the aptamer’ s IC90 against the lowest potency VEGF-A variant.
[00205] In all of the above cases, the patient is treated once every 4 weeks or once every 8 weeks. Treatment of the patient with the pan-variant-specific anti- VEGF-A aptamer results in an absence of recurrence of stage 3+ ROP in one or both eyes in zone I at 54 weeks’ postmenstrual age. These clinical outcomes are significant improvements when compared to an untreated patient and to clinical outcomes when compared to a patient who has been treated with conventional laser therapy. Importantly, the patient’s risk of adverse neurodevelopmental outcomes is substantially reduced when compared to a patient who has been treated with an anti- VEGF-A antibody fragment therapy due to the reduced exposure of the patient to systemic and tissue inhibition of VEGF-A.
Example 9. Aptamers previously reported to bind to VEGF- do not
Figure imgf000087_0001
inhibit VEGF-A activity
[00206] Previous studies have focused on the generation and optimization of aptamers that bind to both VEGF-Aies and VEGF-Am for the puipose of making biosensors to detect and quantify levels of VEGF-A in biological fluids and tissues for use in the diagnosis of cancer (Nonaka Y., Sode K., and Ikebukuro, K. "Screening and improvement of an ami -VEGF DNA
aptamer." Molecules 15.1 (2010): 215-225; Nonaka K., Yoshida W., Abe K , Ferri S., Schulze H., Bachman T.T., and Ikebukuro, K. "Affinity improvement of a VEGF aptamer by in silico maturation for a sensitive VEGF -detection system " Analytical chemistry 85.2 (2012): 1132- 1137). Nonaka et al. (2010) describes the generation of DNA aptamers comprising G-quartets (t eg:, Aptamer Vap7 and VEaP121; Table 9), and report that the isolated aptamers bind to both VEGF-A] 65 and VEGF-A· 2iwi†h low nM affinity. G-quartets are a unique, often potassium- dependent nucleic acid structure in which four guanine bases associate through Hoogsteen hydrogen bonding to form a planar guanine tetrad structure, and two or more of the guanine tetrads stack on top of each oilier to form a G-quartet. Importantly, as the intended use of these molecules was as biosensors, inhibition of VEGF-A activity is not a requirement for a diagnostic application, and the ability of the aptamers described in this publication to inhibit VEGF-A activity was not assessed by the authors. Further, the ability of these molecules to hind VEGF- Auowas not assessed, so it is not clear if these aptamers bind to the receptor binding domain of VEGF-A (residues 1-109 of VEGF-A) or to residues present in VEGF-A 2i and VEGF-Ai65 outside of the receptor binding domain portion of VEGF-A. Nonaka et al. (2012) describes the further efforts to improve the affinity of Aptamer VEaP12I for VEGF-A to optimize its use as a biosensor, and provide Aptamer 31102 (Table 9). Aptamer 3R02 is also a DNA, G-quartet aptamer, with a reported affinity to VEGF-A of approximately 300 pM. The isoform of VF.GF- A used in the data reported in Nonaka et al. (2012) is not specified in the publication. As in the prior publication, as the intended use of these aptamers is as diagnostics, their ability to inhibit VEGF-A activity was not determined. [00207] We sought to determine whether these aptamers inhibited VEGF-A activity. Aptamers Vap7, VEaP121 and 3R02 were synthesized and purified as described in Nonaka et al. 2010 and No aka et al. 2012, To confirm that the synthesized molecules adopted the proper G-quartet conformation, circular dichroism (CD) studies were performed on Aptamers Vap7, VEaP121 and 3R02 folded in the presence of potassium under the conditions described by Nonaka et al. 2010. Consistent with the CD spectra provided in Nonaka et al. 2010, a negative band at approximately 240 run and positive bands at approximately 220 and 270 nrn (FIG. 11) were observed, confirming the synthesized sequence formed the appropriate G-quartet structure.
Table 9. DNA aptamers to VEGF-A
Figure imgf000088_0001
[00208] To determine if Aptamers Vap7, VEaP121, or 3R02 w?ere capable of inhibiting VEGF- A activity, the aptamers were tested for their ability to inhibit KDR phosphorylation induced by either VEGF-Aies or VEGF-Am as compared to a clone of an anti-VEGF antibody (Table 8). The KDR phosphorylation assay was performed as described in Example 4, except that the concentration of VEGF-A 155 or VEGF-Am used to induce KDR phosphorylation was 1 nM, and an ELISA was used to detect phosphorylation of KDR. Prior to use, aptamers were renatured as described by Nonaka et al. 2010 by heating to 95°C for 3 minutes and then allowed to cool to room temperature in TS buffer (TS buffer pH 7.5 (10 mM Tris-HCl; 100 mM NaCl; 5.7 mM KC1; 1 mM MgCC; 1 mM CaCI ; ) Aptamers were tested at a final concentration of 500 nM, and anti-VEGF mAh at a final concentration of 160 nM. As shown in FIG. 12, despite being tested at concentrations 500- to 1000-fold above their reported ¾ for VEGF 65 or VEGFm, Aptamers Vap7, VEaP121 and 3R02 showed no inhibition of VEGF-A induced phosphorylation of KDR. This is in contrast to the anti-VEGF mAh, which completely inhibited KDR phosphorylation by both VEGF-Ai65 and VEGF-A . Based on these results, it is concluded that these DNA aptamers do not inhibit VEGF-A activity and do not bind to the receptor binding face of VEGF- A. Furthermore, given their lack of activity in the KDR phosphorylation assay despite confirmation of proper folding, it is concluded these aptamers bind outside of the receptor binding domain of VEGF-A.
[00209] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. An aptamer comprising a nucleic acid sequence that selectively binds to and inhibits at least one of VEGF-A and VEGF-Ano, wherein less than 50% of pyrimidines present in said nucleic acid sequence comprise a C-5 modified pyrimidine.
2. The aptamer of claim 1, wherein less than 25% of pyrimidines present in said nucleic acid sequence comprise a C-5 modified pyrimidine.
3. The aptamer of claims 1 or 2, wherein less than 10% of pyrimidines present in said nucleic acid sequence comprise a C-5 modified pyrimidine.
4. The aptamer of any one of claims 1-3, wherein said nucleic acid sequence does not comprise any C-5 modified pyrimidines.
5. The aptamer of any one of claims 1-4, wherein said C-5 modified pyrimidine comprises a C-5 modified cytosine or a C-5 modified uridine.
6. The aptamer of any one of claims 1 -5, wherein said C-5 modified py rimidine comprises a C-5 hydrophobic modification.
7. An aptamer comprising a nucleic acid sequence that selectively binds to and inhibits at least one of VEGF-Am and VEGF-Ano, wherein less than 100% of uridines present in said nucleic acid sequence comprise a C-5 modified uridine.
8. The aptamer of claim 7, wherein less than 50% of uridines present in said nucleic acid sequence comprise a C-5 modified uridine.
9. The aptamer of claim 7 or 8, wherein less than 25% of uridines present in said nuclei c acid sequence comprise a C-5 modified uridine.
10. The aptamer of any one of claims 7-9, wherein less than 10% of uridines present in said nucleic acid sequence comprise a C-5 modified uridine.
1 1. The aptamer of any one of claims 7-10, wherein no uridines present in said nucleic acid sequence comprise a C-5 modified uridine.
12. The aptamer of any one of claims 7-1 1, wherein said C-5 modified uridine comprises a C-5 hydrophobic modification .
13. The aptamer of any one of claims 7-12, wherein said C-5 modified uridine is selected from the group consisting of: 5-(N-benzylcarboxyamide)-2'-deoxyuridine (Bnd!J), 5-(N- benzylcarboxyamide)-2'-0-methyluridine, 5-(N-benzylcarboxyamide)-2'-fluorouridine, 5-(N- phenethy icarboxyamide)-2'-deoxyuri dine (PEdU), 5-(N~thiophenylraethy icarboxyami de)-2'- deoxyuridine (ThdU), 5-(N-isobutylcarboxyamide)-2'-deoxyuridine (iBudU), 5-(N- tyrosylcarboxyatnide)-2'-deoxyuridine (TyrdU), 5-(Ni-3,4-methylenedioxybenzylcarboxyamide)-2'- deoxyuridine (MBndU), 5-(N-4-fluorobenzylcarboxyamide)-2'-deoxyuridine (FBndU), 5-(N-3- phenylpropylcarboxyamide)-2'-deoxyuridine (PPdU), 5-(N-imidizolylethylcarboxyamide)-2'- deoxyuridine (ImdU), 5-(N-isobutylcarboxyamide)-2'-0-methyluridine, 5-(N- isobutylcarboxyamide)-2'-fiuorouridine, 5-(N-tryptaminocarboxyamide)-2'-deoxyuridine (TrpdU), 5-(N-R-threoninylcarboxyamide)-2'-deoxyuridine (ThrdU), 5-(N-tryptaminocarboxyamide)-2'-0- methyluridine, 5-(N-tryptaminocarboxyamide)-2'-fluorouridine, 5-(N-[l-(3- trimethylanioniuni)propyl]carboxyamide)-2'-deoxyuridine chloride, 5-(N- naphthylmethylcarboxyamide)-2'-deoxyuridine (NapdU), 5-(N-naphthylmethylcarboxyamide)-2'-0- methyluridine, 5-(N-naphthylmethylcarboxyamide)-2'-fluorouridine, 5-(N-[l-(2,3- dihydroxypropyl)]carboxyamide)-2'-deoxyuridine), 5-(N-2-naphthylmethylcarboxyamide)-2'- deoxyuridine (2NapdU), 5-(N-2-naphthylmethylcarboxyamide)-2'-0-methyluridine, 5-(N-2- naphthylmethylcarboxyamide)-2'-fluorouridine, 5-(N-l-naphthylethylcarboxyamide)-2'- deoxyuridine (NEdU), 5-(N- 1 -naphthyl ethylcarboxyamide)-2'-0-methyluri dine, 5-(N- 1 - naphtby!e†hydcarboxyaniide)~2'-fluorouridine, 5-(N-2-naphthylethylcarboxyamide)-2'-deoxyuridine (2NEdU), 5-(N-2-naphthylethylcarboxyamide)-2'-0-methyluridine, 5-(N-2- naphthylethylcarboxyamide)-2'-fluorouridine, 5-(N-3-benzofuranylethylcarboxyamide)-2'- deoxyuridine (BFdU), 5-(N-3-benzofuranylethylcarboxyamide)-2'-0-methyluridine, 5-(N-3- benzofuranylethylcarboxyamide)-2'-fluorouridine, 5-(N-3 -benzothi opheny lethylcarboxy amide)-2 deoxyuridine (BTdU), 5-(N-3-benzothiophenylethylcarboxyamide)-2'-0-methyluridine, and 5-(N-3- benzothiophenylethylcarboxyamide)-2'-fluorouridine.
14. The aptamer of any one of claims 7-13, wherein said C-5 modified uridine is 5-(N- naphthylmethylcarboxyamide)-2'-deoxyuridine (NapdU).
15. The aptamer of any one of claims 1-14, wherein said aptamer selectively hinds to a receptor binding face or receptor binding domain of VEGF-A121 or VEGF-Ano.
16. The aptamer of claim 15, wherein said receptor binding domain comprises at least one of residues 1-109 of SEQ ID NO: 1.
17. The aptamer of claim 15 or 16, wherein said receptor binding domain comprises at least one of residues Phel7, He43, He46, Glu64, Gln79, He83, Lys84, Pro85, Arg82, His86, Asp63, and Glu67 of SEQ ID NO: 1.
18. The aptamer of any one of claims 1-17, wherein said aptamer inhibits VEGF-Am, VEGF-Ano, or both, with an IC50 of less than about 50 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation AlphaLISA*1 assay, or an in vitro model of VEGF -A-induced angiogenesis.
19. The aptamer of any one of claims 1-18, wherein said aptamer inhibits VEGF- A 121, VEGF-Ano, or both, with an IC50 of less than about 25 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation AlphaLISA^ assay, or an in vitro model of VEGF- A-induced angiogenesis.
20. The aptamer of any one of claims 1-19, wherein said aptamer inhibits VEGF- A 121, VEGF-Ano, or both, with an IC50 of less than about 10 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation AlphaLISA^ assay, or an in vitro model of VEGF -A-induced angiogenesis.
21. The aptamer of any one of claims 1-20, wherein said aptamer inhibits VEGF-Am, VEGF-Ano, or both, with an IC50 of less than about 5 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation AlphaLIS 8’ assay, or an in vitro model of VEGF -A-induced angiogenesis
22. The aptamer of any one of claims 1-21, wherein said aptamer inhibits VEGF-Am, VEGF-Ano, or both, with an IC50 of less than about 1 nM as measured by a VEGF-A:KDR competition binding assay, a KDR phosphorylation AlphaLiSA^ assay, or an in vitro model of VEGF-A-induced angiogenesis.
23. The aptamer of any one of claims 1-22, wherein said aptamer binds to VEGF-Am, VEGF-Ano, or both, with a Kd of less than about 50 nM as measured by surface plasmon resonance assay.
24. The aptamer of any one of claims 1-23, wherein said aptamer binds to VEGF-Am, VEGF-Ano, or both, with a Kd of less than about 25 nM as measured by surface plasmon resonance assay.
25. The aptamer of any one of claims 1-24, wherein said aptamer binds to VEGF-Am, VEGF-Ano, or both, with a Kd of less than about 10 nM as measured by surface plasmon resonance assay.
26. The aptamer of any one of claims 1-25, wherein said aptamer binds to VEGF-Am, VEGF-Ano, or both, with a Kd of less than about 5 nM as measured by surface plasmon resonance assay.
27. The aptamer of any one of claims 1-26, wherein said aptamer binds to VEGF-Am, VEGF-Ano, or both, with a Kd of less than about 1 nM as measured by surface plasmon resonance assay.
28. The aptamer of any one of claims 1-27, wherein said aptamer further selectively binds to and inhibits at least one ofVEGF-A^s, VEGF-AiS9, and VEGF-A2o6.
29. The aptamer of any one of claims 1-28, wherein said aptamer inhibits or reduces an interaction of VEGF-A with KDR.
30. The aptamer of any one of claims 1-29, wherein said aptamer inhibits or reduces VEGF-A- induced KDR phosphorylation.
31. The aptamer of any one of claims 1-30, wherein said aptamer comprises RNA or sugar-modified
RNA.
32. The aptamer of any one of claims 1-30, wherein said aptamer comprises DNA or sugar-modified DNA.
33. The aptamer of any one of claims 1-32, wherein at least 50% of said nucleic acid sequence comprises sugar-modified nucleotides.
34. The aptamer of any one of claims 1-33, wherein 100% of said nucleic acid sequence comprises sugar-modi fi ed nucl eotides.
35. The aptamer of claim 34, wherein said sugar-modified nucleotides comprise a 2’F-modified nucleotide, a 2’OMe-modified nucleotide, or both.
36. The aptamer of claim 34 or 35, wherein said sugar-modified nucleotides are selected form the group consisting of: 2 -G, 2’OMe-G, 2’OMe-U, 2’OMe-A, 2’OMe-C, and any combination thereof.
37. The aptamer of any one of claims 1-36, wherein said aptamer further comprises a 3’ terminal inverted deoxythymidine.
38. The aptamer of any one of claims 1-37, wherein said aptamer comprises a nuclease-stabilized nucleic acid backbone.
39. The aptamer of any one of claims 1-38, wherein said nucleic acid sequence comprises from about 30 to about 90 nucleotides, wherein said nucleotides are unmodified nucleotides, modified nucleotides, or a combination of modified nucleotides and unmodified nucleotides.
40. The aptamer of any one of claims 1-39, wherein said aptamer is conjugated to a polyethylene glycol (PEG) molecule.
41. The aptamer of claim 40, wherein said PEG molecule has a molecular weight selected from the group consisting of: less than about 5 kDa, less than about 10 kDa, less than about 20 kDa, less than about 40 kDa, less than about 60 kDa, and less than about 80 kDa.
42. An aptamer of any one of claims 1-41 , for use in treating an ocular disease or disorder in a subject in need thereof.
43. The aptamer of claim 42, wherein one or more symptoms of said ocular disease or disorder are treated.
44. A method of treating an ocular disease or disorder in a subject in need thereof, comprising administering to said subject an aptamer of any one of claims 1-43, thereby treating said ocular disease or disorder.
45. The method of claim 44, wherein said ocular disease or disorder is selected from the group consisting of: diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, macular edema, choroidal neovascularization, neovascular age-related macular degeneration, myopic choroidal neovascularization, punctate inner choroidopathy, ocular histoplasmosis syndrome, familial exudative vitreoretinopathy, and retinoblastoma.
46. The method of claim 44 or 45, wherein said ocular disease or disorder exhibits elevated levels of
VEGF-A.
47. Use of an aptamer of any one of claims 1-43 in a formulation of a medicament for treatment of an ocular disease or disorder.
48. Use of an aptamer of any one of claims 1-43 for treatment of an ocular disease or disorder.
49. A method for modulating vascular endothelial growth factor- A (VEGF-A) in a biological system, said method comprising: administering to said biological system an aptamer according to any one of claims 1-43, thereby modulating VEGF-A in said biological sy stem.
50. The method of claim 49, wherein said biological system compri ses a biological tissue or biological cells.
51. The method of claim 49, wherein said biological system is a subject.
52. The method of claim 51, wherein said subject is a human.
53. The method of any one of claims 49-52, wherein said modulating comprises inhibiting a function associated with VEGF-A.
54. The method of any one of claims 49-53, wherein said modulating comprises preventing or reducing an association of VEGF-A with one or more of Fit- 1 , KDR, or Nrp-1.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170247702A1 (en) * 2013-09-09 2017-08-31 Somalogic, Inc. PDGF and VEGF Aptamers Having Improved Stability and Their Use in Treating PDGF and VEGF Mediated Diseases and Disorders
US20180051287A1 (en) * 2016-01-20 2018-02-22 Vitrisa Therapeutics, Inc. Compositions and methods for inhibiting factor d

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170247702A1 (en) * 2013-09-09 2017-08-31 Somalogic, Inc. PDGF and VEGF Aptamers Having Improved Stability and Their Use in Treating PDGF and VEGF Mediated Diseases and Disorders
US20180051287A1 (en) * 2016-01-20 2018-02-22 Vitrisa Therapeutics, Inc. Compositions and methods for inhibiting factor d

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