Aptamers and use thereof

[0001] The instant application contains a Sequence Listing in ASCII plain text file format as a file named “Sequence_Listing.txt”, created on Oct. 26, 2021, of 64,687 bytes in size, and which is hereby incorporated by reference in its entirety.

[0002] Neovascularization (NV) and vascular leakage are hallmarks of multiple neovascular retinal diseases such as Diabetic Macular Edema (DME), wet Age-related Macular Degeneration (AMD), and Diabetic Retinopathy (DR).

[0003] VEGF plays a pivotal role in regulating angiogenesis which is the main mechanism of ocular NV, and vascular permeability which determines the extent of leakage, and thus is an effective target for various diseases.

[0004] The Ang2/Tie2 pathway is proven by extensive research to be an important complementary player of VEGF, holding great potential as a novel target for the treatment of various diseases, such as retinal diseases. As reported, Tie2 is a tyrosine kinase receptor for both Angiopoietin-1 (Ang1) and Angiopoietin-2 (Ang2), located on the surface of vascular endothelial cells. Ang2 is only expressed under pathological conditions. It complements VEGF's function by promoting vascular permeability and stimulating the secretion of proangiogenic cytokines, which are required for VEGF-induced ocular NV and vascular leakage.

[0005] Current anti-VEGF drugs, despite their success, still suffer from limited efficacy and duration. Increasing the dosage of anti-VEGF agents may improve both drug potency and duration. Nucleic acid aptamers possess a high solubility and a small molecular weight (~15 kDa), and thus may achieve a much higher molar dosage.

[0006] However, the previous anti-VEGF aptamer, e.g., Macugen® cannot realize this potential. Macugen® fails to recognize all VEGFA isoforms and accordingly has inferior potency. Thus, there is an urgent need to develop aptamers that are more effective than those in the art.

[0007] There is ongoing need to develop aptamers targeting VEGF and/or Ang2 that are more specific, with higher affinity, and/or more effective.

[0008] The present disclosure provides an anti-VEGF aptamer, an anti-Ang2 aptamer, a bispecific aptamer and a composition comprising the anti-VEGF aptamer and/or the anti-Ang2 aptamer, and uses thereof.

[0009] The anti-VEGF aptamer provided in the present disclosure is capable of at least one of the followings: 1) binding to all VEGFA isoforms (e.g. VEGF-121, VEGF-165 and VEGF 189) with a KD value of about 20 nanomolar (nM) or less; 2) forming a specific secondary structure, comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop; 3) being formulated at a concentration of as high as 200 mg/ml or more; 4) being nuclease resistant; 5) binding to VEGFA isoforms from various species, such as human VEGFs, mouse VEGFs, rat VEGFs, rabbit VEGFs, and monkey VEGFs; 6) inhibiting an interaction between a VEGF and a VEGF receptor; and 7) preventing, treating, and/or ameliorating a neovascular disease, disorder or condition.

[0010] The anti-Ang2 aptamer provided in the present disclosure is capable of at least one of the followings: 1) binding to Ang2 with a KD value of about 500 picomolar (pM) or less; 2) not substantially binding to human Ang1; 3) forming a specific secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop; 4) binding to both a human Ang2 and a mouse Ang2; 5) being nuclease resistant; 6) inhibiting an interaction between Ang2 and Tie2; and 7) preventing, treating, and/or ameliorating a neovascular disease, disorder or condition.

[0011] The bispecific aptamer and/or the composition provided in the present disclosure is capable of at least one of the followings: 1) binding to all VEGFA isoforms (e.g. VEGF-121, VEGF-165 and VEGF 189) with a KD value of about 20 nanomolar (nM) or less; 2) binding to Ang2 with a KD value of about 500 picomolar (pM) or less; 3) being nuclease resistant; 4) inhibiting an interaction between Ang2 and Tie2; 5) inhibiting an interaction between a VEGF and a VEGF receptor; and 6) preventing, treating, and/or ameliorating a neovascular disease, disorder or condition.

Anti-VEGF Aptamer

[0012] In one aspect, the present disclosure provides an anti-VEGF aptamer, which binds to VEGF-121 with a KD value of about 20 nanomolar (nM) or less, and binds to VEGF-165 with a K_D value of about 20 nanomolar (nM) or less.

[0013] In some embodiments, the anti-VEGF aptamer binds to VEGF-121 with a K_D value of about 2 nanomolar (nM) or less. In some embodiments, the anti-VEGF aptamer binds to VEGF-165 with a KD value of about 2 nanomolar (nM) or less.

[0014] In some embodiments, the VEGF-121 is a human VEGF-121, a mouse VEGF-120, a monkey VEGF-121, a rabbit VEGF-121 and/or a rat VEGF-120. In some embodiments, the VEGF-165 is a human VEGF-165, a mouse VEGF-164, a monkey VEGF-165, a rabbit VEGF-165 and/or a rat VEGF-164. In some embodiments, the anti-VEGF aptamer binds to VEGF-189, such as the rabbit VEGF-189.

[0015] In some embodiments, the anti-VEGF aptamer specifically binds to both a VEGF receptor-binding domain of VEGF-121 or a fragment thereof and a VEGF receptor-binding domain of VEGF-165 or a fragment thereof.

[0016] In some embodiments, the VEGF receptor-binding domain of VEGF-121 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 118-122, 128, 129, and 133. In some embodiments, the VEGF receptor-binding domain of VEGF-165 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 118-122, 128, and 129.

[0017] In some embodiments, the anti-VEGF aptamer inhibits an interaction between the VEGF-121 and VEGF-R1. In some embodiments, the anti-VEGF aptamer inhibits an interaction between the VEGF-121 and VEGF-R2.

[0018] In some embodiments, the anti-VEGF aptamer inhibits an interaction between the VEGF-165 and VEGF-R1. In some embodiments, the anti-VEGF aptamer inhibits an interaction between the VEGF-165 and VEGF-R2.

[0019] In some embodiments, the anti-VEGF aptamer is capable of reducing and/or ameliorating a lesion and/or a leakage in a laser-induced choroidal neovascularization (CNV) rat model.

[0020] In some embodiments, the anti-VEGF aptamer is highly soluble.

[0021] In some embodiments, the anti-VEGF aptamer is an RNA aptamer, a DNA aptamer, or a combination thereof. In some embodiments, the anti-VEGF aptamer comprises one or more modified nucleotide. In some embodiments, the anti-VEGF aptamer is nuclease resistant. In some embodiments, all the nucleotides of the anti-VEGF aptamer are modified nucleotides. In some embodiments, such modified nucleotide comprises a chemical substitution or modification at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position. In some embodiments, the modified nucleotide comprises one or more modifications independently selected from a 2′-position sugar modification, a 2′-amino (2′-NH₂) modification, a 2′-fluoro (2′-F) modification, a 2′-O-methyl(2′-OMe) modification, a 2′-O-(2-Methoxyethyl) (2′-O-MOE) modification, a 5-position modified pyrimidine, a modification at a cytosine exocyclic amine, a substitution with 5′-bromouracil, a substitution with 5′-bromodeoxyuridine, a substitution with 5′-bromodeoxycytidine, a backbone modification, a locked nucleic acid (LNA), a methylation, a 3′ cap, and a 5′ cap. In some embodiments, the 5-position modified pyrimidine is selected from 5-carboxy-2′-deoxyuridine, 5-aminoallyl-2′-deoxyuridine, 5-[(3-indolyl) propionamide-N-allyl]-2′-deoxyuridine, 5-carboxy-2′-deoxycytidine, 5-aminoallyl-2′-deoxycytidine, biotin-16-aminoallyl-2′-deoxycytidine, 5-(N-benzylcarboxyamide)-2′-deoxyuridine, 5-(N-isobutylcarboxyamide)-2′-deoxyuridine, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine, and 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine.

[0022] In some embodiments, the anti-VEGF aptamer comprises at least one 2′-modified nucleotides. In some embodiments, all the nucleotides of the anti-VEGF aptamer are 2′-modified nucleotides. In some embodiments, the 2′-modified nucleotide is selected from a 2′-amino-modified nucleotide, a 2′-fluoro-modified nucleotide, and a 2′-O-methyl-modified nucleotide, and a 2′-O-(2-methoxyethyl)-modified nucleotide. In some embodiments, the aptamer comprises at least one 2′-fluoro-modified nucleotide, at least one 2′-O-methyl-modified nucleotide, and/or at least one LNA.

[0023] In some embodiments, all the cytidines of the anti-VEGF aptamer are 2′-modified cytidines. In some embodiments, all the cytidines of the anti-VEGF aptamer are 2′-fluoro-modified cytidines and/or 2′-O-methyl-modified cytidines. In some embodiments, all the cytidines of the anti-VEGF aptamer are 2′-deoxy-2′-fluorocytidines. In some embodiments, all the uridines of the anti-VEGF aptamer are 2′-modified uridines. In some embodiments, all the uridines of the anti-VEGF aptamer are 2′-fluoro-modified uridines and/or 2′-O-methyl-modified uridines. In some embodiments, all the uridines of the anti-VEGF aptamer are 2′-deoxy-2′-fluorouridines. In some embodiments, all the adenosines of the anti-VEGF aptamer are 2′-modified adenosines. In some embodiments, all the adenosines of the anti-VEGF aptamer are 2′-fluoro-modified adenosines and/or 2′-O-methyl-modified adenosines. In some embodiments, all the adenosines of the anti-VEGF aptamer are 2′-deoxy-2′-fluoroadenosines. In some embodiments, all the guanosines of the anti-VEGF aptamer are 2′-modified guanosines. In some embodiments, all the guanosines of the anti-VEGF aptamer are 2′-fluoro-modified guanosines and/or 2′-O-methyl-modified guanosines. In some embodiments, all the guanosines of the anti-VEGF aptamer are 2′-deoxy-2′-fluoroguanosines.

[0024] In some embodiments, all the nucleotides of the anti-VEGF aptamer are 2′-fluoro-modified nucleotides and/or 2′-O-methyl-modified nucleotides.

[0025] In some embodiments, all the nucleotides of the anti-VEGF aptamer are 2′-fluoro-modified nucleotides.

[0026] In some embodiments, the anti-VEGF aptamer does not comprise any natural nucleotide.

[0027] In some embodiments, the anti-VEGF aptamer comprises about 10 to about 150 nucleotides.

[0028] In some embodiments, the anti-VEGF aptamer competes with an anti-VEGF reference aptamer for binding to the VEGF-165 and/or to the VEGF-121, wherein the anti-VEGF reference aptamer comprises a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0029] In some embodiments, the anti-VEGF reference aptamer comprises a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0030] In some embodiments, the anti-VEGF reference aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 79-90. In some embodiments, the anti-VEGF reference aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 91-100.

[0031] In some embodiments, the first consensus sequence is comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF reference aptamer. In some embodiments, the first consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-VEGF reference aptamer. In some embodiments, the second consensus sequence is comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF reference aptamer. In some embodiments, the second consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-VEGF reference aptamer.

[0032] In some embodiments, the anti-VEGF reference aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 1-62 or a variant or truncate thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 1-62. In some embodiments, the variant comprises a nucleotide sequence as set forth in any of SEQ ID NO: 1-62 with one or more nucleotide addition, deletion, and/or substitution. In some embodiments, the anti-VEGF reference aptamer comprises about 10 to about 150 nucleotides.

[0033] In some embodiments, the anti-VEGF reference aptamer comprises the first consensus sequence and the second consensus sequence and the first consensus sequence is located 5′ to second consensus sequence.

[0034] In embodiments, the anti-VEGF reference aptamer comprises the first consensus sequence and the second consensus sequence and the second consensus sequence is located 5′ to the first consensus sequence.

[0035] In some embodiments, the anti-VEGF aptamer comprises a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop. In some embodiments, the anti-VEGF aptamer comprises a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0036] In some embodiments, the anti-VEGF aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 79-90. In some embodiments, the anti-VEGF aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 91-100.

[0037] In some embodiments, the first consensus sequence is comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF aptamer. In some embodiments, the first consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-VEGF aptamer. In some embodiments, the second consensus sequence is comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF aptamer. In some embodiments, the second consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-VEGF aptamer.

[0038] The first loop of the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise 2-30 nucleotides.

[0039] In some embodiments, the anti-VEGF aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, or a variant or truncate thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 1-62. In some embodiments, the variant comprises a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, with one or more nucleotide addition, deletion, and/or substitution.

[0040] In some embodiments, the anti-VEGF aptamer comprises the first consensus sequence and the second consensus sequence, and the first consensus sequence is located 5′ to the second consensus sequence. In some embodiments, the anti-VEGF aptamer comprises the first consensus sequence and the second consensus sequence, and the second consensus sequence is located 5′ to the first consensus sequence.

[0041] In some embodiments, the anti-VEGF aptamer is used for modulating the biological activity of a VEGF or a VEGF receptor.

[0042] In some embodiments, the anti-VEGF aptamer is used for preventing, treating, and/or ameliorating a VEGF related disease, disorder or condition. In some embodiments, the VEGF related disease, disorder or condition is a neovascular disease, disorder or condition. In some embodiments, the VEGF related disease, disorder or condition is an ocular neovascular disease, disorder or condition. In some embodiments, the VEGF related disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. In some embodiments, the VEGF related disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0043] In one aspect, the present disclosure provides an anti-VEGF agent, comprising the anti-VEGF aptamer of the present disclosure. In some embodiments, the anti-VEGF agent further comprises a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety is conjugated to the 5′ terminus of the aptamer.

[0044] In one aspect, the present disclosure provides a composition, comprising the anti-VEGF aptamer, or the anti-VEGF agent of the present disclosure.

[0045] In some embodiment, the composition is a pharmaceutical composition. In some embodiment, the composition comprises a pharmaceutically acceptable excipient or carrier. In some embodiment, the composition comprises a therapeutically effective amount of the anti-VEGF aptamer or the anti-VEGF agent.

[0046] In one aspect, the present disclosure provides a method for modulating the biological activity of a VEGF and/or a VEGF receptor, comprising administering to a subject in need thereof an effective amount of the anti-VEGF aptamer or the anti-VEGF agent of the present disclosure.

[0047] In one aspect, the present disclosure provides a method for preventing, treating, and/or ameliorating a VEGF related disease, disorder or condition, comprising administering to a subject in need thereof an effective amount of the anti-VEGF aptamer or the anti-VEGF agent of the present disclosure. In some embodiments, the VEGF related disease, disorder or condition is a neovascular disease, disorder or condition. In some embodiments, the VEGF related disease, disorder or condition is an ocular neovascular disease, disorder or condition. In some embodiment, the VEGF related disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. In some embodiments, the VEGF related disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0048] In one aspect, the present disclosure provides a use of the anti-VEGF aptamer, or the anti-VEGF agent of the present disclosure in the manufacture of an agent for modulating the biological activity of a VEGF or a VEGF receptor.

[0049] In one aspect, the present disclosure provides a use of the anti-VEGF aptamer, or the anti-VEGF agent of the present disclosure, in the manufacture of a medicament for preventing, treating, and/or ameliorating a VEGF related disease, disorder or condition. In some embodiments, the VEGF related disease, disorder or condition is a neovascular disease, disorder or condition. In some embodiments, the VEGF related disease, disorder or condition is an ocular neovascular disease, disorder or condition. In some embodiments, the VEGF related disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. In some embodiments, the VEGF related disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

Anti-Ang2 Aptamer

[0050] In one aspect, the present disclosure provides an anti-Ang2 aptamer that specifically binds to Ang2 with a KD value of about 500 picomolar (pM) or less.

[0051] In some embodiments, the anti-Ang2 aptamer binds to Ang2 with a K_D value of about 100 picomolar (pM) or less.

[0052] In some embodiments, the Ang2 is a human Ang2, a mouse Ang2, a monkey Ang2, a rabbit Ang 2, and/or a rat Ang2.

[0053] In some embodiments, the anti-Ang2 aptamer does not substantially bind to human Ang1.

[0054] In some embodiments, the anti-Ang2 aptamer inhibits an interaction between the Ang2 and Tie2.

[0055] In some embodiments, the anti-Ang2 aptamer is capable of reducing neovascularization and/or reducing vascular permeability in a mouse oxygen-induced ischemic retinopathy (OIR) model.

[0056] In some embodiments, the anti-Ang2 aptamer is highly soluble.

[0057] In some embodiments, the anti-Ang2 aptamer is an RNA aptamer, a DNA aptamer, or a combination thereof. In some embodiments, the anti-Ang2 aptamer comprises one or more modified nucleotides. In some embodiments, the anti-Ang2 aptamer is nuclease resistant. In some embodiments, all the nucleotides of the anti-Ang2 aptamer are modified nucleotides. In some embodiments, the modified nucleotide comprises a chemical substitution or modification at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position. In some embodiments, the modified nucleotide comprises one or more modifications independently selected from a 2′-position sugar modification, a 2′-amino (2′-NH₂) modification, a 2′-fluoro (2′-F) modification, a 2′-O-methyl(2′-OMe) modification, a 2′-O-(2-Methoxyethyl) (2′-O-MOE) modification, a 5-position modified pyrimidine, a modification at a cytosine exocyclic amine, a substitution with 5′-bromouracil, a substitution with 5′-bromodeoxyuridine, a substitution with 5′-bromodeoxycytidine, a backbone modification, a locked nucleic acid (LNA), a methylation, a 3′ cap, and a 5′ cap. In some embodiments, the 5-position modified pyrimidine is selected from 5-carboxy-2′-deoxyuridine, 5-aminoallyl-2′-deoxyuridine, 5-[(3-indolyl) propionamide-N-allyl]-2′-deoxyuridine, 5-carboxy-2′-deoxycytidine, 5-aminoallyl-2′-deoxycytidine, biotin-16-aminoallyl-2′-deoxycytidine, 5-(N-benzylcarboxyamide)-2′-deoxyuridine, 5-(N-isobutylcarboxyamide)-2′-deoxyuridine, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine, and 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine.

[0058] In some embodiments, the anti-Ang2 aptamer comprises at least one 2′-modified nucleotides. In some embodiments, all the nucleotides of the anti-Ang2 aptamer are 2′-modified nucleotides. In some embodiments, the 2′-modified nucleotide is selected from a 2′-amino-modified nucleotide, a 2′-fluoro-modified nucleotide, and a 2′-O-methyl-modified nucleotide and a 2′-O-methoxyl-modified nucleotide.

[0059] In some embodiments, the anti-Ang2 aptamer comprises at least one 2′-fluoro-modified nucleotide, at least one 2′-O-methyl-modified nucleotide, and/or at least one LNA. In some embodiments, all the cytidines of the anti-Ang2 aptamer are 2′-modified cytidines. In some embodiments, all the cytidines of the anti-Ang2 aptamer are 2′-fluoro-modified cytidines and/or 2′-O-methyl-modified cytidines. In some embodiments, all the cytidines of the anti-Ang2 aptamer are 2′-deoxy-2′-fluorocytidines. In some embodiments, all the uridines of the anti-Ang2 aptamer are 2′-modified uridines. In some embodiments, all the uridines of the anti-Ang2 aptamer are 2′-fluoro-modified uridines and/or 2′-O-methyl-modified uridines. In some embodiments, all the uridines of the anti-Ang2 aptamer are 2′-deoxy-2′-fluorouridines. In some embodiments, all the adenosines of the anti-Ang2 aptamer are 2′-modified adenosines. In some embodiments, all the adenosines of the anti-Ang2 aptamer are 2′-fluoro-modified adenosines and/or 2′-O-methyl-modified adenosines. In some embodiments, all the adenosines of the anti-Ang2 aptamer are 2′-deoxy-2′-fluoroadenosines. In some embodiments, all the guanosines of the anti-Ang2 aptamer are 2′-modified guanosines. In some embodiments, all the guanosines of the anti-Ang2 aptamer are 2′-fluoro-modified guanosines and/or 2′-O-methyl-modified guanosines. In some embodiments, all the guanosines of the anti-Ang2 aptamer are 2′-deoxy-2′-fluoroguanosines. In some embodiments, all the nucleotides of the anti-Ang2 aptamer are 2′-fluoro-modified nucleotides and/or 2′-O-methyl-modified nucleotides. In some embodiments, all the nucleotides of the anti-Ang2 aptamer are 2′-fluoro-modified nucleotides.

[0060] In some embodiments, the anti-Ang2 aptamer does not comprise any natural nucleotide. In some embodiments, the anti-Ang2 aptamer comprises about 10 to about 150 nucleotides.

[0061] In some embodiments, the anti-Ang2 aptamer competes with an anti-Ang2 reference aptamer for binding to the Ang2, wherein the anti-Ang2 reference aptamer comprises a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0062] In some embodiments, the anti-Ang2 reference aptamer comprises a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0063] In some embodiments, the anti-Ang2 reference aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 101-108. In some embodiments, the anti-Ang2 reference aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 109-116.

[0064] In some embodiments, the first consensus sequence is comprised in the nucleotide sequence forming the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 reference aptamer. In some embodiments, the first consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-Ang2 reference aptamer.

[0065] In some embodiments, the second consensus sequence is comprised in the nucleotide sequence forming the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 reference aptamer. In some embodiments, the second consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-Ang2 reference aptamer.

[0066] In some embodiments, the anti-Ang2 reference aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 63-74. In some embodiments, the variant comprises a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, with one or more nucleotide addition, deletion, and/or substitution. In some embodiments, the anti-Ang2 reference aptamer comprises about 10 to about 150 nucleotides.

[0067] In some embodiments, the anti-Ang2 reference aptamer comprises the first consensus sequence and the second consensus sequence, and the first consensus sequence is located 5′ to the second consensus sequence.

[0068] In some embodiments, the anti-Ang2 aptamer comprises a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop. In some embodiments, the anti-Ang2 aptamer comprises a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0069] In some embodiments, the anti-Ang2 aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 101-108. In some embodiments, the anti-Ang2 aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 109-116.

[0070] In some embodiments, the first consensus sequence is comprised in the nucleotide sequence forming the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 aptamer. In some embodiments, the first consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-Ang2 aptamer. In some embodiments, the second consensus sequence is comprised in the nucleotide sequence forming the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 aptamer. In some embodiments, the second consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-Ang2 aptamer.

[0071] The first loop of the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprises 2-30 nucleotides.

[0072] In some embodiments, the anti-Ang2 aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 63-74. In some embodiments, the variant comprises a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, with one or more nucleotide addition, deletion, and/or substitution.

[0073] In some embodiments, the anti-Ang2 aptamer comprises the first consensus sequence and the second consensus sequence, and the first consensus sequence is located 5′ to the second consensus sequence.

[0074] In some embodiments, the anti-Ang2 aptamer comprises the first consensus sequence and the second consensus sequence, and the second consensus sequence is located 5′ to the first consensus sequence.

[0075] In some embodiments, the anti-Ang2 aptamer is used for modulating the biological activity of Ang2 or Tie2.

[0076] In some embodiments, the anti-Ang2 aptamer is used for preventing, treating, and/or ameliorating an Ang2 related disease, disorder or condition. In some embodiments, the Ang2 related disease, disorder or condition is a neovascular disease, disorder or condition. In some embodiments, the Ang2 related disease, disorder or condition is an ocular neovascular disease, disorder or condition. In some embodiments, the Ang2 related disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. In some embodiments, the Ang2 related disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy.

[0077] In one aspect, the present disclosure provides an anti-Ang2 agent, comprising the anti-Ang2 aptamer of the present disclosure. In some embodiments, the anti-Ang2 agent further comprises a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety is conjugated to the 3′ terminus of the anti-Ang2 aptamer.

[0078] In one aspect, the present disclosure provides a composition, comprising the anti-Ang2 aptamer, or the anti-Ang2 agent of the present disclosure.

[0079] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutically acceptable excipient or carrier. In some embodiments, the composition comprises an effective amount of the anti-Ang2 aptamer or the anti-Ang2 agent.

[0080] In some embodiments, the composition further comprises an anti-VEGF agent. In some embodiments, the anti-VEGF agent comprises Aflibercept.

[0081] In one aspect, the present disclosure provides a method for modulating the biological activity of Ang2 or Tie2, comprising administering to a subject in need thereof an effective amount of the anti-Ang2 aptamer, or the anti-Ang2 agent of the present disclosure.

[0082] In one aspect, the present disclosure provides a method for preventing, treating, and/or ameliorating an Ang2 related disease, disorder or condition, comprising administering to a subject in need thereof an effective amount of the anti-Ang2 aptamer or the anti-Ang2 agent of the present disclosure.

[0083] In some embodiments, the Ang2 related disease, disorder or condition is a neovascular disease, disorder or condition. In some embodiments, the Ang2 related disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. In some embodiments, the Ang2 related disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy. In some embodiments, the method further comprises administering to the subject an anti-VEGF agent. In some embodiments, the anti-VEGF agent comprises Aflibercept.

[0084] In one aspect, the present disclosure provides a use of the anti-Ang2 aptamer, or the anti-Ang2 agent of the present disclosure in the manufacture of an agent for modulating the biological activity of Ang2 or Tie2.

[0085] In one aspect, the present disclosure provides a use of the anti-Ang2 aptamer, or the anti-Ang2 agent of the present disclosure in the manufacture of a medicament for preventing, treating, and/or ameliorating an Ang2 related disease, disorder or condition.

[0086] In some embodiments, the Ang2 related disease, disorder or condition is a neovascular disease, disorder or conditions. In some embodiments, the Ang2 related disease, disorder or condition is an ocular neovascular disease, disorder or condition. In some embodiments, the Ang2 related disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. In some embodiments, the Ang2 related disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0087] In one aspect, the present disclosure provides a use of 1) the anti-Ang2 aptamer, or the anti-Ang2 agent of the present disclosure in combination with 2) an anti-VEGF agent in the manufacture of a medicament for preventing, treating, and/or ameliorating a neovascular disease, disorder or condition. In some embodiments, the anti-VEGF agent comprises Aflibercept. In some embodiments, the neovascular disease or disorder is an ocular neovascular disease, disorder or condition. In some embodiments, the neovascular disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. In some embodiments, the neovascular disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

Bispecific Aptamer

[0088] In another aspect, the present disclosure provides a bispecific aptamer comprising an anti-Ang2 aptamer and an anti-VEGF aptamer. The bispecific aptamer may be comprised in the composition of the present disclosure.

[0089] In some embodiments, the anti-VEGF aptamer binds to VEGF-121 with a K_D value of about 20 nanomolar (nM) or less, and binds to VEGF-165 with a K_D value of about 20 nanomolar (nM) or less.

[0090] In some embodiments, the anti-Ang2 aptamer specifically binds to Ang2 with a K_D value of about 500 picomolar (pM) or less.

[0091] In some embodiments, the anti-VEGF aptamer binds to VEGF-121 with a K_D value of about 2 nanomolar (nM) or less. In some embodiments, the anti-VEGF aptamer binds to VEGF-165 with a K_D value of about 2 nanomolar (nM) or less.

[0092] In some embodiments, the VEGF-121 is a human VEGF-121, a mouse VEGF-120, a monkey VEGF-121, a rabbit VEGF-121, and or a rat VEGF-120. In some embodiments, the VEGF-165 is a human VEGF-165, a mouse VEGF-164, a monkey VEGF-165, a rabbit VEGF-165, or a rat VEGF-164.

[0093] In some embodiments, the anti-VEGF aptamer specifically binds to both a VEGF receptor-binding domain of VEGF-121 and a VEGF receptor-binding domain of VEGF-165.

[0094] In some embodiments, the VEGF receptor-binding domain of VEGF-121 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 118-122, 128, 129, and 133. In some embodiments, the VEGF receptor-binding domain of VEGF-165 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 118-122, 128, and 129.

[0095] In some embodiments, the anti-VEGF aptamer inhibits an interaction between the VEGF-121 and VEGF-R1. In some embodiments, the anti-VEGF aptamer inhibits an interaction between the VEGF-121 and VEGF-R2. In some embodiments, the anti-VEGF aptamer inhibits an interaction between the VEGF-165 and VEGF-R1. In some embodiments, the anti-VEGF aptamer inhibits an interaction between the VEGF-165 and VEGF-R2.

[0096] In some embodiments, the anti-VEGF aptamer is capable of reducing and/or ameliorating a lesion and/or a leakage in a laser-induced choroidal neovascularization (CNV) rat model.

[0097] In some embodiments, the anti-VEGF aptamer is highly soluble.

[0098] In some embodiments, the anti-VEGF aptamer is an RNA aptamer, a DNA aptamer, or a combination thereof. In some embodiments, the anti-VEGF aptamer is nuclease resistant. In some embodiments, the anti-VEGF aptamer comprises one or more modified nucleotide. In some embodiments, all the nucleotides of the anti-VEGF aptamer are modified nucleotides. In some embodiments, the modified nucleotide comprises a chemical substitution or modification at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position. In some embodiments, the modified nucleotide comprises one or more modifications independently selected from a 2′-position sugar modification, a 2′-amino (2′-NH₂) modification, a 2′-fluoro (2′-F) modification, a 2′-O-methyl(2′-OMe) modification, a 2′-O-(2-Methoxyethyl) (2′-O-MOE) modification, a 5-position modified pyrimidine, a modification at a cytosine exocyclic amine, a substitution with 5′-bromouracil, a substitution with 5′-bromodeoxyuridine, a substitution with 5′-bromodeoxycytidine, a backbone modification, a locked nucleic acid (LNA), a methylation, a 3′ cap, and a 5′ cap. In some embodiments, the 5-position modified pyrimidine is selected from 5-carboxy-2′-deoxyuridine, 5-aminoallyl-2′-deoxyuridine, 5-[(3-indolyl) propionamide-N-allyl]-2′-deoxyuridine, 5-carboxy-2′-deoxycytidine, 5-aminoallyl-2′-deoxycytidine, biotin-16-aminoallyl-2′-deoxycytidine, 5-(N-benzylcarboxyamide)-2′-deoxyuridine, 5-(N-isobutylcarboxyamide)-2′-deoxyuridine, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine, and 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine.

[0099] In some embodiments, the anti-VEGF aptamer comprises at least one 2′-modified nucleotide. In some embodiments, all the nucleotides of the anti-VEGF aptamer are 2′-modified nucleotides. In some embodiments, the 2′-modified nucleotide is selected from a 2′-amino-modified nucleotide, a 2′-fluoro-modified nucleotide, and a 2′-O-methyl-modified nucleotide and a 2′-O-(2-methoxyethyl)-modified nucleotide. In some embodiments, the anti-VEGF aptamer comprises at least one 2′-fluoro-modified nucleotide. In some embodiments, the anti-VEGF aptamer comprises at least one 2′-fluoro-modified nucleotide, at least one 2′-O-methyl-modified nucleotide, and/or at least one LNA.

[0100] In some embodiments, all the cytidines of the anti-VEGF aptamer are 2′-modified cytidines. In some embodiments, all cytidines of the anti-VEGF aptamer are 2′-fluoro-modified cytidines and/or 2′-O-methyl-modified cytidines. In some embodiments, all the cytidines of the anti-VEGF aptamer are 2′-deoxy-2′-fluorocytidines. In some embodiments, all the uridines of the anti-VEGF aptamer are 2′-modified uridines. In some embodiments, all the uridines of the anti-VEGF aptamer are 2′-fluoro-modified uridines and/or 2′-O-methyl-modified uridines. In some embodiments, all the uridines of the anti-VEGF aptamer are 2′-deoxy-2′-fluorouridines. In some embodiments, all the adenosines of the anti-VEGF aptamer are 2′-modified adenosines. In some embodiments, all the adenosines of the anti-VEGF aptamer are 2′-fluoro-modified adenosines and/or 2′-O-methyl-modified adenosines. In some embodiments, all the adenosines of the anti-VEGF aptamer are 2′-deoxy-2′-fluoroadenosines. In some embodiments, all the guanosines are 2′-modified guanosines. In some embodiments, all the guanosines of the anti-VEGF aptamer are 2′-fluoro-modified guanosines and/or 2′-O-methyl-modified guanosines. In some embodiments, all the guanosines of the anti-VEGF aptamer are 2′-deoxy-2′-fluoroguanosines.

[0101] In some embodiments, all the nucleotides of the anti-VEGF aptamer are 2′-fluoro-modified nucleotides and/or 2′-O-methyl-modified nucleotides.

[0102] In some embodiments, all the nucleotides of the anti-VEGF aptamer are 2′-fluoro-modified nucleotides.

[0103] In some embodiments, the anti-VEGF aptamer does not comprise any natural nucleotide.

[0104] In some embodiments, the anti-VEGF aptamer comprises about 10 to about 150 nucleotides.

[0105] In some embodiments, the anti-VEGF aptamer competes with an anti-VEGF reference aptamer for binding to the VEGF-165 and/or to the VEGF-121, and the anti-VEGF reference aptamer comprises a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0106] In some embodiments, the anti-VEGF reference aptamer comprises a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0107] In some embodiments, the anti-VEGF reference aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 79-90. In some embodiments, the anti-VEGF reference aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 91-100.

[0108] In some embodiments, the first consensus sequence is comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF reference aptamer. In some embodiments, the first consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-VEGF reference aptamer. In some embodiments, the second consensus sequence is comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF reference aptamer. In some embodiments, the second consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-VEGF reference aptamer.

[0109] In some embodiments, the anti-VEGF reference aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, or a variant or truncate thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 1-62. In some embodiments, the variant comprises a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, with one or more nucleotide addition, deletion, and/or substitution. In some embodiments, the anti-VEGF reference aptamer comprises about 10 to about 150 nucleotides.

[0110] In some embodiments, the anti-VEGF reference aptamer comprises the first consensus sequence and the second consensus sequence, and the first consensus sequence is located 5′ to the second consensus sequence

[0111] In some embodiments, the anti-VEGF reference aptamer comprises the first consensus sequence and the second consensus sequence, wherein the second consensus sequence is located 5′ to the first consensus sequence.

[0112] In some embodiments, the anti-VEGF aptamer comprises a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop. In some embodiments, the anti-VEGF aptamer comprises a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0113] In some embodiments, the anti-VEGF aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 79-90. In some embodiments, the anti-VEGF aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 91-100.

[0114] In some embodiments, the first consensus sequence is comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF aptamer. In some embodiments, the first consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-VEGF aptamer. In some embodiments, the second consensus sequence is comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF aptamer. In some embodiments, the second consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-VEGF aptamer.

[0115] In some embodiments, the anti-VEGF aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, or a variant or truncate thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 1-62. In some embodiments, the variant comprises a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, with one or more nucleotide addition, deletion, and/or substitution.

[0116] In some embodiments, the anti-VEGF aptamer comprises the first consensus sequence and the second consensus sequence, and the first consensus sequence is located 5′ to the second consensus sequence.

[0117] In some embodiments, the anti-VEGF aptamer comprises the first consensus sequence and the second consensus sequence, and the second consensus sequence is located 5′ to the consensus sequence.

[0118] In some embodiments, the anti-Ang2 aptamer binds to Ang2 with a K_D value of about 100 picomolar (pM) or less.

[0119] In some embodiments, the Ang2 is a human Ang2, a mouse Ang2, a monkey Ang2, a rabbit Ang2, and/or a rat Ang2.

[0120] In some embodiments, the anti-Ang2 aptamer does not substantially bind to human Ang1.

[0121] In some embodiments, the anti-Ang2 aptamer inhibits an interaction between the Ang2 and Tie2.

[0122] In some embodiments, the anti-Ang2 aptamer is capable of reducing neovascularization and/or reducing vascular permeability in a mouse oxygen-induced ischemic retinopathy (OIR) model.

[0123] In some embodiments, the anti-Ang2 aptamer is highly soluble.

[0124] In some embodiments, the anti-Ang2 aptamer is an RNA aptamer, a DNA aptamer, or a combination thereof. In some embodiments, the anti-Ang2 aptamer comprises one or more modified nucleotide. In some embodiments, the anti-Ang2 aptamer is nuclease resistant. In some embodiments, all the nucleotides of the anti-Ang2 aptamer are modified nucleotides. In some embodiments, the modified nucleotide comprises a chemical substitution or modification at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position. In some embodiments, the modified nucleotide comprises one or more modifications independently selected from a 2′-position sugar modification, a 2′-amino (2′-NH₂) modification, a 2′-fluoro (2′-F) modification, a 2′-O-methyl(2′-OMe) modification, a 2′-O-(2-Methoxyethyl) (2′-O-MOE) modification, a 5-position modified pyrimidine, a modification at a cytosine exocyclic amine, a substitution with 5′-bromouracil, a substitution with 5′-bromodeoxyuridine, a substitution with 5′-bromodeoxycytidine, a backbone modification, a locked nucleic acid (LNA), a methylation, a 3′ cap, and a 5′ cap. In some embodiments, the 5-position modified pyrimidine is selected from 5-carboxy-2′-deoxyuridine, 5-aminoallyl-2′-deoxyuridine, 5-[(3-indolyl) propionamide-N-allyl]-2′-deoxyuridine, 5-carboxy-2′-deoxycytidine, 5-aminoallyl-2′-deoxycytidine, biotin-16-aminoallyl-2′-deoxycytidine, 5-(N-benzylcarboxyamide)-2′-deoxyuridine, 5-(N-isobutylcarboxyamide)-2′-deoxyuridine, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine, and 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine.

[0125] In some embodiments, the anti-Ang2 aptamer comprises at least one 2′-modified nucleotides. In some embodiments, all the nucleotides of the anti-Ang2 aptamer are 2′-modified nucleotides. In some embodiments, the 2′-modified nucleotide is selected from a 2′-amino modified nucleotide, a 2′-fluoro-modified nucleotide, a 2′-O-methyl-modified nucleotide, and a 2′-O-(2-methoxyethyl)-modified nucleotide. In some embodiments, the anti-Ang2 aptamer comprises at least one 2′-fluoro-modified nucleotide, at least one 2′-O-methyl-modified nucleotide, and/or at least one LNA.

[0126] In some embodiments, the anti-Ang2 aptamer comprises at least one 2′-fluoro-modified nucleotide. In some embodiments, all the cytidines of the anti-Ang2 aptamer are 2′-modified cytidines. In some embodiments, all the cytidines of the anti-Ang2 aptamer are 2′-fluoro-modified cytidines and/or 2′-O-methyl-modified cytidines. In some embodiments, all the cytidines of the anti-Ang2 aptamer are 2′-deoxy-2′-fluorocytidines. In some embodiments, all the uridines of the anti-Ang2 aptamer are 2′-modified uridines. In some embodiments, all the uridines of the anti-Ang2 aptamer are 2′-fluoro-modified uridines and/or 2′-O-methyl-modified uridines. In some embodiments, all the uridines of the anti-Ang2 aptamer are 2′-deoxy-2′-fluorouridines. In some embodiments, all the adenosines of the anti-Ang2 aptamer are 2′-modified adenosines. In some embodiments, all the adenosines of the anti-Ang2 aptamer are 2′-fluoro-modified adenosines and/or 2′-O-methyl-modified adenosines. In some embodiments, all the adenosines of the anti-Ang2 aptamer are 2′-deoxy-2′-fluoroadenosines. In some embodiments, all the guanosines of the anti-Ang2 aptamer are 2′-modified guanosines. In some embodiments, all the all guanosines of the anti-Ang2 aptamer are 2′-fluoro-modified guanosines and/or 2′-O-methyl-modified guanosines. In some embodiments, all the guanosines of the anti-Ang2 aptamer are 2′-deoxy-2′-fluoroguanosines. In some embodiments, all the nucleotides of the anti-Ang2 aptamer are 2′-fluoro-modified nucleotides. In some embodiments, all the nucleotides of the anti-Ang2 aptamer are 2′-fluoro-modified nucleotides and/or 2′-O-methyl-modified nucleotides.

[0127] In some embodiments, the anti-Ang2 aptamer does not comprise any natural nucleotide. In some embodiments, the anti-Ang2 aptamer comprises about 10 to about 150 nucleotides.

[0128] In some embodiments, the anti-Ang2 aptamer competes with an anti-Ang2 reference aptamer for binding to the Ang2, wherein the anti-Ang2 reference aptamer comprises a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0129] In some embodiments, the anti-Ang2 reference aptamer comprises a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0130] In some embodiments, the anti-Ang2 reference aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 101-108. In some embodiments, the anti-Ang2 reference aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 109-116.

[0131] In some embodiments, the first consensus sequence is comprised in the nucleotide sequence forming the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 reference aptamer. In some embodiments, the first consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-Ang2 reference aptamer.

[0132] In some embodiments, the second consensus sequence is comprised in the nucleotide sequence forming the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 reference aptamer. In some embodiments, the second consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-Ang2 reference aptamer.

[0133] In some embodiments, the anti-Ang2 reference aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 63-74. In some embodiments, the variant comprises a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, with one or more nucleotide addition, deletion, and/or substitution. In some embodiments, the anti-Ang2 reference aptamer comprises about 10 to about 150 nucleotides.

[0134] In some embodiments, the anti-Ang2 reference aptamer comprises the first consensus sequence and the second consensus sequence, and the first consensus sequence is located 5′ to the second consensus sequence.

[0135] In some embodiments, the anti-Ang2 reference aptamer comprises the first consensus sequence and the second consensus sequence, and the second consensus sequence is located 5′ to the first consensus sequence.

[0136] In some embodiments, the anti-Ang2 aptamer comprises a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop. In some embodiments, the aptamer comprises a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0137] In some embodiments, the anti-Ang2 aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 101-108. In some embodiments, the anti-Ang2 aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 109-116.

[0138] In some embodiments, the first consensus sequence is comprised in the nucleotide sequence forming the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 aptamer. In some embodiments, the first consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-Ang2 aptamer. In some embodiments, the second consensus sequence is comprised in the nucleotide sequence forming the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 aptamer. In some embodiments, the second consensus sequence is not comprised in the nucleotide sequence forming first loop of the anti-Ang2 aptamer.

[0139] In some embodiments, the anti-Ang2 aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 63-74. In some embodiments, the variant comprises a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, with one or more nucleotide addition, deletion, and/or substitution.

[0140] In some embodiments, the anti-Ang2 aptamer comprises the first consensus sequence and the second consensus sequence, and the first consensus sequence is located 5′ to the second consensus sequence.

[0141] In some embodiments, the anti-Ang2 aptamer comprises the first consensus sequence and the second consensus sequence, and the second consensus sequence is located 5′ to the first consensus sequence.

[0142] In some embodiments, the anti-VEGF aptamer is conjugated to a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety is conjugated to the 5′ terminus of the anti-VEGF aptamer. In some embodiments, the PEG moiety is conjugated to the 3′ terminus of the anti-VEGF aptamer. In some embodiments, the anti-Ang2 aptamer is conjugated to a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety is conjugated to the 5′ terminus of the anti-Ang2 aptamer. In some embodiments, the PEG moiety is conjugated to the 3′ terminus of the anti-Ang2 aptamer.

[0143] In some embodiments, the bispecific aptamer is of the formula A1-(L) n-A2, wherein A1 is the anti-VEGF aptamer and A2 is the anti-Ang2 aptamer, or A1 is the anti-Ang2 aptamer and A2 is the anti-VEGF aptamer; L is a linker; and n is a number of at least 0. In some embodiments, n is 0 to 20. In some embodiments, n is 0 to 10.

[0144] In some embodiments, the bispecific aptamer is capable of specifically binding to both VEGF and Ang2.

[0145] In some embodiments, the bispecific aptamer binds to VEGF-121 with a K_D value of about 500 μM or less, and binds to Ang2 with a K_D value of about 500 μM or less.

[0146] In some embodiments, the bispecific aptamer binds to VEGF-121 with a K_D value of about 200 μM or less, and binds to Ang2 with a K_D value of about 200 μM or less.

[0147] In some embodiments, the bispecific aptamer binds to VEGF-121 with a K_D value of about 100 μM or less, and binds to Ang2 with a K_D value of about 100 μM or less.

[0148] In some embodiments of the bispecific aptamer, L is selected from a naturally occurring nucleotide linker, a modified nucleotide linker, a hydrocarbon linker, a polyethylene glycol linker and a combination thereof. In some embodiments, at least one L is a polyethylene glycol linker. In some embodiments, at least one L is a hexaethylene glycol (H or PEG6) linker. In some embodiments, L is a hexaethylene glycol linker, and n is 0 to 10.

[0149] In some embodiments of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, or a variant or truncate thereof; and A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof.

[0150] In some embodiments of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof; and A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, or a variant or truncate thereof.

[0151] In some embodiments of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 45, or a truncate thereof, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0152] In some embodiments of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 55, or a truncate thereof, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0153] In some embodiments of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, or a truncate thereof, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0154] In some embodiments of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 45, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0155] In some embodiments of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 55, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0156] In some embodiments of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0157] In some embodiments, the bispecific aptamer is selected from r-AMSB101, r-AMSB102, r-AMSB103, r-AMSB104, r-AMSB105, r-AMSB106, r-AMSB103.1, r-AMSB103.2, r-AMSB103.3, r-AMSB103.4, r-AMSB103.5, r-AMSB103.6, AMSB101, AMSB102, AMSB103, AMSB104, AMSB105, AMSB106, AMSB103.1, AMSB103.2, AMSB103.3, AMSB103.4, AMSB103.5, and AMSB103.6.

[0158] In some embodiments, the bispecific aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 75-78 and 138-157.

Composition

[0159] In one aspect, the present disclosure provides a composition comprising the anti-Ang2 aptamer of the present disclosure, the anti-VEGF aptamer of the present disclosure, and/or the bispecific aptamer of the present disclosure.

[0160] In some embodiments, the composition is a pharmaceutical composition.

[0161] In some embodiments, the composition comprises a pharmaceutically acceptable excipient or carrier.

[0162] In some embodiments, the composition is for preventing, treating and/or ameliorating a neovascular disease, disorder or condition. The neovascular disease, disorder or condition may be an ocular neovascular disease, disorder or condition. In some embodiments, the neovascular disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. In some embodiments, the neovascular disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

Medical Use and Methods

[0163] In another aspect, the present disclosure provides a method for preventing, treating and/or ameliorating a neovascular disease, disorder or condition, comprising administering to a subject in need thereof an effective amount of the anti-Ang2 aptamer of the present disclosure, an effective amount of the anti-VEGF aptamer of the present disclosure, an effective amount of the bispecific aptamer of the present disclosure, and/or an effective amount of the composition of the present disclosure.

[0164] In some embodiments, the neovascular disease, disorder or condition is an ocular neovascular disease, disorder or condition. In some embodiments, the neovascular disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. In some embodiments, the neovascular disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0165] In another aspect, the present disclosure provides use of the anti-Ang2 aptamer of the present disclosure, the anti-VEGF aptamer of the present disclosure, the bispecific aptamer of the present disclosure, and/or the composition of the present disclosure in the manufacture of a medicament for preventing, treating and/or ameliorating a neovascular disease, disorder or condition.

[0166] In some embodiments, the neovascular disease, disorder or condition is an ocular neovascular disease, disorder or condition. In some embodiments, the neovascular disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. In some embodiments, the neovascular disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0167] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0168] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0181] While various embodiments of the 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 may 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.

[0182] The term “aptamer”, as used herein, generally refers to a non-naturally occurring nucleic acid that has a desirable action on a target molecule. A desirable action may comprise, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way that modifies or alters the target or the functional activity of the target, covalently attaching to the target (as in a suicide inhibitor), and facilitating the reaction between the target and another molecule. In some embodiments, the action may be specific binding affinity for a target molecule, such target molecule being a three-dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which is independent of Watson/Crick base pairing or triple helix formation, wherein the aptamer is not a nucleic acid having the known physiological function of being bound by the target molecule. Aptamers to a given target include nucleic acids that may be identified from a candidate mixture of nucleic acids, where the aptamer may be a ligand of the target, by a method comprising: (a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to other nucleic acids in the candidate mixture can be partitioned from the remainder of the candidate mixture; (b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby aptamers of the target molecule are identified. It is recognized that affinity interactions are a matter of degree; however, in this context, the “specific binding affinity” of an aptamer for its target means that the aptamer may bind to its target generally with a much higher degree of affinity than it binds to other, non-target, components in a mixture or sample. An “aptamer” or “nucleic acid ligand” is a set of copies of one type or species of nucleic acid molecule that has a particular nucleotide sequence. An aptamer may include any suitable number of nucleotides. Aptamers may be DNA and/or RNA (or a combination of DNA and RNA) and may be single stranded, double stranded, or contain double stranded or triple stranded regions. In some cases, an aptamer may also comprise one or more (e.g., 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) non-nucleotide substitutions. For example, one or more nucleotides of the aptamer may be substituted with a polyethylene glycol moiety or a linker comprising multiple (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) polyethylene glycol moieties. In some cases, the linker may be a hexaethylene glycol (H or PEG6) linker. Such substitution may occur to any nucleotide of the aptamer, as long as it does not substantially affect the activity (e.g., binding activity, or inhibition activity) and/or function of the aptamer. In some cases, the substitution does not substantially affect the secondary structure of the aptamer, for example, the substitution may occur to a nucleotide comprised in a bulge or a loop region of the aptamer secondary structure. In some cases, the substitution does not occur to any nucleotide comprised in a stem region of the aptamer secondary structure.

[0183] The term “anti-VEGF aptamer”, as used herein, generally refers to a nucleic acid molecule having a specific binding affinity for VEGF. In some cases, the anti-VEGF aptamer may comprise one or more (e.g., 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) non-nucleotide substitutions, as defined herein. For example, one or more nucleotides of the anti-VEGF aptamer may be substituted with a polyethylene glycol moiety or a linker comprising multiple (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) polyethylene glycol moieties. In some cases, the linker may be a hexaethylene glycol (H or PEG6) linker. Such substitution may occur to any nucleotide of the anti-VEGF aptamer, as long as it does not substantially affect the activity (e.g., binding activity, or inhibition activity) and/or function of the anti-VEGF aptamer. In some cases, the substitution does not substantially affect the secondary structure of the anti-VEGF aptamer, for example, the substitution may occur to a nucleotide comprised in a bulge or a loop region of the anti-VEGF aptamer secondary structure. In some cases, the substitution does not occur to any nucleotide comprised in a stem region of the anti-VEGF aptamer secondary structure.

[0184] The term “VEGF”, as used herein, generally refers to vascular endothelial growth factor A (VEGFA or VEGF-A) as well as splice variants and isoforms thereof. VEGF can be found as four different splice variants known as VEGF121 (or VEGF-121), VEGF165 (or VEGF-165), VEGF189 (or VEGF-189) and VEGF206 (or VEGF-206; the number refers to the number of amino acids in the polypeptide). All four isoforms exist as disulfide-linked homodimers. The secretion patterns of the isoforms are different in various cell types, although VEGF-165 is the most common isoform observed. The isoforms bind with high affinity to two receptors, Flt-1 and Flk-1/KDR, but they differ in their binding affinity for heparin and extracellular matrix. The number of human VEGF in NCBI database can be NP_001273973.1, NP_001028928.1, NP_001020540.2, NP_001020539.2, NP_001020538.2, NP_003367.4, or NP_001020537.2.

[0185] The term “K_D value”, as used herein, generally refers to a dissociation constant, measured by a technique appropriate for the aptamer and target pair, for example by using Ligand Binding Assays with radioactive or fluorescent measurement, Surface Plasmon Resonance (SPR), Bio-Layer Interferometry (BLI, e.g., the Octet® Systems), SRU biosystems BIND®, Isothermal Titration calorimetry (ITC), or MicroscaleThermophoresis (MST). In some embodiments, the K_D value is determined using a standard fluorescence-based ligand binding assay and saturation analysis. In one example, various concentrations of fluorescently labeled target molecules were incubated with a particle of the present disclosure for at least 3 hours at room temperature with gentle rotation. Each sample was then washed, and the remaining bound target was quantified by measuring the fluorescence of each particle using a flow cytometer. The background-subtracted fluorescence values were then fit to a saturation binding curve, e.g. by using an equilibrium binding model (for example, according to the law of mass action).

[0186] The term “specific binding” or “specifically binds to” or “specific for” are used interchangeably herein and generally refer to the binding of an agent (e.g., a nucleic acid agent, such as an aptamer) to a target molecule (e.g., a protein or a part thereof), and the binding is measurably and/or statistically different from a non-specific interaction (e.g., a non-specific interaction may be binding to a reference molecule or a random molecule). Specific binding can be measured, for example, by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target, in this case, specific binding is indicated if the binding of the labeled target to a candidate agent is competitively-inhibited by excess unlabeled target. Specific binding may be exhibited, for example, by a molecule having a K_D for the target of at least about 1 nM, at least about 900 μM, at least about 800 μM, at least about 700 μM, at least about 600 μM, at least about 500 μM, at least about 400 μM, at least about 300 μM, at least about 200 μM, at least about 100 μM, at least about 90 μM, at least about 80 μM, at least about 70 μM, at least about 60 μM, at least about 50 μM, at least about 40 μM, at least about 30 μM, at least about 20 μM, at least about 10 μM, at least about 5 pM, at least about 1 μM, or greater.

[0187] The term “VEGF receptor-binding domain”, as used herein, generally refers to a domain of VEGF that binds to or is recognized by a corresponding VEGF receptor.

[0188] The term “VEGF-R” or “VEGF receptor” as used herein, generally refers to receptors that bind VEGF or VEGF family members, including their splice variants and isoforms. VEGFRs may include: (i) Flt-1 (fins-like tyrosine kinase), which is also known as VEGF-R1 (Shibuya et al., Oncogene (1990), 5:519-524; De Vries et al., Science (1992), 255:989-991); (ii) Flk-1 (fetal liver kinase), the mouse RTK (Quinn et al., Proc Natl Acad Sci USA (1993), 90:7533-7537; Millauer et al., Cell (1993), 72:835-846) and its human homolog, KDR (kinase insert domain-containing receptor which is also known as VEGF-R2; Terman et al., Biochem Biophys Res Comm (1992), 187:1579-1586); and (iii) Flt-4, which is expressed on lymphatic endothelium, but not vascular endothelium (Pajusola et al., Cancer Res (1992), 52:5738-43).

[0189] The term “RNA aptamer”, as used herein, generally refers to an aptamer comprising ribonucleotide units. “RNA aptamer” is also meant to encompass RNA analogs as defined herein.

[0190] The term “DNA aptamer”, as used herein, generally refers to an aptamer comprising deoxyribonucleotide units. “DNA aptamer” is also meant to encompass DNA analogs as defined herein.

[0191] The term “highly soluble”, as used herein, generally refers to a feature of an agent that is soluble in a solvent (e.g., water or a proper buffer) with a relatively high degree (e.g. 80% of the whole weight or more, 85% of the whole weight or more, 90% of the whole weight or more, 95% of the whole weight or more, 99% of the whole weight or more).

[0192] The term “nuclease resistant”, as used herein, generally refers to a feature of an agent that is resistant to nuclease cleavage. In some cases, nucleic acid molecules may be destroyed by exonucleases acting at either the 5′ or 3′ terminus of the nucleic acid. In addition, endonucleases may cleave the DNA or RNA at internal phosphodiester linkages between individual nucleotides. However, when the molecule (e.g., DNA, or RNA) is nuclease resistant (e.g., after certain modification), the exonuclease or endonuclease may not be able to destroy or digest it anymore.

[0193] The term “modified nucleotide”, as used herein, generally refers to a nucleotide that does not exist naturally, but is an analog or ester of a naturally occurring nucleotide. When used in the context of an oligonucleotide or a nucleic acid molecule (such as an aptamer), it generally means that at least one of the four constituent nucleotide (i.e., A, G, T/U, and C) of the oligonucleotide is an analog or ester of a naturally occurring nucleotide. In some embodiments, the modified nucleotide may confer nuclease resistance to the oligonucleotide. In some embodiments, the modified nucleotides may lead to predominantly hydrophobic interactions of an aptamer with its protein targets resulting in high binding efficiency and stable co-crystal complexes. A pyrimidine with a substitution at the C-5 position is an example of a modified nucleotide. Modifications may include backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine, and the like. Modifications may also include 3′ and 5′ modifications, such as capping. Other modifications may include substitution of one or more of the natural nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those comprising chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those comprising alkylators, and those with modified linkages (e.g., alpha anomeric nucleic acids, etc.). Further, any of the hydroxyl groups ordinarily present on the sugar of a nucleotide may be replaced by a phosphonate group or a phosphate group; protected by standard protecting groups; or activated to prepare additional linkages to additional nucleotides or to a solid support. The 5′ and 3′ terminal OH groups can be phosphorylated or substituted with amines, organic capping group moieties of from about 1 to about 20 carbon atoms, polyethylene glycol (PEG) polymers in some embodiments ranging from about 10 to about 80 kDa, PEG polymers in some embodiments ranging from about 20 to about 60 kDa, or other hydrophilic or hydrophobic biological or synthetic polymers. In some embodiments, modifications may be of the C-5 position of pyrimidines. These modifications may be produced through an amide linkage directly at the C-5 position or by other types of linkages.

[0194] The term “2′-modified nucleotide”, as used herein, generally refers to a nucleotide having a modification of the 2′ carbon atom. For example, a cytidine, a uridine, an adenosine, a guanosine may have a modification of the 2′ carbon atom.

[0195] The term “2′-fluoro-modified nucleotide”, as used herein, generally refers to a nucleotide having a 2′-fluoro substituent group. 2′-Deoxy-2′-fluoro modified oligonucleotides may be prepared by methods described in U.S. Pat. No. 6,531,584.

[0196] The term “cytidine”, as used herein, generally refers to a nucleoside molecule that is formed when cytosine is attached to a ribose ring (also known as a ribofuranose) via a β-N1-glycosidic bond. A cytidine may have the following structure:

[0197]

[0198] The term “2′-modified cytidine”, as used herein, generally refers to a cytidine having a modification of the 2′ carbon atom.

[0199] The term “2′-deoxy-2′-fluorocytidine”, as used herein, generally refers to a chemical having a structure as the following:

[0200]

[0201] The term “2′-modified uridine”, as used herein, generally refers to a uridine having a modification of the 2′ carbon atom. Uridine is a glycosylated pyrimidine-analog containing uracil attached to a ribose ring (or more specifically, a ribofuranose) via a β-N1-glycosidic bond. A uridine may have a structure as the following:

[0202]

[0203] The term “2′-deoxy-2′-fluorouridine”, as used herein, generally refers to a chemical having a structure as the following:

[0204]

[0205] The term “2′-modified adenosine”, as used herein, generally refers to an adenosine having a modification of the 2′ carbon atom. The adenosine is a purine nucleoside composed of a molecule of adenine attached to a ribose sugar molecule (ribofuranose) moiety via a β-N9-glycosidic bond. An adenosine may have a structure as the following:

[0206]

[0207] The term “2′-deoxy-2′-fluoroadenosine”, as used herein, generally refers to a chemical having a structure as the following:

[0208]

[0209] The term “2′-modified guanosine”, as used herein, generally refers to a guanosine having a modification of the 2′ carbon atom. The guanosine is a purine nucleoside comprising guanine attached to a ribose (ribofuranose) ring via a β-N9-glycosidic bond. A guanosine may have a structure as the following:

[0210]

[0211] The term “2′-deoxy-2′-fluoroguanosine”, as used herein, generally refers to a chemical having a structure as the following:

[0212]

[0213] The term “nucleotide”, as used herein, generally refers to an organic molecule that serves as the monomer unit for forming the nucleic acid polymer deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The nucleotide is composed of three subunit molecules: a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. A nucleoside is composed of a nitrogenous base and a 5-carbon sugar. Thus, a nucleoside with a phosphate group yields a nucleotide.

[0214] The term “natural nucleotide”, as used herein, generally refers to a nucleotide occurring in nature. For example, the natural nucleotide may comprise cytidine, adenosine, guanosine, uridine. In some embodiments, “natural nucleic acid” also comprises synthesized or modified nucleotides not impeding amplification and/or sequencing.

[0215] The term “reference aptamer”, as used herein, generally refers to an aptamer which competitively binds to a target as the aptamer of the present disclosure. In some embodiments, the reference aptamer may bind to the same target as that of the aptamer of the present disclosure.

[0216] The term “competes”, as used herein, generally refers to one chemical substance inhibiting the effect of another by competing with it for binding or bonding. For example, a reference aptamer may competitively bind to the target with the aptamer of the present disclosure. As another example, an aptamer of the present disclosure may competitively bind to the target with a receptor or a ligand of the target.

[0217] The term “secondary structure”, as used herein, generally refers to a higher-ordered structure of a nucleic acid molecule. The secondary structure may be formed through base pairing interactions within a single nucleic acid polymer or between two polymers. It can be represented as a list of bases which are paired in a nucleic acid molecule. The secondary structure may be generally divided into helices (contiguous base pairs), and various kinds of loops (unpaired nucleotides surrounded by helices). Frequently, these elements, or combinations of them, can be further classified into additional categories comprising stems, bulges, loops, and helix. In the present disclosure, the secondary structure may be determined by using the NUPACK software suite.

[0218] The term “5′-3′”, as used herein, generally refers to the order from the 5′-end to the 3′-end of a DNA or an RNA strand. The 5′-end may refer to the end of the DNA or RNA strand that has the fifth carbon in the sugar-ring of the deoxyribose or ribose at its terminus. A phosphate group attached to the 5′-end permits ligation of two nucleotides, i.e., the covalent binding of a 5′-phosphate to the 3′-hydroxyl group of another nucleotide, to form a phosphodiester bond. The 3′-end may refer to a termination at the hydroxyl group of the third carbon in the sugar-ring, and is also known as the tail end. The 3′-hydroxyl is often required in the synthesis of new nucleic acid molecules as it is ligated (joined) to the 5′-phosphate of a separate nucleotide, allowing the formation of strands of linked nucleotides.

[0219] The term “stem”, as used herein, generally refers to a region of a nucleic acid secondary structure, and a stem comprises base-paired helix.

[0220] The term “bulge”, as used herein, generally refers to a region of a nucleic acid secondary structure in which one strand of a helix has one or more bases with no complementary counterpart in the opposite strand in the secondary structure of a nucleic acid molecule.

[0221] The term “loop”, as used herein, generally refers to a short series of consecutive unpaired bases in a longer paired helix in the secondary structure of the nucleic acid.

[0222] The term “consensus sequence”, as used herein, generally refers to certain positions, not necessarily contiguous, of an oligonucleotide that are specified. By “specified”, it is meant that the composition of the position may be other than completely random. Not all oligonucleotides in a mixture may have the same nucleotide at such position; for example, the consensus sequence may comprise a known ratio of particular nucleotides.

[0223] The term “truncate”, as used herein, generally refers to an aptamer that has been truncated by deletion of nucleotides but still possesses a desired or even improved binding characteristic. Truncates may vary in length in accordance with the length of the starting aptamer and as defined herein for the term “aptamer”. Truncations in the truncate may occur in fixed or variable regions, or both fixed and variable regions, of the starting aptamer.

[0224] The term “modulating the biological activity”, as used herein, generally refers to adjusting (e.g., increasing or decreasing) a biological activity of a target molecule (e.g., a protein, such as a receptor or a ligand). For example, the anti-VEGF aptamer of the present disclosure may modulate the biological activity of a VEGF and/or a VEGF receptor.

[0225] The term “neovascular disease or disorder”, as used herein, generally refers to a disease or a disorder related to neovascularization. A neovascular disease or disorder may comprise ocular neovascular disease (e.g., retinal and choroidal neovascular diseases), rubeotic glaucoma, pterygia, solid tumor cancers, osteoarthritis, rheumatoid arthritis, vascular anomalies and malformations (e.g., hemangiomas, lymphangiomas, and the like), and psoriasis.

[0226] The term “ocular neovascular disease”, as used herein, generally refers to a disease characterized by ocular neovascularization, i.e., the development of abnormal blood vessels in the eye of a subject.

[0227] The term “VEGF related disease, disorder or condition”, as used herein, generally refers to disease, disorder or condition associated with an abnormal activity or loss of activity of VEGF. For example, the VEGF related disease, disorder or condition may comprise aberrant angiogenesis. For example, the VEGF related disease, disorder or condition may comprise cancer, rheumatoid arthritis, the bullous diseases (including bullous pemphigoid, dermatitis herpetiformis, and erythema multiforme), and psoriasis.

[0228] The term “age-related macular degeneration”, or “AMD”, as used herein, generally refers to a macular degeneration which is related to aging. The macular degeneration may affect a central region of the retina known as the macula, and it may lead to a gradual or rapid loss of vision to the level of 20/200 or less. And the AMD may affect central vision, such as for reading and watching television, resulting lost while peripheral vision remains relatively intact.

[0229] The term “diabetic retinopathy”, or “DR”, as used herein, generally refers to a late microvascular complication of diabetes, for example, diabetes Type II. The diabetic retinopathy is a retinal microvascular disease that may be manifested as a cascade of stages with increasing levels of severity and worsening prognoses for vision. DR may be broadly classified into 2 major clinical stages: nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR), where the term “proliferative” refers to the presence of retinal neovascularization.

[0230] The term “diabetic macular edema”, as used herein, generally refers to a symptom attached at any stage of diabetic retinopathy causing a marked reduction of central visual acuity.

[0231] The term “retinal vein occlusion”, as used herein, generally refers to a fully or partially occluded retinal vessel that limits the flow of blood through the retinal tissue. The site of the occlusion typically occurs on the venous side and may occur in either the branch vessel (branch retinal vein occlusion-BRVO) or in the central retinal vein (central retinal vein occlusion-CRVO).

[0232] The term “myopic choroidal neovascularization”, as used herein, generally refers to disease causing visual impairment in persons with pathologic myopia. When vessels are newly generated in the macular area, pigmented fibrous scars, which result in scotoma in the center of vision, are often formed. The myopic choroidal neovascularization belongs to choroidal neovascularization.

[0233] The term “anti-VEGF agent”, as used herein, generally refers to a compound that inhibits the activity or production of vascular endothelial growth factor (“VEGF”).

[0234] The term “composition”, as used herein, generally refers to a distribution of individual substances that constitute a mixture. The individual substances can be named as components.

[0235] The term “pharmaceutical composition”, as used herein, generally refers to a formulation prepared in a form suitable for pharmaceutical administration. A pharmaceutical composition may be typically formulated to be compatible with its intended route of administration. Examples of routes of administration may include, but are not limited to, oral and parenteral, e.g., intravenous, intradermal, subcutaneous, inhalation, topical, transdermal, transmucosal, and rectal administration.

[0236] The term “pharmaceutically acceptable excipient or carrier”, as used herein, generally refers to any excipient or carrier approved by a regulatory agency of a federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and, more particularly, in humans. The term “carrier” may refer to a diluent, adjuvant, excipient, or vehicle with which therapeutic is administered and includes, but is not limited to, such sterile liquids as water and oils.

[0237] The term “effective amount”, as used herein, generally refers to an amount necessary to ameliorate at least one symptom of a disorder or condition to be prevented, reduced, or treated as described herein. The phrase “therapeutically effective amount” as it relates to an anti-VEGF aptamer, an anti-Ang2 aptamer, or a bispecific aptamer, a composition or an agent of the present disclosure means that the amount of the aptamer shall provide the desired specific pharmacological response after being administered in a significant number of individuals in need of such treatment. It is emphasized that a therapeutically effective amount of an aptamer that is administered to a particular individual in a particular instance will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art.

[0238] The term “Ang2”, as used herein, generally refers to Angiopoietin-2, which is an angiogenesis factor belonging to the E2F family. Ang2 is thought to be a naturally occurring antagonist for Tie2. Not only human Ang2, but also Ang2 from various species shall be considered to be covered by the term, unless explicitly specified otherwise. The term “Ang2” also encompasses functional fragments and components of the full length Ang2. Human Ang2 is also known as Ensembl: ENSG00000091879, or MIM:601922.

[0239] The term “an interaction between said Ang2 and Tie2”, as used herein, generally refers to an activity of an Ang2 binding to a Tie2. Tie2 is an endothelial receptor tyrosine kinase (RTK) that is involved in both embryonic vascular development and pathological angiogenesis.

[0240] The term “does not substantially bind”, as used herein, generally refers to little or almost no binding to a particular substance. For example, very few or almost no (e.g., less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01%) aptamer of the present disclosure may bind to the target thereof.

[0241] The term “anti-Ang2 aptamer”, as used herein, generally refers to a nucleic acid molecule having a specific binding affinity for Ang2. In some cases, the anti-Ang2 aptamer may comprise one or more (e.g., 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) non-nucleotide substitutions, as defined herein. For example, one or more nucleotides of the anti-Ang2 aptamer may be substituted with a polyethylene glycol moiety or a linker comprising multiple (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) polyethylene glycol moieties. In some cases, the linker may be a hexaethylene glycol (H or PEG6) linker. Such substitution may occur to any nucleotide of the anti-VEGF aptamer, as long as it does not substantially affect the activity (e.g., binding activity, or inhibition activity) and/or function of the anti-Ang2 aptamer. In some cases, the substitution does not substantially affect the secondary structure of the anti-Ang2 aptamer, for example, the substitution may occur to a nucleotide comprised in a bulge or a loop region of the anti-Ang2 aptamer secondary structure. In some cases, the substitution does not occur to any nucleotide comprised in a stem region of the anti-Ang2 aptamer secondary structure.

[0242] The term “anti-Ang2 agent”, as used herein, generally refers to an agent that inhibits the activity or production of Ang2.

[0243] The term “Aflibercept”, as used herein, generally refers to a recombinant fusion protein consisting of vascular endothelial growth factor (VEGF)-binding portions from the extracellular domains of human VEGF receptors 1 and 2, that are fused to the Fc portion of the human IgG1 immunoglobulin.

[0244] The term “linker”, as used herein, generally refers to an agent which is designed to facilitate the functional connection of two parts in to a linked one. For example, the linker may be used to make a single chain to achieve the desired biological activity. For example, the linker may be used to make a single chain to confer a degree of stability required for the resulted linked nucleotides.

[0245] The term “nucleotide linker”, as used herein, generally refers to a linker made of nucleotides. The nucleotide linker that connects adjacent nucleotides can vary in length.

[0246] The term “modified nucleotide linker”, as used herein, generally refers to a nucleotide linker with one or more modifications.

[0247] The term “hydrocarbon linker”, as used herein, generally refers to a linker made of hydrocarbon. The hydrocarbon is an organic compound consisting entirely of hydrogen and carbon. Examples of hydrocarbons include but are not limited to, e.g., methane, ethane, propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane.

[0248] The term “polyethylene glycol linker”, as used herein, generally refers to a linker made of one or more polyethylene glycol (PEG) subunits.

[0249] The term “a fragment thereof”, as used herein, generally refers to a partial region obtainable from an intact parent molecule (e.g., an aptamer of the present disclosure). The fragment may still retain the properties of the parent molecule that it is derived from. In some case, the fragments may offer a number of advantages over the intact parent molecule for manufacturing and/or medical use.

[0250] The term “variant”, as used herein, generally refers to an aptamer molecule that differs from a parent molecule (e.g., an aptamer) by at least one nucleotide acid. A variant may refer to the molecule itself, a composition comprising the molecule. It may also refer to the nucleotide acid sequence of the molecule, when such a molecule is a DNA or RNA. In some cases, a variant differs from its parent molecule (e.g., an aptamer) by an addition, deletion or substitution of one or more nucleotide acid, such as 1-50, 1-40, 1-30, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 nucleotide acid. In some cases, the variant may possess at least about 80% (e.g., at least 85%, 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%, at least 99% or higher) sequence homology/identity with the nucleotide acid sequence of its parent molecule.

[0251] The term “reducing neovascularization”, as used herein, generally refers to that the degree of neovascularization is reduced. Neovascularization is the natural formation of new blood vessels, usually in the form of functional microvascular networks, may be capable of perfusion by red blood cells, that form to serve as collateral circulation in response to local poor perfusion or ischemia. In the present disclosure, it may be interchangeably used with “angiogenesis”.

[0252] The term “reducing vascular permeability”, as used herein, generally refers to that the degree of vascular permeability in a tissue is reduced. Alteration of vascular permeability is thought to play a role in both normal and pathological physiological processes (Cullinan-Bove et al, 1993, Endocrinology 133:829-837; Senger et al, 1993, Cancer and Metastasis Reviews, 12:303-324). And disease states associated with angiogenesis and/or increased vascular permeability may be cancer, diabetes, psoriasis, rheumatoid arthritis, Kaposi's sarcoma, haemangioma, acute and chronic nephropathies, atheroma, arterial restenosis, autoimmune diseases, acute inflammation, excessive scar formation and adhesions, endometriosis, dysfunctional uterine bleeding and ocular diseases with retinal vessel proliferation.

[0253] The term “mouse oxygen-induced ischemic retinopathy (OIR) model”, as used herein, generally refers to an animal model developing oxygen-induced retinopathy in the mouse with reproducible and quantifiable proliferative retinal neovascularization. The model may be suitable for examining pathogenesis and therapeutic intervention for retinal neovascularization in retinopathy of prematurity (ROP) and other vascular pathologies. The preparation method of the model is described in, e.g., Smith L E, et al. Oxygen-induced retinopathy in the mouse. Invest. Opthalmol. Vis. Sci. 1994; 35:101-111.

[0254] The term “Ang2 related disease, disorder or condition”, as used herein, generally refers to a disease, disorder or condition that is associated with an abnormal activity or loss of activity of Ang2. For example, the Ang2 related disease, disorder or condition may have effects on angiogenesis. Examples for such disease, disorder or condition may be cancer or cancerous diseases, eye diseases such as age-related macular degeneration and diabetic retinopathy, and/or chronic kidney diseases such as diabetic nephropathy, postrenal failure, prerenal azotemia and intrinsic renal failure.

[0255] As used herein, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a particle” includes a plurality of such particles and reference to “the sequence” includes reference to one or more said sequences and equivalents thereof known to those skilled in the art, and so forth.

[0256] The term “about”, as used herein, generally refers to an approximation to a given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. For example, it may refer to a value that is no more than 10% above or below the value being modified by the term.

[0257] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0258] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. This is intended to provide support for all such combinations.

Anti-VEGF Aptamer

[0259] In one aspect, the present disclosure provides an anti-VEGF aptamer, which binds to VEGF-121 with a K_D value of about 20 nanomolar (nM) or less, and binds to VEGF-165 with a K_D value of about 20 nanomolar (nM) or less.

[0260] For example, the anti-VEGF aptamer may bind to VEGF-121 with a K_D value of about 20 nanomolar (nM) or less, 18 nanomolar (nM) or less, 16 nanomolar (nM) or less, 14 nanomolar (nM) or less, 12 nanomolar (nM) or less, 10 nanomolar (nM) or less, 8 nanomolar (nM) or less, 6 nanomolar (nM) or less, 4 nanomolar (nM) or less, 2 nanomolar (nM) or less, 1 nanomolar (nM) or less, 0.5 nanomolar (nM) or less, 0.4 nanomolar (nM) or less, 0.2 nanomolar (nM) or less, 0.1 nanomolar (nM) or less, 0.09 nanomolar (nM) or less, 0.08 nanomolar (nM) or less, 0.07 nanomolar (nM) or less, 0.06 nanomolar (nM) or less, 0.05 nanomolar (nM) or less, 0.04 nanomolar (nM) or less, or 0.03 nanomolar (nM) or less.

[0261] For example, the anti-VEGF aptamer may bind to VEGF-165 with a K_D value of about 20 nanomolar (nM) or less, 18 nanomolar (nM) or less, 16 nanomolar (nM) or less, 14 nanomolar (nM) or less, 12 nanomolar (nM) or less, 10 nanomolar (nM) or less, 8 nanomolar (nM) or less, 6 nanomolar (nM) or less, 4 nanomolar (nM) or less, 2 nanomolar (nM) or less, 1 nanomolar (nM) or less, 0.5 nanomolar (nM) or less, 0.4 nanomolar (nM) or less, 0.2 nanomolar (nM) or less, 0.18 nanomolar (nM) or less, 0.15 nanomolar (nM) or less, 0.12 nanomolar (nM) or less, 0.1 nanomolar (nM) or less, 0.09 nanomolar (nM) or less, 0.08 nanomolar (nM) or less, 0.07 nanomolar (nM) or less, 0.06 nanomolar (nM) or less, 0.05 nanomolar (nM) or less, 0.04 nanomolar (nM) or less, or 0.03 nanomolar (nM) or less.

[0262] The VEGF-121 may be a human VEGF-121, a mouse VEGF-120, a monkey VEGF-121, a rabbit VEGF-121, and/or a rat VEGF-120. The VEGF-165 may be a human VEGF-165, a mouse VEGF-164, a monkey VEGF-165, a rabbit VEGF-165, and/or a rat VEGF-164.

[0263] The human VEGF-121 (NCBI No. P15692-9) may comprise an amino acid sequence as set forth in SEQ ID NO: 117. The mouse VEGF-120 (NCBI No. Q00731-3) may comprise an amino acid sequence as set forth in SEQ ID NO: 132. The rat VEGF-120 (NCBI No. P16612-4) may comprise an amino acid sequence as set forth in SEQ ID NO: 130.

[0264] The human VEGF-165 (NCBI No. P15692-4) may comprise an amino acid sequence as set forth in SEQ ID NO: 123. The mouse VEGF-164 (NCBI No. Q00731-2) may comprise an amino acid sequence as set forth in SEQ ID NO: 131. The monkey VEGF-165 (NCBI No. H9EQI6) may comprise an amino acid sequence as set forth in SEQ ID NO: 123. The rat VEGF-164 (NCBI No. P16612-2) may comprise an amino acid sequence as set forth in SEQ ID NO: 127.

[0265] The rabbit VEGF-189 (NCBI No. G1T1L9) may comprise an amino acid sequence as set forth in SEQ ID NO: 124.

[0266] The anti-VEGF aptamer may specifically bind to both 1) a VEGF receptor-binding domain of VEGF-121 or a fragment thereof; and 2) a VEGF receptor-binding domain of VEGF-165 or a fragment thereof.

[0267] The VEGF receptor-binding domain of VEGF-121 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 118-122, 128, 129, and 133. The VEGF receptor-binding domain of VEGF-165 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 118-122, 128, and 129.

[0268] The receptor binding domain 1 of human VEGF-121 may comprise an amino acid sequence as set forth in SEQ ID NO: 118. The receptor binding domain 2 of human VEGF-121 may comprise an amino acid sequence as set forth in SEQ ID NO: 119. The receptor binding domain 3 of human VEGF-121 may comprise an amino acid sequence as set forth in SEQ ID NO: 120. The receptor binding domain 4 of human VEGF-121 may comprise an amino acid sequence as set forth in SEQ ID NO: 121. The receptor binding domain 5 of human VEGF-121 may comprise an amino acid sequence as set forth in SEQ ID NO: 122.

[0269] The receptor binding domain 1 of human VEGF-165 may comprise an amino acid sequence as set forth in SEQ ID NO: 118. The receptor binding domain 2 of human VEGF-165 may comprise an amino acid sequence as set forth in SEQ ID NO: 119. The receptor binding domain 3 of human VEGF-165 may comprise an amino acid sequence as set forth in SEQ ID NO: 120. The receptor binding domain 4 of human VEGF-165 may comprise an amino acid sequence as set forth in SEQ ID NO: 121. The receptor binding domain 5 of human VEGF-165 may comprise an amino acid sequence as set forth in SEQ ID NO: 122.

[0270] The receptor binding domain 1 of Monkey VEGF-165 may comprise an amino acid sequence as set forth in SEQ ID NO: 118. The receptor binding domain 2 of Monkey VEGF-165 may comprise an amino acid sequence as set forth in SEQ ID NO: 119. The receptor binding domain 3 of Monkey VEGF-165 may comprise an amino acid sequence as set forth in SEQ ID NO: 120. The receptor binding domain 4 of Monkey VEGF-165 may comprise an amino acid sequence as set forth in SEQ ID NO: 121. The receptor binding domain 5 of Monkey VEGF-165 may comprise an amino acid sequence as set forth in SEQ ID NO: 122.

[0271] The receptor binding domain 1 of rabbit VEGF-189 may comprise an amino acid sequence as set forth in SEQ ID NO: 125. The receptor binding domain 2 of rabbit VEGF-189 may comprise an amino acid sequence as set forth in SEQ ID NO: 119. The receptor binding domain 3 of rabbit VEGF-189 may comprise an amino acid sequence as set forth in SEQ ID NO: 126. The receptor binding domain 4 of rabbit VEGF-189 may comprise an amino acid sequence as set forth in SEQ ID NO: 121. The receptor binding domain 5 of rabbit VEGF-189 may comprise an amino acid sequence as set forth in SEQ ID NO:122.

[0272] The receptor binding domain 1 of rat VEGF-164 may comprise an amino acid sequence as set forth in SEQ ID NO: 118. The receptor binding domain 2 of rat VEGF-164 may comprise an amino acid sequence as set forth in SEQ ID NO: 119. The receptor binding domain 3 of rat VEGF-164 may comprise an amino acid sequence as set forth in SEQ ID NO: 128. The receptor binding domain 4 of rat VEGF-164 may comprise an amino acid sequence as set forth in SEQ ID NO: 121. The receptor binding domain 5 of rat VEGF-164 may comprise an amino acid sequence as set forth in SEQ ID NO: 129.

[0273] The receptor binding domain 1 of rat VEGF-120 may comprise an amino acid sequence as set forth in SEQ ID NO: 118. The receptor binding domain 2 of rat VEGF-120 may comprise an amino acid sequence as set forth in SEQ ID NO:119. The receptor binding domain 3 of rat VEGF-120 may comprise an amino acid sequence as set forth in SEQ ID NO: 128. The receptor binding domain 4 of rat VEGF-120 may comprise an amino acid sequence as set forth in SEQ ID NO:121. The receptor binding domain 5 of rat VEGF-120 may comprise an amino acid sequence as set forth in SEQ ID NO: 129.

[0274] The receptor binding domain 1 of mouse VEGF-164 may comprise an amino acid sequence as set forth in SEQ ID NO: 118. The receptor binding domain 2 of mouse VEGF-164 may comprise an amino acid sequence as set forth in SEQ ID NO:119. The receptor binding domain 3 of mouse VEGF-164 may comprise an amino acid sequence as set forth in SEQ ID NO: 128. The receptor binding domain 4 of mouse VEGF-164 may comprise an amino acid sequence as set forth in SEQ ID NO: 121x. The receptor binding domain 5 of mouse VEGF-164 may comprise an amino acid sequence as set forth in SEQ ID NO: 129.

[0275] The receptor binding domain 1 of mouse VEGF-120 may comprise an amino acid sequence as set forth in SEQ ID NO: 118. The receptor binding domain 2 of mouse VEGF-120 may comprise an amino acid sequence as set forth in SEQ ID NO: 119. The receptor binding domain 3 of mouse VEGF-120 may comprise an amino acid sequence as set forth in SEQ ID NO: 128. The receptor binding domain 4 of mouse VEGF-120 may comprise an amino acid sequence as set forth in SEQ ID NO: 133. The receptor binding domain 5 of mouse VEGF-120 may comprise an amino acid sequence as set forth in SEQ ID NO: 129.

[0276] The anti-VEGF aptamer may inhibit an interaction between the VEGF-121 and VEGF-R1. The anti-VEGF aptamer may inhibit an interaction between the VEGF-121 and VEGF-R2. The anti-VEGF aptamer may inhibit an interaction between the VEGF-165 and VEGF-R1. The anti-VEGF aptamer may also or in addition inhibit an interaction between the VEGF-165 and VEGF-R2.

[0277] The anti-VEGF aptamer of the present disclosure may be capable of reducing and/or ameliorating neovascularization and/or leakage in an animal model, such as an ocular animal model.

[0278] The animal model may be a vertebrate animal model, in some cases, the animal model may be a mammal model. The animal may be a rat, a mouse, a rabbit, a goat, a sheep, a non-human primate, or any other suitable animal.

[0279] In some embodiments, the anti-VEGF aptamer of the present disclosure may be capable of reducing and/or ameliorating a lesion and/or a leakage in a laser-induced choroidal neovascularization (CNV) model (such as a rat model or a non-human primate (NHP) model).

[0280] The anti-VEGF aptamer of the present disclosure may be highly soluble. For example, the anti-VEGF aptamer may be dissolved in the solvent with a relatively high degree (e.g. 80% of the whole weight or more, 85% of the whole weight or more, 90% of the whole weight or more, 95% of the whole weight or more, 99% of the whole weight or more). For example, the anti-VEGF aptamer may be formulated at a concentration of 100 mg/ml or more, or at a concentration of 150 mg/ml or more, such as at a concentration of 200 mg/ml or more.

[0281] The anti-VEGF aptamer may be an RNA aptamer, a DNA aptamer, or a combination thereof. For example, the anti-VEGF aptamer may comprise primarily RNA, it may comprise primarily DNA, or it may comprise both RNA and DNA.

[0282] The anti-VEGF aptamer may comprise one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) modified nucleotide. In some cases, all of the nucleotides of the anti-VEGF aptamer may be modified nucleotides. For instance, the anti-VEGF aptamer may have 20 nucleotides, and all of these 20 nucleotides may be modified nucleotide.

[0283] In some cases, the anti-VEGF aptamer of the present disclosure may be nuclease resistant.

[0284] The modified nucleotide may comprise a chemical substitution or modification at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position. The modified nucleotide may comprise one or more modifications independently selected from a 2′-position sugar modification, a 2′-amino (2′-NH₂) modification, a 2′-fluoro (2′-F) modification, a 2′-O-methyl(2′-OMe) modification, a 2′-O-(2-Methoxyethyl) (2′-O-MOE) modification, a 5-position modified pyrimidine, a modification at a cytosine exocyclic amine, a substitution with 5′-bromouracil, a substitution with 5′-bromodeoxyuridine, a substitution with 5′-bromodeoxycytidine, a backbone modification, a locked nucleic acid (LNA), a methylation, a 3′ cap, and a 5′ cap. The 5-position modified pyrimidine is selected from 5-carboxy-2′-deoxyuridine, 5-aminoallyl-2′-deoxyuridine, 5-[(3-indolyl) propionamide-N-allyl]-2′-deoxyuridine, 5-carboxy-2′-deoxycytidine, 5-aminoallyl-2′-deoxycytidine, biotin-16-aminoallyl-2′-deoxycytidine, 5-(N-benzylcarboxyamide)-2′-deoxyuridine, 5-(N-isobutylcarboxyamide)-2′-deoxyuridine, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine, and 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine.

[0285] The anti-VEGF aptamer may comprise at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) 2′-modified nucleotide. In some embodiments, all the nucleotides may be 2′-modified nucleotides. The 2′-modified nucleotide may be selected from a 2′-amino modified nucleotide, a 2′-fluoro-modified nucleotide, a 2′-O-methyl-modified nucleotide, and a 2′-O-(2-methoxyethyl)-modified nucleotide.

[0286] In some cases, all the cytidines may be 2′-modified cytidines. In some cases, all the cytidines may be 2′-fluoro-modified cytidines and/or 2′-O-methyl-modified cytidines. In some cases, all the cytidines may be 2′-deoxy-2′-fluorocytidines. In some cases, all the uridines may be 2′-modified uridines. In some cases, all the uridines may be 2′-fluoro-modified uridines and/or 2′-O-methyl-modified uridines. In some cases, all the uridines may be 2′-deoxy-2′-fluorouridines. In some cases, all the adenosines may be 2′-modified adenosines. In some cases, all the adenosines may be 2′-fluoro-modified adenosines and/or 2′-O-methyl-modified adenosines. In some cases, all the adenosines may be 2′-deoxy-2′-fluoroadenosines. In some cases, all the guanosines may be 2′-modified guanosines. In some cases, all the guanosines may be 2′-fluoro-modified guanosines and/or 2′-O-methyl-modified guanosines. In some cases, all the guanosines may be 2′-deoxy-2′-fluoroguanosines.

[0287] For example, the anti-VEGF aptamer comprising the at least one 2′-modified nucleotide. In some embodiments, all the nucleotides of the anti-VEGF aptamer may be 2′-modified nucleotides. In some embodiments, all the nucleotides of the anti-VEGF aptamer may be 2′-fluoro-modified nucleotides and/or 2′-O-methyl-modified nucleotides. For example, the 2′-fluoro-modified nucleotides may be 2′-fluoro (2′-F)-modified nucleotides or 2′-fluoro-2′-deoxy-modified nucleotides. For example, the 2′-fluoro-modified nucleotides may comprise the following structure:

[0288]

[0289] In some cases, the anti-VEGF aptamer does not comprise any natural nucleotide (e.g., unmodified A, C, T, U or G).

[0290] The anti-VEGF aptamer may comprise about 10 to about 150 nucleotides. For example, the anti-VEGF aptamer may comprise about 10 to about 150 nucleotides, about 10 to about 140 nucleotides, about 10 to about 130 nucleotides, about 10 to about 120 nucleotides, about 10 to about 110 nucleotides, about 10 to about 100 nucleotides, about 10 to about 90 nucleotides, about 10 to about 80 nucleotides, about 10 to about 70 nucleotides, about 10 to about 60 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides.

[0291] The anti-VEGF aptamer may compete with an anti-VEGF reference aptamer for binding to the VEGF-165 and/or to the VEGF-121.

[0292] The anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a nucleotide sequence with or without modifications. For example, the aptamer may comprise natural and/or modified nucleotides. When an aptamer is defined as comprising or consisting of a nucleotide sequence as set forth in a particular SEQ ID NO, the nucleotides within the sequence may be further modified.

[0293] According to the present disclosure, the anti-VEGF aptamer r-AMS0401 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 1; the anti-VEGF aptamer AMS0401 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 31. Thus, an aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 1 may encompass both r-AMS0401 and AMS0401, but an aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 31 may not encompass r-AMS0401.

[0294] Similarly, the anti-VEGF aptamer r-AMS0402 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 2; and the anti-VEGF aptamer AMS0402 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 32. The anti-VEGF aptamer r-AMS0403 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 3; and the anti-VEGF aptamer AMS0403 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 33. The anti-VEGF aptamer r-AMS0404 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 4; and the anti-VEGF aptamer AMS0404 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 34. The anti-VEGF aptamer r-AMS0405 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 5; and the anti-VEGF aptamer AMS0405 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 35. The anti-VEGF aptamer r-AMS0406 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 6; and the anti-VEGF aptamer AMS0406 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 36. The anti-VEGF aptamer r-AMS0407 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 7; and the anti-VEGF aptamer AMS0407 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 37. The anti-VEGF aptamer r-AMS0408 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 8; and the anti-VEGF aptamer AMS0408 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 38. The anti-VEGF aptamer r-AMS0409 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 9; and the anti-VEGF aptamer AMS0409 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 39. The anti-VEGF aptamer r-AMS0410 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 10; and the anti-VEGF aptamer AMS0410 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 40. The anti-VEGF aptamer r-AMS0411 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 11; and the anti-VEGF aptamer AMS0411 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 41. The anti-VEGF aptamer r-AMS0416 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 12; and the anti-VEGF aptamer AMS0416 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 42. The anti-VEGF aptamer r-AMS0419 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 13; and the anti-VEGF aptamer AMS0419 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 43. The anti-VEGF aptamer r-AMS0420 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 14; and the anti-VEGF aptamer AMS0420 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 44. The anti-VEGF aptamer r-AMS0421 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 15; and the anti-VEGF aptamer AMS0421 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 45. The anti-VEGF aptamer r-AMS0425 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 16; and the anti-VEGF aptamer AMS0425 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 46. The anti-VEGF aptamer r-AMS0421.1 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 17; and the anti-VEGF aptamer AMS0421.1 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 47. The anti-VEGF aptamer r-AMS0427 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 18; and the anti-VEGF aptamer AMS0427 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 48. Further, the anti-VEGF aptamer r-AMS0427 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 18; and the anti-VEGF aptamers AMS0427.1 and AMS0427.2 have the corresponding nucleotides with various modifications, and their nucleotide sequences are as set forth in SEQ ID NO: 49 and SEQ ID NO: 50, respectively. The anti-VEGF aptamer r-AMS0421.3 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 19; and the anti-VEGF aptamer AMS0421.3 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 51. The anti-VEGF aptamer r-AMS0421.4 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 20; and the anti-VEGF aptamer AMS0421.4 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 52. The anti-VEGF aptamer r-AMS0421.5 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 21; and the anti-VEGF aptamer AMS0421.5 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 53. The anti-VEGF aptamer r-AMS0425.1 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 22; and the anti-VEGF aptamer AMS0425.1 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 54. The anti-VEGF aptamer r-AMS0426 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 23; and the anti-VEGF aptamer AMS0426 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 55. The anti-VEGF aptamer r-AMS0425.3 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 24; and the anti-VEGF aptamer AMS0425.3 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 56. The anti-VEGF aptamer r-AMS0425.8 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 25; and the anti-VEGF aptamer AMS0425.8 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 57. The anti-VEGF aptamer r-AMS0425.10 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 26; and the anti-VEGF aptamer AMS0425.10 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 58. The anti-VEGF aptamer r-AMS0425.11 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 27; and the anti-VEGF aptamer AMS0425.11 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 59. The anti-VEGF aptamer r-AMS0425.12 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 28; and the anti-VEGF aptamer AMS0425.12 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 60. The anti-VEGF aptamer r-AMS0425.16 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 29; and the anti-VEGF aptamer AMS0425.16 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 62. The anti-VEGF aptamer r-AMS0430 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 30; and the anti-VEGF aptamer AMS0430 has the corresponding 2′-fluoro-modified nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 61.

[0295] The anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop. The anti-VEGF aptamer may comprise a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0296] The anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 79-90.

[0297] Alternatively, or in addition, the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 91-100.

[0298] For example, the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 79, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 91; the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 80, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 92; the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 81, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 93; the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 82, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 93; the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 83, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 94; the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 84, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 95; the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 86, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 97; the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 87, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 98; the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 88, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 98; the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 89, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 99; the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 90, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 100.

[0299] The first consensus sequence may be comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure. In some cases, the first consensus sequence may not be comprised in the nucleotide sequence forming the first loop of the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure. For example, the first consensus sequence may be present anywhere within the aptamer, except in the region forming the first loop.

[0300] For example, in some cases, the first consensus sequence may be comprised in the second stem, the second bulge and the third stem. In some cases, the first consensus sequence may be comprised in the second stem, the second bulge, the third stem, and the third bulge. In some cases, the first consensus sequence may be comprised in the second stem, the second bulge, the third stem, the third bulge, and the fourth stem.

[0301] The second consensus sequence may be comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure. In some cases, the second consensus sequence may not be comprised in the nucleotide sequence forming the first loop of the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure. For example, the second consensus sequence may be present anywhere within the aptamer, except in the region forming the first loop.

[0302] In some cases, the second consensus sequence may be comprised in the fourth stem, the third bulge, the third stem, the second bulge, and the second stem. In some cases, the second consensus sequence may be comprised in the third bulge, the third stem, the second bulge, and the second stem. In some cases, the second consensus sequence may be comprised in the third stem, the second bulge, and the second stem.

[0303] The first consensus sequence and the second consensus sequence may be paired to make sure that the secondary structure of the aptamer is properly formed. For example, when a first consensus sequence is chosen, those skilled in the art may be able to adjust the nucleotide sequence of the second consensus sequence, as long as the secondary structure of the aptamer can be properly formed.

[0304] The anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise the first consensus sequence and the second consensus sequence, wherein the first consensus sequence may be located 5′ to the second consensus sequence.

[0305] In some cases, the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise the first consensus sequence and the second consensus sequence, wherein the second consensus sequence may be located 5′ to the first consensus sequence.

[0306] The first loop of the anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprises 2-30 nucleotides (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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, or at least 30 nucleotides).

[0307] The anti-VEGF reference aptamer or the anti-VEGF aptamer of the present disclosure may comprise a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, or a variant or truncate thereof. The variant may comprise a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 1-62. The variant may comprise a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, with one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, or more) nucleotide addition, deletion, and/or substitution.

[0308] The anti-VEGF reference aptamer may comprise about 10 to about 150 nucleotides. For example, the anti-VEGF reference aptamer may comprise about 10 to about 150 nucleotides, about 10 to about 140 nucleotides, about 10 to about 130 nucleotides, about 10 to about 120 nucleotides, about 10 to about 110 nucleotides, about 10 to about 100 nucleotides, about 10 to about 90 nucleotides, about 10 to about 80 nucleotides, about 10 to about 70 nucleotides, about 10 to about 60 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, or about 10 to about 30 nucleotides, about 10 to about 20 nucleotides.

[0309] The anti-VEGF aptamer may be used for modulating the biological activity of a VEGF or a VEGF receptor.

[0310] The anti-VEGF aptamer may be used for preventing, treating, and/or ameliorating a VEGF related disease, disorder or condition. For example, the VEGF related disease, disorder or condition may be a neovascular disease, disorder or condition. For example, the VEGF related disease, disorder or condition may be an ocular neovascular disease, disorder or condition. For example, the VEGF related disease, disorder or condition may be selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. For example, the VEGF related disease, disorder or condition may be selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0311] In one aspect, the present disclosure provides an anti-VEGF agent, comprising the aptamer of the present disclosure. The anti-VEGF agent may further comprise a polyethylene glycol (PEG) moiety. For example, the PEG moiety may be conjugated to the 5′ terminus and/or the 3′ terminus of the anti-VEGF aptamer.

[0312] In another aspect, the present disclosure provides a composition comprising the anti-VEGF aptamer, and/or the anti-VEGF agent of the present application.

[0313] In some cases, the composition may be a pharmaceutical composition. For example, the composition may comprise a pharmaceutically acceptable excipient or carrier.

[0314] In some cases, the composition may be presented as discrete dosage forms, with each dosage containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid. Such dosage forms can be prepared by any of the methods known to a skilled person, for example, it may include the step of bringing the active ingredient into association with the carrier, which constitutes one or more other ingredients. In general, the compositions are prepared by uniformly and intimately mixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

[0315] The composition may comprise a therapeutically effective amount of the active composition (e.g., the anti-VEGF aptamer or the anti-VEGF agent of the present disclosure). A therapeutically effective amount may be an amount of the subject composition capable of preventing and/or curing (at least partially) a neovascular disease or disorder and/or any complications thereof in a subject suffering from or having a risk of developing the condition or disorder. The specific amount/concentration of the active agent comprised may vary according to the method of administration and the need of a patient, and can be determined based on e.g., volume, viscosity, and/or body weight of a patient etc. For example, an appropriate dosage may be about 0.1 mg or 1 mg/kg/day to about 50 mg/kg/day; sometimes, the dosage can be even higher. It shall be understood that these specific doses may be conveniently adjusted by a skilled person in the art (e.g., a doctor or a pharmacist) based on conditions of a specific patient, formulation, and/or disease.

[0316] The composition may further comprise an effective amount of an additional therapeutically active component, for example, an additional therapeutically active component for preventing, treating and/or ameliorating a neovascular disease or disorder. Each of the active components may be present in the pharmaceutical composition in a pharmaceutically effective amount. The anti-VEGF aptamer of the present application may or may not be associated with the additional active component.

[0317] Described below are non-limiting exemplary compositions and methods for preparing such a composition. The composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The composition may be in unit dosage forms suitable for single administration of precise dosages. In some embodiments, the composition may be a liquid pharmaceutical composition.

[0318] In the composition, the anti-VEGF aptamer may be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration.

[0319] The composition may further include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, without limitation, detackifiers, anti-foaming compositions, buffering compositions, polymers, antioxidants, preservatives, chelating compositions, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending compositions, binders, fillers, plasticizers, lubricants, and/or mixtures thereof.

[0320] In another aspect, the present disclosure provides a method for modulating the biological activity of a VEGF or a VEGF receptor, comprising administering to a subject in need thereof an effective amount of the anti-VEGF aptamer or the anti-VEGF agent of the present disclosure.

[0321] In another aspect, the present disclosure provides a method for preventing, treating, and/or ameliorating a VEGF related disease, disorder or condition, comprising administering to a subject in need thereof an effective amount of the anti-VEGF aptamer, or the anti-VEGF agent of the present disclosure.

[0322] For example, the VEGF related disease, disorder or condition may be a neovascular disease, disorder or condition. For example, the VEGF related disease, disorder or condition may be an ocular neovascular disease, disorder or condition. For example, the VEGF related disease, disorder or condition may be selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. For example, the VEGF related disease, disorder or condition may be selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0323] In one aspect, the present disclosure provides a use of the anti-VEGF aptamer, or the anti-VEGF agent of the present disclosure in the manufacture of an agent for modulating the biological activity of a VEGF or a VEGF receptor.

[0324] In one aspect, the present disclosure provides a use of the aptamer of the present disclosure, or the anti-VEGF agent of the present disclosure, in the manufacture of a medicament for preventing, treating, and/or ameliorating a VEGF related disease, disorder or condition.

[0325] The VEGF related disease, disorder or condition may be a neovascular disease, disorder or condition. For example, the VEGF related disease, disorder or condition may be an ocular neovascular disease, disorder or condition. For example, the VEGF related disease, disorder or condition may be selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. For example, the VEGF related disease, disorder or condition may be selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

Anti-Ang2 Aptamer

[0326] In one aspect, the present disclosure provides an aptamer that specifically binds to Ang2 with a K_D value of about 500 picomolar (pM) or less. For example, the anti-Ang2 aptamer may bind to Ang2 with a K_D value of about 500 picomolar (pM) or less, about 400 picomolar (pM) or less, about 300 picomolar (pM) or less, about 200 picomolar (pM) or less, about 100 picomolar (pM) or less, about 90 picomolar (pM) or less, about 80 picomolar (pM) or less, about 70 picomolar (pM) or less, about 60 picomolar (pM) or less, about 50 picomolar (pM) or less, about 40 picomolar (pM) or less, about 30 picomolar (pM) or less, or about 20 picomolar (pM) or less.

[0327] The Ang2 may be a human Ang2, a mouse Ang2, a monkey Ang2, a rabbit Ang2, and/or a rat Ang2.

[0328] The anti-Ang2 aptamer may not substantially bind to human Ang1. For example, the anti-Ang2 aptamer may bind to Ang1 with an affinity that is significantly lower (e.g. at least 1-fold lower, at least 2 folds lower, at least 3 folds lower, at least 4 or more folds lower) than its affinity for Ang2. In some cases, the anti-Ang2 aptamer may bind to Ang1 with a K_D value that is not measurable using Ligand Binding Assays with radioactive or fluorescent measurement, Surface Plasmon Resonance (SPR), Bio-Layer Interferometry (BLI, e.g., the Octet® Systems), SRU biosystems BIND®, Isothermal Titration calorimetry (ITC), or MicroscaleThermophoresis (MST).

[0329] The anti-Ang2 aptamer may inhibit an interaction between the Ang2 and Tie2.

[0330] The anti-Ang2 aptamer of the present disclosure may be capable of reducing and/or ameliorating neovascularization and/or vascular permeability in an animal model, such as in an ocular animal model. For example, the ocular animal model may be an oxygen-induced ischemic retinopathy (OIR) model. The animal model may be a vertebrate animal model, in some cases, the animal model may be a mammal model. The animal may be a rat, a mouse, a rabbit, a goat, a sheep, a non-human primate, or any other suitable animal.

[0331] The anti-Ang2 aptamer of the present disclosure may be capable of reducing neovascularization and/or reducing vascular permeability in a mouse oxygen-induced ischemic retinopathy (OIR) model.

[0332] The anti-Ang2 aptamer of the present disclosure may be highly soluble. For example, the anti-Ang2 aptamer may be soluble in the solvent with a relatively high degree (e.g. 80% of the whole weight or more, 85% of the whole weight or more, 90% of the whole weight or more, 95% of the whole weight or more, 99% of the whole weight or more). For example, the anti-Ang2 aptamer may be formulated at a concentration of 100 mg/ml or more, at a concentration of 150 mg/ml or more, such as at least 200 mg/ml.

[0333] The anti-Ang2 aptamer may be an RNA aptamer, a DNA aptamer, or a combination thereof. For example, the anti-VEGF aptamer may comprise primarily RNA, it may comprise primarily DNA, or it may comprise both RNA and DNA.

[0334] The anti-Ang2 aptamer may comprise one or more modified nucleotide. In some cases, all the nucleotides of the anti-Ang2 aptamer may be modified nucleotides.

[0335] For example, the modified nucleotide may comprise a chemical substitution or modification at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position. The modified nucleotide may comprise one or more modifications independently selected from a 2′-position sugar modification, a 2′-amino (2′-NH₂) modification, a 2′-fluoro (2′-F) modification, a 2′-O-methyl(2′-OMe) modification, a 2′-O-(2-Methoxyethyl) (2′-O-MOE) modification, a 5-position modified pyrimidine, a modification at a cytosine exocyclic amine, a substitution with 5′-bromouracil, a substitution with 5′-bromodeoxyuridine, a substitution with 5′-bromodeoxycytidine, a backbone modification, a locked nucleic acid (LNA), a methylation, a 3′ cap, and a 5′ cap. The 5-position modified pyrimidine may be selected from 5-carboxy-2′-deoxyuridine, 5-aminoallyl-2′-deoxyuridine, 5-[(3-indolyl) propionamide-N-allyl]-2′-deoxyuridine, 5-carboxy-2′-deoxycytidine, 5-aminoallyl-2′-deoxycytidine, biotin-16-aminoallyl-2′-deoxycytidine, 5-(N-benzylcarboxyamide)-2′-deoxyuridine, 5-(N-isobutylcarboxyamide)-2′-deoxyuridine, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine, and 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine.

[0336] The anti-Ang2 aptamer may comprise at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) 2′-modified nucleotide. In some cases, all the nucleotides of the anti-Ang2 aptamer may be 2′-modified nucleotides. The 2′-modified nucleotide may be selected from a 2′-amino modified nucleotide, a 2′-fluoro-modified nucleotide, a 2′-O-methyl-modified nucleotide, and a 2′-O-(2-methoxyethyl)-modified nucleotide. For example, the 2′-fluoro-modified nucleotides may be 2′-fluoro (2′-F)-modified nucleotides or 2′-fluoro-2′-deoxy-modified nucleotides. For example, the 2′-fluoro-modified nucleotides may comprise the following structure:

[0337]

[0338] The anti-Ang2 aptamer may comprise at least one 2′-fluoro-modified nucleotide, at least one 2′-O-methyl-modified nucleotide, and/or at least one LNA.

[0339] In some cases, all the cytidines of the anti-Ang2 aptamer may be 2′-modified cytidines. In some cases, all the cytidines may be 2′-fluoro-modified cytidines and/or 2′-O-methyl-modified cytidines. In some cases, all the cytidines may be 2′-deoxy-2′-fluorocytidines. In some cases, all the uridines may be 2′-modified uridines. In some cases, all the uridines may be 2′-fluoro-modified uridines and/or 2′-O-methyl-modified uridines. In some cases, all the uridines may be 2′-deoxy-2′-fluorouridines. In some cases, all the adenosines may be 2′-modified adenosines. In some cases, all the adenosines may be 2′-fluoro-modified adenosines and/or 2′-O-methyl-modified adenosines. In some cases, all the adenosines may be 2′-deoxy-2′-fluoroadenosines. In some cases, all the guanosines may be 2′-modified guanosines. In some cases, all the guanosines may be 2′-fluoro-modified guanosines and/or 2′-O-methyl-modified guanosines. In some cases, all the guanosines may be 2′-deoxy-2′-fluoroguanosines. In some cases, all the nucleotides may be 2′-fluoro-modified nucleotides and/or 2′-O-methyl-modified nucleotides. In some cases, all the nucleotides may be 2′-fluoro-modified nucleotides.

[0340] In some cases, the anti-Ang2 aptamer does not comprise any natural nucleotide (e.g., unmodified A, C, T, U or G).

[0341] The anti-Ang2 aptamer may comprise about 10 to about 150 nucleotides. For example, the anti-Ang2 aptamer may comprise about 10 to about 150 nucleotides, about 10 to about 140 nucleotides, about 10 to about 130 nucleotides, about 10 to about 120 nucleotides, about 10 to about 110 nucleotides, about 10 to about 100 nucleotides, about 10 to about 90 nucleotides, about 10 to about 80 nucleotides, about 10 to about 70 nucleotides, about 10 to about 60 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, or about 10 to about 20 nucleotides.

[0342] The anti-Ang2 aptamer may compete with an anti-Ang2 reference aptamer for binding to the Ang2.

[0343] The anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a nucleotide sequence with or without modifications. For example, the aptamer may comprise natural and/or modified nucleotides. When an aptamer is defined as comprising or consisting of a nucleotide sequence as set forth in a particular SEQ ID NO, the nucleotides within the sequence may be further modified.

[0344] According to the present disclosure, the anti-Ang2 aptamer r-AMS0526 has natural nucleotides, and the nucleotide sequence is as set forth in SEQ ID NO: 64; and the anti-Ang2 aptamer AMS0526 has the corresponding 2′-fluoro-modified nucleotides, and the nucleotide sequence is as set forth in SEQ ID NO: 68. Thus, an aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64 may encompass both r-AMS0526 and AMS0526, but an aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68 may not encompass r-AMS0526.

[0345] Similarly, the anti-Ang2 aptamer r-AMS0525 has natural nucleotides, and the nucleotide sequence is as set forth in SEQ ID NO: 63; and the anti-Ang2 aptamer AMS0525 has the corresponding 2′-fluoro-modified nucleotides, and the nucleotide sequence is as set forth in SEQ ID NO: 67. The anti-Ang2 aptamer r-AMS0525.3 has natural nucleotides, and the nucleotide sequence is as set forth in SEQ ID NO: 65; and the anti-Ang2 aptamer AMS0525.3 has the corresponding 2′-fluoro-modified nucleotides, and the nucleotide sequence is as set forth in SEQ ID NO: 69. The anti-Ang2 aptamer r-AMS0525.4 has natural nucleotides, and the nucleotide sequence is as set forth in SEQ ID NO: 66; and the anti-Ang2 aptamer AMS0525.4 has the corresponding 2′-fluoro-modified nucleotides, and the nucleotide sequence is as set forth in SEQ ID NO: 70. The anti-Ang2 aptamer r-AMS0525 has natural nucleotides, and its nucleotide sequence is as set forth in SEQ ID NO: 63; and the anti-Ang2 aptamers AMS0525.5, AMS0525.6, AMS0525.7, or AMS0525.8 have the corresponding nucleotides with various modifications, and their nucleotide sequences are as set forth in SEQ ID NO: 71-74.

[0346] The anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop. The anti-Ang2 aptamer may comprise a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.

[0347] The anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 101-108.

[0348] Alternatively, or in addition, the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 109-116.

[0349] For example, the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 101, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 109; the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 102, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 110; the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 103, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 111; the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 104, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 112; the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 105, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 113; the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 106, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 114; the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 107, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 115; the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 108, as well as a second consensus sequence comprising a nucleotide sequence as set forth is as set forth in SEQ ID NO: 116.

[0350] The first consensus sequence may be comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 reference aptamer of the anti-Ang2 aptamer of the present disclosure. In some cases, the first consensus sequence may not be comprised in the nucleotide sequence forming the first loop of the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure. For example, the first consensus sequence may be present anywhere within the aptamer, except in the region forming the first loop.

[0351] In some cases, the first consensus sequence may be comprised in the first stem, the first bulge and the second stem. In some cases, the first consensus sequence may be comprised in the first stem, the first bulge, the second stem, and the second bulge. In some cases, the first consensus sequence may be comprised in the first stem, the first bulge, the second stem, the second bulge and the third stem. In some cases, the first consensus sequence may be comprised in the first stem, the first bulge, the second stem, the second bulge, the third stem, and the third bulge. In some cases, the first consensus sequence may be comprised in the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge, and the fourth stem. In some cases, the first consensus sequence may be comprised in the second stem, the second bulge and the third stem. In some cases, the first consensus sequence may be comprised in the second stem, the second bulge, the third stem, and the third bulge. In some cases, the first consensus sequence may be comprised in the second stem, the second bulge, the third stem, the third bulge and the fourth stem. In some cases, the first consensus sequence may be comprised in the third stem, the third bulge and the fourth stem.

[0352] The second consensus sequence may be comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure. In some cases, the second consensus sequence may not be comprised in the nucleotide sequence forming the first loop of the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure. For example, the second consensus sequence may be present anywhere within the aptamer, except in the region forming the first loop.

[0353] In some cases, the second consensus sequence may be comprised in the first stem, the first bulge and the second stem. In some cases, the second consensus sequence may be comprised in the first stem, the first bulge, the second stem, and the second bulge. In some cases, the second consensus sequence may be comprised in the first stem, the first bulge, the second stem, the second bulge and the third stem. In some cases, the second consensus sequence may be comprised in the first stem, the first bulge, the second stem, the second bulge, the third stem, and the third bulge. In some cases, the second consensus sequence may be comprised in the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge, and the fourth stem. In some cases, the second consensus sequence may be comprised in the second stem, the second bulge and the third stem. In some cases, the second consensus sequence may be comprised in the second stem, the second bulge, the third stem, and the third bulge. In some cases, the second consensus sequence may be comprised in the second stem, the second bulge, the third stem, the third bulge and the fourth stem. In some cases, the second consensus sequence may be comprised in the third stem, the third bulge and the fourth stem.

[0354] The first consensus sequence and the second consensus sequence may be paired to make sure that the secondary structure of the aptamer is properly formed. For example, when a first consensus sequence is chosen, those skilled in the art may be able to adjust the nucleotide sequence of the second consensus sequence, as long as the secondary structure of the aptamer can be properly formed.

[0355] The anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise the first consensus sequence and the second consensus sequence, wherein the first consensus sequence may be located 5′ to the second consensus sequence.

[0356] In some cases, the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise the first consensus sequence and the second consensus sequence, wherein the second consensus sequence may be located 5′ to the first consensus sequence.

[0357] For example, 1) the first consensus sequence may be comprised in the second stem, the second bulge, the third stem, the third bulge and the fourth stem; while the second consensus sequence may be comprised in the fourth stem, the third bulge, the third stem, the second bulge and the second stem; 2) the first consensus sequence may be in first bulge, the second stem, the second bulge, the third stem, the third bulge and the fourth stem; while the second consensus sequence may be comprised in the in the fourth stem, the third bulge, the third stem, the second bulge, the second stem and the first bulge; or, 3) the first consensus sequence may be comprised in the nucleotide sequence forming the first stem, the second stem, the second bulge, the third stem, the third bulge and the fourth stem; while the second consensus sequence may be comprised in the fourth stem, the third bulge, the third stem, the second bulge and the second stem, the first bulge and the first stem.

[0358] For example, 1) the second consensus sequence may be comprised in the second stem, the second bulge, the third stem, the third bulge and the fourth stem; while the first consensus sequence may be comprised in the fourth stem, the third bulge, the third stem, the second bulge and the second stem; 2) the second consensus sequence may be in first bulge, the second stem, the second bulge, the third stem, the third bulge and the fourth stem; while the first consensus sequence may be comprised in the in the fourth stem, the third bulge, the third stem, the second bulge, the second stem and the first bulge; or, 3) the second consensus sequence may be comprised in the nucleotide sequence forming the first stem, the second stem, the second bulge, the third stem, the third bulge and the fourth stem; while the first consensus sequence may be comprised in the fourth stem, the third bulge, the third stem, the second bulge and the second stem, the first bulge and the first stem.

[0359] The first loop of the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprises 2-30 nucleotides (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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, or at least 30 nucleotides).

[0360] The anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof. The variant may comprise a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 63-74. The variant may comprise a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, with one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, or more) nucleotide addition, deletion, and/or substitution.

[0361] The anti-Ang2 reference aptamer may comprise about 10 to about 150 nucleotides. For example, the anti-Ang2 reference aptamer may comprise about 10 to about 150 nucleotides, about 10 to about 140 nucleotides, about 10 to about 130 nucleotides, about 10 to about 120 nucleotides, about 10 to about 110 nucleotides, about 10 to about 100 nucleotides, about 10 to about 90 nucleotides, about 10 to about 80 nucleotides, about 10 to about 70 nucleotides, about 10 to about 60 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, or about 10 to about 20 nucleotides.

[0362] The anti-Ang2 aptamer may be used for modulating the biological activity of Ang2 or Tie2.

[0363] The anti-Ang2 aptamer may be used for preventing, treating, and/or ameliorating a Ang2 related disease, disorder or condition. For example, the Ang2 related disease, disorder or condition may be a neovascular disease, disorder or condition. For example, the Ang2 related disease, disorder or condition may be an ocular neovascular disease, disorder or condition. For example, the Ang2 related disease, disorder or condition may be selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. For example, the Ang2 related disease, disorder or condition may be selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0364] In one aspect, the present disclosure provides an anti-Ang2 agent, comprising the aptamer of the present disclosure. The anti-Ang2 agent may further comprise a polyethylene glycol (PEG) moiety. For example, the PEG moiety may be conjugated to the 5′ terminus and/or the 3′ terminus of the anti-Ang2 aptamer.

[0365] In another aspect, the present disclosure provides a composition comprising the anti-Ang2 aptamer, and/or the anti-Ang2 agent of the present application.

[0366] In some cases, the composition may be a pharmaceutical composition. For example, the composition may comprise a pharmaceutically acceptable excipient or carrier.

[0367] In some cases, the composition may be presented as discrete dosage forms, with each dosage containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid. Such dosage forms can be prepared by any of the methods known to a skilled person, for example, it may include the step of bringing the active ingredient into association with the carrier, which constitutes one or more other ingredients. In general, the compositions are prepared by uniformly and intimately mixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

[0368] The composition may comprise a therapeutically effective amount of the active composition (e.g., the anti-Ang2 aptamer or the anti-Ang2 agent of the present disclosure). A therapeutically effective amount may be an amount of the subject composition capable of preventing and/or curing (at least partially) a neovascular disease or disorder and/or any complications thereof in a subject suffering from or having a risk of developing the condition or disorder. The specific amount/concentration of the active agent comprised may vary according to the method of administration and the need of a patient, and can be determined based on e.g., volume, viscosity, and/or body weight of a patient etc. For example, an appropriate dosage may be about 0.1 mg or 1 mg/kg/day to about 50 mg/kg/day; sometimes, the dosage can be even higher. It shall be understood that these specific doses may be conveniently adjusted by a skilled person in the art (e.g., a doctor or a pharmacist) based on conditions of a specific patient, formulation, and/or disease.

[0369] The composition may further comprise an effective amount of an additional therapeutically active component, for example, an additional therapeutically active component for preventing, treating and/or ameliorating a neovascular disease or disorder. Each of the active components may be present in the pharmaceutical composition in a pharmaceutically effective amount. The anti-Ang2 aptamer of the present application may or may not be associated with the additional active component.

[0370] In some cases, the additional therapeutically active component may be an anti-VEGF agent. The anti-VEGF agent may comprise Aflibercept. The anti-VEGF agent may also comprise the anti-VEGF aptamer of the present disclosure.

[0371] Described below are non-limiting exemplary compositions and methods for preparing such a composition. The composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The composition may be in unit dosage forms suitable for single administration of precise dosages. In some embodiments, the composition may be a liquid pharmaceutical composition.

[0372] In the composition, the anti-Ang2 aptamer may be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration.

[0373] The composition may further include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, without limitation, detackifiers, anti-foaming compositions, buffering compositions, polymers, antioxidants, preservatives, chelating compositions, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending compositions, binders, fillers, plasticizers, lubricants, and/or mixtures thereof.

[0374] In one aspect, the present disclosure provides a method for modulating the biological activity of Ang2 or Tie2, comprising administering to a subject in need thereof an effective amount of the anti-Ang2 aptamer, or the anti-Ang2 agent of the present disclosure.

[0375] In one aspect, the present disclosure provides a method for preventing, treating, and/or ameliorating an Ang2 related disease, disorder or condition, comprising administering to a subject in need thereof an effective amount of the anti-Ang2 aptamer, or the anti-Ang2 agent of the present disclosure.

[0376] For example, the Ang2 related disease, disorder or condition may be a neovascular disease, disorder or condition may be a neovascular disease, disorder or condition. The Ang2 related disease, disorder or condition may be an ocular neovascular disease, disorder or condition. The Ang2 related disease, disorder or condition may be selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. The Ang2 related disease, disorder or condition may be selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0377] The method may further comprise administering to the subject an anti-VEGF agent. The anti-VEGF agent may comprise Aflibercept and/or the anti-VEGF aptamer of the present disclosure.

[0378] In another aspect, the present disclosure provides a use of the anti-Ang2 aptamer, or the anti-Ang2 agent of the present disclosure in the manufacture of an agent for modulating the biological activity of Ang2 or Tie2.

[0379] In one aspect, the present disclosure provides a use of the aptamer of the present disclosure, or the anti-Ang2 agent of the present disclosure, in the manufacture of a medicament for preventing, treating, and/or ameliorating an Ang2 related disease, disorder or condition.

[0380] For example, the Ang2 related disease, disorder or condition may be a neovascular disease, disorder or condition may be a neovascular disease, disorder or condition. The Ang2 related disease, disorder or condition may be an ocular neovascular disease, disorder or condition. The Ang2 related disease, disorder or condition may be selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. The Ang2 related disease, disorder or condition may be selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0381] In one aspect, the present disclosure provides a use of 1) the anti-Ang2 aptamer, or the anti-Ang2 agent of the present disclosure in combination with 2) an anti-VEGF agent (e.g., Aflibercept and/or an anti-VEGF aptamer of the present disclosure) in the manufacture of a medicament for preventing, treating, and/or ameliorating a neovascular disease, disorder or condition.

[0382] The anti-Ang2 aptamer, or the anti-Ang2 agent of the present disclosure may be administered simultaneously or sequentially with the anti-VEGF agent.

[0383] The neovascular disease, disorder or condition may be an ocular neovascular disease, disorder or condition. The neovascular disease, disorder or condition may be selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. The neovascular disease, disorder or condition may be selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

Bispecific Aptamer

[0384] In one aspect, the present disclosure provides a bispecific aptamer comprising an anti-Ang2 aptamer of the present disclosure and an anti-VEGF aptamer of the present disclosure. The bispecific aptamer may be comprised in the composition of the present disclosure.

[0385] The bispecific aptamer may be of the formula A1-(L) n-A2, wherein: A1 is an anti-VEGF aptamer of the present disclosure and A2 is an anti-Ang2 aptamer of the present disclosure; or A1 is an anti-Ang2 aptamer of the present disclosure and A2 is an anti-VEGF aptamer of the present disclosure; L is a linker; and n is a number of at least 0. For example, n may be 0 to 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0386] The bispecific aptamer may be capable of specifically binding to both VEGF and Ang2.

[0387] For example, the bispecific aptamer may bind to VEGF-121 with a K_D value of about 500 pM or less (for example, the bispecific aptamer may bind to VEGF-121 with a K_D value of about 500 picomolar (pM) or less, about 400 picomolar (pM) or less, about 300 picomolar (pM) or less, about 200 picomolar (pM) or less, about 100 picomolar (pM) or less, about 90 picomolar (pM) or less, about 80 picomolar (pM) or less, about 70 picomolar (pM) or less, about 60 picomolar (pM) or less, about 50 picomolar (pM) or less, about 40 picomolar (pM) or less, about 30 picomolar (pM) or less, or about 20 picomolar (pM) or less), and the bispecific aptamer may bind to Ang2 with a K_D value of about 500 pM or less (for example, the bispecific aptamer may bind to Ang2 with a K_D value of about 500 picomolar (pM) or less, about 400 picomolar (pM) or less, about 300 picomolar (pM) or less, about 200 picomolar (pM) or less, about 100 picomolar (pM) or less, about 90 picomolar (pM) or less, about 80 picomolar (pM) or less, about 70 picomolar (pM) or less, about 60 picomolar (pM) or less, about 50 picomolar (pM) or less, about 40 picomolar (pM) or less, about 30 picomolar (pM) or less, or about 20 picomolar (pM) or less).

[0388] The L may be selected from a naturally occurring nucleotide linker, a modified nucleotide linker, a hydrocarbon linker, a polyethylene glycol linker and a combination thereof.

[0389] In some cases, at least one L may be a polyethylene glycol linker. In some cases, at least one L may be a hexaethylene glycol (also indicated as H or PEG6) linker. For example, the L may be a hexaethylene glycol linker, and n may be 0 to 10 (for example, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0390] For example, A1 may be an anti-VEGF aptamer comprising a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, or a variant or truncate thereof; and A2 may be an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof.

[0391] As another example, A1 may be an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof; and A2 may be an anti-VEGF aptamer comprising a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, or a variant or truncate thereof.

[0392] In some cases of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 15, or a truncate thereof, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0393] In some cases of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 18, or a truncate thereof, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0394] In some cases of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 23, or a truncate thereof, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0395] In some cases of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, or a truncate thereof, A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 15, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0396] In some cases of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, or a truncate thereof, A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 18, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0397] In some cases of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, or a truncate thereof, A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 23, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0398] In some cases of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 45, or a truncate thereof, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0399] In some cases of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, or a truncate thereof, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0400] In some cases of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 55, or a truncate thereof, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0401] In some cases of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 45, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0402] In some cases of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0403] In some cases of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 55, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0404] In some cases, the bispecific aptamer may comprise a nucleotide sequence as set forth in any of SEQ ID NO: 75-78 and 138-157.

[0405] The bispecific aptamer may be selected from r-AMSB101, r-AMSB102, r-AMSB103, r-AMSB104, r-AMSB105, r-AMSB106, r-AMSB103.1, r-AMSB103.2, r-AMSB103.3, r-AMSB103.4, r-AMSB103.5, r-AMSB103.6, AMSB101, AMSB102, AMSB103, AMSB104, AMSB105, AMSB106, AMSB103.1, AMSB103.2, AMSB103.3, AMSB103.4, AMSB103.5 and AMSB103.6.

[0406] In the r-AMSB101, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 15 A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, L is a hexaethylene glycol linker, and n is 0.

[0407] In the r-AMSB102, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 23, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, L is a hexaethylene glycol linker, and n is 0.

[0408] In the r-AMSB103, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 18, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, L is a hexaethylene glycol linker, and n is 0.

[0409] In the r-AMSB104, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 15, L is a hexaethylene glycol linker, and n is 0.

[0410] In the r-AMSB105, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 23, L is a hexaethylene glycol linker, and n is 0.

[0411] In the r-AMSB106, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 18, L is a hexaethylene glycol linker, and n is 0.

[0412] In the r-AMSB103.1, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 18, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, L is a hexaethylene glycol linker, and n is 1.

[0413] In the r-AMSB103.2, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 18, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, L is a hexaethylene glycol linker, and n is 2.

[0414] In the r-AMSB103.3, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 18, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, L is a hexaethylene glycol linker, and n is 3.

[0415] In the r-AMSB103.4, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 18, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, L is a hexaethylene glycol linker, and n is 4.

[0416] In the r-AMSB103.5, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 18, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, L is a hexaethylene glycol linker, and n is 5.

[0417] In the r-AMSB103.6, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 18, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 64, L is a hexaethylene glycol linker, and n is 6.

[0418] In the AMSB101, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 45, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, L is a hexaethylene glycol linker, and n is 0.

[0419] In the AMSB102, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 55, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, L is a hexaethylene glycol linker, and n is 0.

[0420] In the AMSB103, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, L is a hexaethylene glycol linker, and n is 0.

[0421] In the AMSB104, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 45, L is a hexaethylene glycol linker, and n is 0.

[0422] In the AMSB105, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 55, L is a hexaethylene glycol linker, and n is 0.

[0423] In the AMSB106, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, L is a hexaethylene glycol linker, and n is 0.

[0424] In the AMSB103.1, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, L is a hexaethylene glycol linker, and n is 1.

[0425] In the AMSB103.2, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, L is a hexaethylene glycol linker, and n is 2.

[0426] In the AMSB103.3, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, L is a hexaethylene glycol linker, and n is 3.

[0427] In the AMSB103.4, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, L is a hexaethylene glycol linker, and n is 4.

[0428] In the AMSB103.5, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, L is a hexaethylene glycol linker, and n is 5.

[0429] In the AMSB103.6, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, L is a hexaethylene glycol linker, and n is 6.

Compositions

[0430] In one aspect, the present disclosure provides a composition comprising the anti-Ang2 aptamer of the present disclosure, the anti-VEGF aptamer of the present disclosure, and/or the bispecific aptamer of the present disclosure. In some cases, the composition comprises an effective amount of the anti-Ang2 aptamer of the present disclosure and an effective amount of the anti-VEGF aptamer of the present disclosure. In some cases, the composition comprises an effective amount of the bispecific aptamer of the present disclosure.

[0431] The composition may be a pharmaceutical composition. The composition may comprise a pharmaceutically acceptable excipient or carrier.

[0432] The composition may be used for preventing, treating and/or ameliorating a neovascular disease, disorder or condition. The neovascular disease, disorder or condition may be an ocular neovascular disease, disorder or condition. In some embodiments, the neovascular disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. In some embodiments, the neovascular disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0433] The composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The composition may be in unit dosage forms suitable for single administration of precise dosages. In some cases, the composition may be a liquid pharmaceutical composition.

[0434] The pharmaceutically acceptable excipient or carrier may include, without limitation, detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and/or mixtures thereof.

[0435] In some cases, the composition may be presented as discrete dosage forms, with each dosage containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid. Such dosage forms can be prepared by any of the methods known to a skilled person, for example, it may include the step of bringing the active ingredient into association with the carrier, which constitutes one or more other ingredients. In general, the compositions are prepared by uniformly and intimately mixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

[0436] The composition may comprise a therapeutically effective amount of the active agent (e.g., the agent of the present disclosure). A therapeutically effective amount may be an amount of the subject composition capable of preventing and/or curing (at least partially) a neovascular disease or disorder and/or any complications thereof in a subject suffering from or having a risk of developing the condition or disorder. The specific amount/concentration of the active agent comprised may vary according to the method of administration and the need of a patient, and can be determined based on e.g., volume, viscosity, and/or body weight of a patient, etc. For example, an appropriate dosage may be about 0.1 mg or 1 mg/kg/day to about 50 mg/kg/day; sometimes, the dosage can be even higher. It shall be understood that these specific doses may be conveniently adjusted by a skilled person in the art (e.g., a doctor or a pharmacist) based on conditions of a specific patient, formulation, and/or disease.

Medical Use and Method

[0437] In another aspect, the present disclosure provides a method for preventing, treating and/or ameliorating a neovascular disease, disorder or condition, comprising administering to a subject in need thereof an effective amount of the anti-Ang2 aptamer of the present disclosure, an effective amount of the anti-VEGF aptamer of the present disclosure, an effective amount of the bispecific aptamer of the present disclosure, and/or an effective amount of the composition of the present disclosure.

[0438] In another aspect, the present disclosure provides use of the anti-Ang2 aptamer of the present disclosure, the anti-VEGF aptamer of the present disclosure, the bispecific aptamer of the present disclosure, and/or the composition of the present disclosure in the manufacture of a medicament for preventing, treating and/or ameliorating a neovascular disease, disorder or condition.

[0439] The neovascular disease, disorder or condition may be an ocular neovascular disease, disorder or condition. In some embodiments, the neovascular disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. In some embodiments, the neovascular disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

Further Embodiments

[0440] The present disclosure further provides the following embodiments:

• • [0441] 1. An anti-Ang2 aptamer, which specifically binds to Ang2 with a K_D value of about 500 picomolar (pM) or less.
  • [0442] 2. The anti-Ang2 aptamer of embodiment 1, which specifically binds to Ang2 with a K_D value of about 100 picomolar (pM) or less.
  • [0443] 3. The anti-Ang2 aptamer of any one of embodiments 1-2, wherein said Ang2 is a human Ang2, a mouse Ang2, a monkey Ang2, a rabbit Ang2, and/or a rat Ang2.
  • [0444] 4. The anti-Ang2 aptamer of any one of embodiments 1-3, which does not substantially bind to human Ang1.
  • [0445] 5. The anti-Ang2 aptamer of any one of embodiments 1-4, which inhibits an interaction between said Ang2 and Tie2.
  • [0446] 6. The anti-Ang2 aptamer of any one of embodiments 1-5, which is capable of reducing neovascularization and/or reducing vascular permeability in a mouse oxygen-induced ischemic retinopathy (OIR) model.
  • [0447] 7. The anti-Ang2 aptamer of any one of embodiments 1-6, which is highly soluble.
  • [0448] 8. The anti-Ang2 aptamer of any one of embodiments 1-7, which is an RNA aptamer, a DNA aptamer, or a combination thereof.
  • [0449] 9. The anti-Ang2 aptamer of any one of embodiments 1-8, which comprises one or more modified nucleotide, and/or one or more substitutions (such as a substitution with one or more PEGs).

[0450] For example, one or more (e.g., 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) nucleotides of the anti-Ang2 aptamer may be substituted with a polyethylene glycol moiety or a linker comprising multiple (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) polyethylene glycol moieties. In some cases, the linker may be a hexaethylene glycol (H or PEG6) linker. Such substitution may occur to any nucleotide of the anti-Ang2 aptamer, as long as it does not substantially affect the activity (e.g., binding activity, or inhibition activity) and/or function of the anti-Ang2 aptamer. In some cases, the substitution does not substantially affect the secondary structure of the anti-Ang2 aptamer, for example, the substitution may occur to a nucleotide comprised in a bulge or a loop region of the anti-Ang2 aptamer secondary structure. In some cases, the substitution does not occur to any nucleotide comprised in a stem region of the anti-Ang2 aptamer secondary structure.

• • [0451] 10. The anti-Ang2 aptamer of any one of embodiments 1-9, which is nuclease resistant.
  • [0452] 11. The anti-Ang2 aptamer of any one of embodiments 1-10, wherein all nucleotides of said anti-Ang2 aptamer are modified nucleotides.
  • [0453] 12. The anti-Ang2 aptamer of any one of embodiments 9-11, wherein said modified nucleotide comprises a chemical substitution or modification at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.
  • [0454] 13. The anti-Ang2 aptamer of any one of embodiments 9-12, wherein said modified nucleotide comprises one or more modifications independently selected from a 2′-position sugar modification, a 2′-amino (2′-NH₂) modification, a 2′-fluoro (2′-F) modification, a 2′-O-methyl(2′-OMe) modification, a 2′-O-(2-Methoxyethyl) (2′-O-MOE) modification, a 5-position modified pyrimidine, a modification at a cytosine exocyclic amine, a substitution with 5′-bromouracil, a substitution with 5′-bromodeoxyuridine, a substitution with 5′-bromodeoxycytidine, a backbone modification, a locked nucleic acid (LNA), a methylation, a 3′ cap, and a 5′ cap.
  • [0455] 14. The anti-Ang2 aptamer of embodiment 13, wherein said 5-position modified pyrimidine is selected from 5-carboxy-2′-deoxyuridine, 5-aminoallyl-2′-deoxyuridine, 5-[(3-indolyl) propionamide-N-allyl]-2′-deoxyuridine, 5-carboxy-2′-deoxycytidine, 5-aminoallyl-2′-deoxycytidine, biotin-16-aminoallyl-2′-deoxycytidine, 5-(N-benzylcarboxyamide)-2′-deoxyuridine, 5-(N-isobutylcarboxyamide)-2′-deoxyuridine, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine, and 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine.
  • [0456] 15. The anti-Ang2 aptamer of any one of embodiments 1-14, which comprises one or more 2′-modified nucleotides.
  • [0457] 16. The anti-Ang2 aptamer of any one of embodiments 1-15, wherein all nucleotides of said anti-Ang2 aptamer are 2′-modified nucleotides.
  • [0458] 17. The anti-Ang2 aptamer of any one of embodiments 15-16, wherein said 2′-modified nucleotide is selected from a 2′-amino modified nucleotide, a 2′-fluoro-modified nucleotide, a 2′-O-methyl-modified nucleotide, and a 2′-O-(2-methoxyethyl)-modified nucleotide.
  • [0459] 18. The anti-Ang2 aptamer of any one of embodiments 1-17, which comprises at least one 2′-fluoro-modified nucleotide, at least one 2′-O-methyl-modified nucleotide, and/or at least one LNA.
  • [0460] 19. The anti-Ang2 aptamer of any one of embodiments 1-18, wherein all cytidines of said anti-Ang2 aptamer are 2′-modified cytidines.
  • [0461] 20. The anti-Ang2 aptamer of any one of embodiments 1-19, wherein all cytidines of said anti-Ang2 aptamer are 2′-fluoro-modified cytidines and/or 2′-O-methyl-modified cytidines.
  • [0462] 21. The anti-Ang2 aptamer of any one of embodiments 1-20, wherein all cytidines of said anti-Ang2 aptamer are 2′-deoxy-2′-fluorocytidines.
  • [0463] 22. The anti-Ang2 aptamer of any one of embodiments 1-21, wherein all uridines of said anti-Ang2 aptamer are 2′-modified uridines.
  • [0464] 23. The anti-Ang2 aptamer of any one of embodiments 1-22, wherein all uridines of said anti-Ang2 aptamer are 2′-fluoro-modified uridines and/or 2′-O-methyl-modified uridines.
  • [0465] 24. The anti-Ang2 aptamer of any one of embodiments 1-23, wherein all uridines of said anti-Ang2 aptamer are 2′-deoxy-2′-fluorouridines.
  • [0466] 25. The anti-Ang2 aptamer of any one of embodiments 1-24, wherein all adenosines of said anti-Ang2 aptamer are 2′-modified adenosines.
  • [0467] 26. The anti-Ang2 aptamer of any one of embodiments 1-25, wherein all adenosines of said anti-Ang2 aptamer are 2′-fluoro-modified adenosines and/or 2′-O-methyl-modified adenosines.
  • [0468] 27. The anti-Ang2 aptamer of any one of embodiments 1-26, wherein all adenosines of said anti-Ang2 aptamer are 2′-deoxy-2′-fluoroadenosines.
  • [0469] 28. The anti-Ang2 aptamer of any one of embodiments 1-27, wherein all guanosines of said anti-Ang2 aptamer are 2′-modified guanosines.
  • [0470] 29. The anti-Ang2 aptamer of any one of embodiments 1-28, wherein all guanosines of said anti-Ang2 aptamer are 2′-fluoro-modified guanosines and/or 2′-O-methyl-modified guanosines.
  • [0471] 30. The anti-Ang2 aptamer of any one of embodiments 1-29, wherein all guanosines of said anti-Ang2 aptamer are 2′-deoxy-2′-fluoroguanosines.
  • [0472] 31. The anti-Ang2 aptamer of any one of embodiments 1-30, wherein all nucleotides of said anti-Ang2 aptamer are 2′-modified nucleotides.
  • [0473] 32. The anti-Ang2 aptamer of any one of embodiments 1-31, wherein all nucleotides of said anti-Ang2 aptamer are 2′-fluoro-modified nucleotides and/or 2′-O-methyl-modified nucleotides.
  • [0474] 33. The anti-Ang2 aptamer of any one of embodiments 1-32, wherein all nucleotides of said anti-Ang2 aptamer are 2′-fluoro-modified nucleotides.
  • [0475] 34. The anti-Ang2 aptamer of any one of embodiments 1-33, which does not comprise any natural nucleotide.
  • [0476] 35. The anti-Ang2 aptamer of any one of embodiments 1-34, which comprises about 10 to about 150 nucleotides.
  • [0477] 36. The anti-Ang2 aptamer of any one of embodiments 1-35, which competes with an anti-Ang2 reference aptamer for binding to said Ang2, wherein said anti-Ang2 reference aptamer comprises a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.
  • [0478] 37. The anti-Ang2 aptamer of embodiment 36, wherein said anti-Ang2 reference aptamer comprises a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.
  • [0479] 38. The anti-Ang2 aptamer of any one of embodiments 36-37, wherein said anti-Ang2 reference aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 101-108.
  • [0480] 39. The anti-Ang2 aptamer of any one of embodiments 36-38, wherein said anti-Ang2 reference aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 109-116.
  • [0481] 40. The anti-Ang2 aptamer of any one of embodiments 38-39, wherein said first consensus sequence is comprised in the nucleotide sequence forming the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 reference aptamer.
  • [0482] 41. The anti-Ang2 aptamer of any one of embodiments 38-40, wherein said first consensus sequence is not comprised in the nucleotide sequence forming the first loop of the anti-Ang2 reference aptamer.
  • [0483] 42. The anti-Ang2 aptamer of any one of embodiments 39-41, wherein said second consensus sequence is comprised in the nucleotide sequence forming the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 reference aptamer.
  • [0484] 43. The anti-Ang2 aptamer of any one of embodiments 39-42, wherein said second consensus sequence is not comprised in the nucleotide sequence forming the first loop of the anti-Ang2 reference aptamer.
  • [0485] 44. The anti-Ang2 aptamer of any one of embodiments 36-43, wherein said first loop of the anti-Ang2 reference aptamer comprises 2-30 nucleotides.
  • [0486] 45. The anti-Ang2 aptamer of any one of embodiments 36-44, wherein said first loop of the anti-Ang2 reference aptamer comprises 3-10 nucleotides.
  • [0487] 46. The anti-Ang2 aptamer of any one of embodiments 36-45, wherein said anti-Ang2 reference aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof.
  • [0488] 47. The anti-Ang2 aptamer of embodiment 46, wherein said variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 63-74.
  • [0489] 48. The anti-Ang2 aptamer of any one of embodiments 46-47, wherein said variant comprises a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, with one or more nucleotide addition, deletion, and/or substitution.
  • [0490] 49. The anti-Ang2 aptamer of any one of embodiments 36-48, wherein said anti-Ang2 reference aptamer comprises about 10 to about 150 nucleotides.
  • [0491] 50. The anti-Ang2 aptamer of any one of embodiments 38-49, wherein said anti-Ang2 reference aptamer comprises said first consensus sequence and said second consensus sequence, and wherein said first consensus sequence is located 5′ to said second consensus sequence.
  • [0492] 51. The anti-Ang2 aptamer of any one of embodiments 38-49, wherein said anti-Ang2 reference aptamer comprises said first consensus sequence and said second consensus sequence, and wherein said second consensus sequence is located 5′ to said first consensus sequence.
  • [0493] 52. The anti-Ang2 aptamer of any one of embodiments 1-51, wherein said anti-Ang2 aptamer comprises a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.
  • [0494] 53. The anti-Ang2 aptamer of any one of embodiments 1-52, wherein said anti-Ang2 aptamer comprises a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.
  • [0495] 54. The anti-Ang2 aptamer of any one of embodiments 1-53, wherein said anti-Ang2 aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 101-108.
  • [0496] 55. The anti-Ang2 aptamer of any one of embodiments 1-54, wherein said anti-Ang2 aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 109-116.
  • [0497] 56. The anti-Ang2 aptamer of any one of embodiments 54-55, wherein said first consensus sequence is comprised in the nucleotide sequence forming the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 aptamer.
  • [0498] 57. The anti-Ang2 aptamer of any one of embodiments 54-56, wherein said first consensus sequence is not comprised in the nucleotide sequence forming the first loop of the anti-Ang2 aptamer.
  • [0499] 58. The anti-Ang2 aptamer of any one of embodiments 55-57, wherein said second consensus sequence is comprised in the nucleotide sequence forming the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-Ang2 aptamer.
  • [0500] 59. The anti-Ang2 aptamer of any one of embodiments 55-58, wherein said second consensus sequence is not comprised in the nucleotide sequence forming the first loop of the anti-Ang2 aptamer.
  • [0501] 60. The anti-Ang2 aptamer of any one of embodiments 52-59, wherein said first loop of the anti-Ang2 aptamer comprises 2-30 nucleotides.
  • [0502] 61. The anti-Ang2 aptamer of any one of embodiments 52-60, wherein said first loop of the anti-Ang2 aptamer comprises 3-10 nucleotides.
  • [0503] 62. The anti-Ang2 aptamer of any one of embodiments 1-61, wherein said anti-Ang2 aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof.
  • [0504] 63. The anti-Ang2 aptamer of embodiment 62, wherein said variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 63-74.
  • [0505] 64. The anti-Ang2 aptamer of any one of embodiments 62-63, wherein said variant comprises a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, with one or more nucleotide addition, deletion, and/or substitution.
  • [0506] 65. The anti-Ang2 aptamer of any one of embodiments 54-64, wherein said anti-Ang2 aptamer comprises said first consensus sequence and said second consensus sequence, and wherein said first consensus sequence is located 5′ to said second consensus sequence.
  • [0507] 66. The anti-Ang2 aptamer of any one of embodiments 54-64, wherein said anti-Ang2 aptamer comprises said first consensus sequence and said second consensus sequence, wherein said second consensus sequence is located 5′ to said first consensus sequence.
  • [0508] 67. The anti-Ang2 aptamer of any one of embodiments 1-66, wherein said anti-Ang2 aptamer is conjugated to a polyethylene glycol (PEG) moiety.
  • [0509] 68. The anti-Ang2 aptamer of embodiment 67, wherein at least one PEG moiety is conjugated to the 5′ terminus of said anti-Ang2 aptamer.
  • [0510] 69. The anti-Ang2 aptamer of any one of embodiments 67-68, wherein at least one PEG moiety is conjugated to the 3′ terminus of said anti-Ang2 aptamer.
  • [0511] 70. An anti-VEGF aptamer, which binds to VEGF-121 with a K_D value of about 20 nanomolar (nM) or less, and binds to VEGF-165 with a K_D value of about 20 nanomolar (nM) or less.
  • [0512] 71. The anti-VEGF aptamer of embodiment 70, which binds to VEGF-121 with a K_D value of about 2 nanomolar (nM) or less.
  • [0513] 72. The anti-VEGF aptamer of any one of embodiments 70-71, which binds to VEGF-165 with a K_D value of about 2 nanomolar (nM) or less.
  • [0514] 73. The anti-VEGF aptamer of any one of embodiments 70-72, wherein said VEGF-121 is a human VEGF-121, a mouse VEGF-120, a monkey VEGF-121, a rabbit VEGF-121, and/or a rat VEGF-120.
  • [0515] 74. The anti-VEGF aptamer of any one of embodiments 70-73, wherein said VEGF-165 is a human VEGF-165, a mouse VEGF-164, a monkey VEGF-165, a rabbit VEGF-165, and/or a rat VEGF-164.
  • [0516] 75. The anti-VEGF aptamer of any one of embodiments 70-74, which specifically binds to both a VEGF receptor-binding domain of VEGF-121 and a VEGF receptor-binding domain of VEGF-165.
  • [0517] 76. The anti-VEGF aptamer of embodiment 75, wherein said VEGF receptor-binding domain of VEGF-121 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 118-122, 128, 129, and 133.
  • [0518] 77. The anti-VEGF aptamer of any one of embodiments 75-76, wherein said VEGF receptor-binding domain of VEGF-165 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 118-122, 128, and 129.
  • [0519] 78. The anti-VEGF aptamer of any one of embodiments 70-77, which inhibits an interaction between said VEGF-121 and VEGF-R1.
  • [0520] 79. The anti-VEGF aptamer of any one of embodiments 70-78, which inhibits an interaction between said VEGF-121 and VEGF-R2.
  • [0521] 80. The anti-VEGF aptamer of any one of embodiments 70-79, which inhibits an interaction between said VEGF-165 and VEGF-R1.
  • [0522] 81. The anti-VEGF aptamer of any one of embodiments 70-80, which inhibits an interaction between said VEGF-165 and VEGF-R2.
  • [0523] 82. The anti-VEGF aptamer of any one of embodiments 70-81, which is capable of reducing and/or ameliorating a lesion and/or a leakage in a laser-induced choroidal neovascularization (CNV) rat model.
  • [0524] 83. The anti-VEGF aptamer of any one of embodiments 70-82, which is highly soluble.
  • [0525] 84. The anti-VEGF aptamer of any one of embodiments 70-83, which is an RNA aptamer, a DNA aptamer, or a combination thereof.
  • [0526] 85. The anti-VEGF aptamer of any one of embodiments 70-84, which is nuclease resistant.
  • [0527] 86. The anti-VEGF aptamer of any one of embodiments 70-85, which comprises one or more modified nucleotides, and/or one or more substitutions (such as a substitution with one or more PEGs). For example, one or more (e.g., 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) nucleotides of the anti-VEGF aptamer may be substituted with a polyethylene glycol moiety or a linker comprising multiple (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) polyethylene glycol moieties. In some cases, the linker may be a hexaethylene glycol (H or PEG6) linker. Such substitution may occur to any nucleotide of the anti-VEGF aptamer, as long as it does not substantially affect the activity (e.g., binding activity, or inhibition activity) and/or function of the anti-VEGF aptamer. In some cases, the substitution does not substantially affect the secondary structure of the anti-VEGF aptamer, for example, the substitution may occur to a nucleotide comprised in a bulge or a loop region of the anti-VEGF aptamer secondary structure. In some cases, the substitution does not occur to any nucleotide comprised in a stem region of the anti-VEGF aptamer secondary structure.
  • [0528] 87. The anti-VEGF aptamer of embodiment 86, wherein all nucleotides of said anti-VEGF aptamer are modified nucleotides.
  • [0529] 88. The anti-VEGF aptamer of any one of embodiments 86-87, wherein said modified nucleotide comprises a chemical substitution or modification at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.
  • [0530] 89. The anti-VEGF aptamer of any one of embodiments 86-88, wherein said modified nucleotide comprises one or more modifications independently selected from a 2′-position sugar modification, a 2′-amino (2′-NH₂) modification, a 2′-fluoro (2′-F) modification, a 2′-O-methyl(2′-OMe) modification, a 2′-O-(2-Methoxyethyl) (2′-O-MOE) modification, a 5-position modified pyrimidine, a modification at a cytosine exocyclic amine, a substitution with 5′-bromouracil, a substitution with 5′-bromodeoxyuridine, a substitution with 5′-bromodeoxycytidine, a backbone modification, a locked nucleic acid (LNA), a methylation, a 3′ cap, and a 5′ cap.
  • [0531] 90. The anti-VEGF aptamer of embodiment 89, wherein said 5-position modified pyrimidine is selected from 5-carboxy-2′-deoxyuridine, 5-aminoallyl-2′-deoxyuridine, 5-[(3-indolyl) propionamide-N-allyl]-2′-deoxyuridine, 5-carboxy-2′-deoxycytidine, 5-aminoallyl-2′-deoxycytidine, biotin-16-aminoallyl-2′-deoxycytidine, 5-(N-benzylcarboxyamide)-2′-deoxyuridine, 5-(N-isobutylcarboxyamide)-2′-deoxyuridine, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine, and 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine.
  • [0532] 91. The anti-VEGF aptamer of any one of embodiments 70-90, which comprises at least one 2′-modified nucleotide.
  • [0533] 92. The anti-VEGF aptamer of embodiment 91, wherein all nucleotides of said anti-VEGF aptamer are 2′-modified nucleotides.
  • [0534] 93. The anti-VEGF aptamer of any one of embodiments 91-92, wherein said 2′-modified nucleotide is selected from a 2′-amino modified nucleotide, a 2′-fluoro-modified nucleotide, a 2′-O-methyl-modified nucleotide, and a 2′-O-(2-methoxyethyl)-modified nucleotide.
  • [0535] 94. The anti-VEGF aptamer of any one of embodiments 70-93, which comprises at least one 2′-fluoro-modified nucleotide, at least one 2′-O-methyl-modified nucleotide, and/or at least one LNA.
  • [0536] 95. The anti-VEGF aptamer of any one of embodiments 70-94, wherein all cytidines of said anti-VEGF aptamer are 2′-modified cytidines.
  • [0537] 96. The anti-VEGF aptamer of any one of embodiments 70-95, wherein all cytidines of said anti-VEGF aptamer are 2′-fluoro-modified cytidines and/or 2′-O-methyl-modified cytidines.
  • [0538] 97. The anti-VEGF aptamer of any one of embodiments 70-96, wherein all cytidines of said anti-VEGF aptamer are 2′-deoxy-2′-fluorocytidines.
  • [0539] 98. The anti-VEGF aptamer of any one of embodiments 70-97, wherein all uridines of said anti-VEGF aptamer are 2′-modified uridines.
  • [0540] 99. The anti-VEGF aptamer of any one of embodiments 70-98, wherein all uridines of said anti-VEGF aptamer are 2′-fluoro-modified uridines and/or 2′-O-methyl-modified uridines.
  • [0541] 100. The anti-VEGF aptamer of any one of embodiments 70-99, wherein all uridines of said anti-VEGF aptamer are 2′-deoxy-2′-fluorouridines.
  • [0542] 101. The anti-VEGF aptamer of any one of embodiments 70-100, wherein all adenosines of said anti-VEGF aptamer are 2′-modified adenosines.
  • [0543] 102. The anti-VEGF aptamer of any one of embodiments 70-101, wherein all adenosines of said anti-VEGF aptamer are 2′-fluoro-modified adenosines and/or 2′-O-methyl-modified adenosines.
  • [0544] 103. The anti-VEGF aptamer of any one of embodiments 70-102, wherein all adenosines of said anti-VEGF aptamer are 2′-deoxy-2′-fluoroadenosines.
  • [0545] 104. The anti-VEGF aptamer of any one of embodiments 70-103, wherein all guanosines of said anti-VEGF aptamer are 2′-modified guanosines.
  • [0546] 105. The anti-VEGF aptamer of any one of embodiments 70-104, wherein all guanosines of said anti-VEGF aptamer are 2′-fluoro-modified guanosines and/or 2′-O-methyl-modified guanosines.
  • [0547] 106. The anti-VEGF aptamer of any one of embodiments 70-105, wherein all guanosines of said anti-VEGF aptamer are 2′-deoxy-2′-fluoroguanosines.
  • [0548] 107. The anti-VEGF aptamer of any one of embodiments 70-106, wherein all nucleotides of said anti-VEGF aptamer are 2′-modified nucleotides.
  • [0549] 108. The anti-VEGF aptamer of any one of embodiments 70-107, wherein all nucleotides of said anti-VEGF aptamer are 2′-fluoro-modified nucleotides and/or 2′-O-methyl-modified nucleotides.
  • [0550] 109. The anti-VEGF aptamer of any one of embodiments 70-108, wherein all nucleotides of said anti-VEGF aptamer are 2′-fluoro-modified nucleotides.
  • [0551] 110. The anti-VEGF aptamer of any one of embodiments 70-109, wherein said anti-VEGF aptamer does not comprise any natural nucleotide.
  • [0552] 111. The anti-VEGF aptamer of any one of embodiments 70-110, which comprises about 10 to about 150 nucleotides.
  • [0553] 112. The anti-VEGF aptamer of any one of embodiments 70-111, wherein said anti-VEGF aptamer competes with an anti-VEGF reference aptamer for binding to said VEGF-165 and to said VEGF-121, wherein said anti-VEGF reference aptamer comprises a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.
  • [0554] 113. The anti-VEGF aptamer of embodiment 112, wherein said anti-VEGF reference aptamer comprises a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.
  • [0555] 114. The anti-VEGF aptamer of any one of embodiments 112-113, wherein said anti-VEGF reference aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO:79-90.
  • [0556] 115. The anti-VEGF aptamer of any one of embodiments 112-114, wherein said anti-VEGF reference aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 91-100.
  • [0557] 116. The anti-VEGF aptamer of any one of embodiments 114-115, wherein said first consensus sequence is comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF reference aptamer.
  • [0558] 117. The anti-VEGF aptamer of any one of embodiments 114-116, wherein said first consensus sequence is not comprised in the nucleotide sequence forming the first loop of the anti-VEGF reference aptamer.
  • [0559] 118. The anti-VEGF aptamer of any one of embodiments 115-117, wherein said second consensus sequence is comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF reference aptamer.
  • [0560] 119. The anti-VEGF aptamer of any one of embodiments 115-118, wherein said second consensus sequence is not comprised in the nucleotide sequence forming the first loop of the anti-VEGF reference aptamer.
  • [0561] 120. The anti-VEGF aptamer of any one of embodiments 112-119, wherein said first loop of the anti-VEGF reference aptamer comprises 2-30 nucleotides.
  • [0562] 121. The anti-VEGF aptamer of any one of embodiments 112-120, wherein said first loop of the anti-VEGF reference aptamer comprises 3-10 nucleotides.
  • [0563] 122. The anti-VEGF aptamer of any one of embodiments 112-121, wherein said anti-VEGF reference aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, or a variant or truncate thereof.
  • [0564] 123. The anti-VEGF aptamer of embodiment 122, wherein said variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 1-62.
  • [0565] 124. The anti-VEGF aptamer of any one of embodiments 122-123, wherein said variant comprises a nucleotide sequence as set forth in any of SEQ ID NO:1-62, with one or more nucleotide addition, deletion, and/or substitution.
  • [0566] 125. The anti-VEGF aptamer of any one of embodiments 112-124, wherein said anti-VEGF reference aptamer comprises about 10 to about 150 nucleotides.
  • [0567] 126. The anti-VEGF aptamer of any one of embodiments 114-125, wherein said anti-VEGF reference aptamer comprises said first consensus sequence and said second consensus sequence, and wherein said first consensus sequence is located 5′ to said second consensus sequence.
  • [0568] 127. The anti-VEGF aptamer of any one of embodiments 114-125, wherein said anti-VEGF reference aptamer comprises said first consensus sequence and said second consensus sequence, and wherein said second consensus sequence is located 5′ to said first consensus sequence.
  • [0569] 128. The anti-VEGF aptamer of any one of embodiments 70-127, wherein said anti-VEGF aptamer comprises a secondary structure comprising, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.
  • [0570] 129. The anti-VEGF aptamer of any one of embodiments 70-128, wherein said anti-VEGF aptamer comprises a secondary structure consisting of, from 5′ to 3′ direction, a first stem, a first bulge, a second stem, a second bulge, a third stem, a third bulge, a fourth stem, and a first loop.
  • [0571] 130. The anti-VEGF aptamer of any one of embodiments 70-129, wherein said anti-VEGF aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 79-90.
  • [0572] 131. The anti-VEGF aptamer of any one of embodiments 70-130, wherein said anti-VEGF aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any of SEQ ID NO: 91-100.
  • [0573] 132. The anti-VEGF aptamer of any one of embodiments 130-131, wherein said first consensus sequence is comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF aptamer.
  • [0574] 133. The anti-VEGF aptamer of any one of embodiments 130-132, wherein said first consensus sequence is not comprised in the nucleotide sequence forming the first loop of the anti-VEGF aptamer.
  • [0575] 134. The anti-VEGF aptamer of any one of embodiments 131-133, wherein said second consensus sequence is comprised in the nucleotide sequence forming the first stem, the first bulge, the second stem, the second bulge, the third stem, the third bulge and/or the fourth stem of the anti-VEGF aptamer.
  • [0576] 135. The anti-VEGF aptamer of any one of embodiments 131-134, wherein said second consensus sequence is not comprised in the nucleotide sequence forming the first loop of the anti-VEGF aptamer.
  • [0577] 136. The anti-VEGF aptamer of any one of embodiments 128-135, wherein said first loop of the anti-VEGF aptamer comprises 2-30 nucleotides.
  • [0578] 137. The anti-VEGF aptamer of any one of embodiments 128-136, wherein said first loop of the anti-VEGF aptamer comprises 3-10 nucleotides.
  • [0579] 138. The anti-VEGF aptamer of any one of embodiments 70-137, wherein said anti-VEGF aptamer comprises a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, or a variant or truncate thereof.
  • [0580] 139. The anti-VEGF aptamer of embodiment 138, wherein said variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as set forth in any of SEQ ID NO: 1-62.
  • [0581] 140. The anti-VEGF aptamer of any one of embodiments 138-139, wherein said variant comprises a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, with one or more nucleotide addition, deletion, and/or substitution.
  • [0582] 141. The anti-VEGF aptamer of any one of embodiments 130-140, wherein said anti-VEGF aptamer comprises said first consensus sequence and said second consensus sequence, and wherein said first consensus sequence is located 5′ to said second consensus sequence.
  • [0583] 142. The anti-VEGF aptamer of any one of embodiments 130-140, wherein said anti-VEGF aptamer comprises said first consensus sequence and said second consensus sequence, wherein said second consensus sequence is located 5′ to said first consensus sequence.
  • [0584] 143. The anti-VEGF aptamer of any one of embodiments 70-142, wherein said anti-VEGF aptamer is conjugated to a polyethylene glycol (PEG) moiety.
  • [0585] 144. The anti-VEGF aptamer of embodiment 143, wherein at least one PEG moiety is conjugated to the 5′ terminus of said anti-VEGF aptamer.
  • [0586] 145. The anti-VEGF aptamer of any one of embodiments 143-144, wherein at least one PEG moiety is conjugated to the 3′ terminus of said anti-VEGF aptamer.
  • [0587] 146. A bispecific aptamer, comprising the anti-Ang2 aptamer of any one of embodiments 1-69, and the anti-VEGF aptamer of any one of embodiments 70-145.
  • [0588] 147. The bispecific aptamer of embodiment 146, which is of the formula A1-(L) n-A2, wherein:
  • [0589] A1 is said anti-VEGF aptamer and A2 is said anti-Ang2 aptamer, or A1 is said anti-Ang2 aptamer and A2 is said anti-VEGF aptamer;
  • [0590] L is a linker; and
  • [0591] n is a number of at least 0.
  • [0592] 148. The bispecific aptamer of any one of embodiments 146-147, which is capable of specifically binding to both VEGF and Ang2.
  • [0593] 149. The bispecific aptamer of any one of embodiments 146-148, which binds to VEGF-121 with a K_D value of about 500 μM or less, and binds to Ang2 with a K_D value of about 500 μM or less.
  • [0594] 150. The bispecific aptamer of any one of embodiments 146-149, which binds to VEGF-121 with a K_D value of about 200 μM or less, and binds to Ang2 with a K_D value of about 200 μM or less.
  • [0595] 151. The bispecific aptamer of any one of embodiments 146-150, which binds to VEGF-121 with a K_D value of about 100 μM or less, and binds to Ang2 with a K_D value of about 100 μM or less.
  • [0596] 152. The bispecific aptamer of any one of embodiments 147-151, wherein n is 0 to 20.
  • [0597] 153. The bispecific aptamer of any one of embodiments 147-152, wherein n is 0 to 10.
  • [0598] 154. The bispecific aptamer of any one of embodiments 147-153, wherein L is selected from a naturally occurring nucleotide linker, a modified nucleotide linker, a hydrocarbon linker, a polyethylene glycol linker and a combination thereof.
  • [0599] 155. The bispecific aptamer of any one of embodiments 147-154, wherein at least one L is a polyethylene glycol linker.
  • [0600] 156. The bispecific aptamer of any one of embodiments 147-155, wherein at least one L is a hexaethylene glycol linker.
  • [0601] 157. The bispecific aptamer of any one of embodiments 147-156, wherein L is a hexaethylene glycol linker, and n is 0 to 10.
  • [0602] 158. The bispecific aptamer of any one of embodiments 147-157, wherein A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in any of SEQ ID NO:1-62, or a variant or truncate thereof; and A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof.
  • [0603] 159. The bispecific aptamer of any one of embodiments 147-158, wherein A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in any of SEQ ID NO: 63-74, or a variant or truncate thereof; and A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in any of SEQ ID NO: 1-62, or a variant or truncate thereof.
  • [0604] 160. The bispecific aptamer of any one of embodiments 147-159, wherein:
  • [0605] 1) A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 45, or a truncate thereof, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10;
  • [0606] 2) A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 55, or a truncate thereof, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10;
  • [0607] 3) A1 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10;
  • [0608] 4) A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 45, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10;
  • [0609] 5) A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 55, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10; or
  • [0610] 6) A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 68, or a truncate thereof, A2 is an anti-VEGF aptamer comprising a nucleotide sequence as set forth in SEQ ID NO: 48, or a truncate thereof, L is a hexaethylene glycol linker, and n is 0 to 10.
  • [0611] 161. The bispecific aptamer of any one of embodiments 146-160, which is selected from r-AMSB101, r-AMSB102, r-AMSB103, r-AMSB104, r-AMSB105, r-AMSB106, r-AMSB103.1, r-AMSB103.2, r-AMSB103.3, r-AMSB103.4, r-AMSB103.5, r-AMSB103.6, AMSB101, AMSB102, AMSB103, AMSB104, AMSB105, AMSB106, AMSB103.1, AMSB103.2, AMSB103.3, AMSB103.4, AMSB103.5, and AMSB103.6.
  • [0612] 162. The bispecific aptamer of any one of embodiments 146-161, which comprises a nucleotide sequence as set forth in any of SEQ ID NO: 75-78 and 138-157.
  • [0613] 163. A composition, comprising the anti-Ang2 aptamer of any one of embodiments 1-69, the anti-VEGF aptamer of any one of embodiments 70-145, and/or the bispecific aptamer of any one of embodiments 146-162.
  • [0614] 164. The composition of embodiment 163, which is a pharmaceutical composition.
  • [0615] 165. The composition of any one of embodiments 163-164, comprising a pharmaceutically acceptable excipient or carrier.
  • [0616] 166. The composition of any one of embodiments 163-165, for preventing, treating and/or ameliorating a neovascular disease, disorder or condition.
  • [0617] 167. The composition of embodiment 166, wherein said neovascular disease, disorder or condition is an ocular neovascular disease, disorder or condition.
  • [0618] 168. The composition of any one of embodiments 166-167, wherein said neovascular disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy.
  • [0619] 169. The composition of any one of embodiments 166-168, wherein said neovascular disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.
  • [0620] 170. A method for preventing, treating and/or ameliorating a neovascular disease, disorder or condition, comprising administering to a subject in need thereof an effective amount of the anti-Ang2 aptamer of any one of embodiments 1-69, an effective amount of the anti-VEGF aptamer of any one of embodiments 70-145, an effective amount of the bispecific aptamer of any one of embodiments 146-162, and/or an effective amount of the composition according to any one of embodiments 163-169.
  • [0621] 171. The method of embodiment 170, wherein said neovascular disease, disorder or condition is an ocular neovascular disease, disorder or condition.
  • [0622] 172. The method of any one of embodiments 170-171, wherein said neovascular disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy.
  • [0623] 173. The method of any one of embodiments 170-172, wherein said neovascular disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.
  • [0624] 174. Use of the anti-Ang2 aptamer of any one of embodiments 1-69, the anti-VEGF aptamer of any one of embodiments 70-145, the bispecific aptamer of any one of embodiments 146-162, and/or the composition of any one of embodiments 163-169 in the manufacture of a medicament for preventing, treating and/or ameliorating a neovascular disease, disorder or condition.
  • [0625] 175. The use of embodiments 174, wherein said neovascular disease, disorder or condition is an ocular neovascular disease, disorder or condition.
  • [0626] 176. The use of any one of embodiments 174-175, wherein said neovascular disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy.
  • [0627] 177. The use of any one of embodiments 174-176, wherein said neovascular disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0628] The present disclosure also relates to the following sequences:

[0629]

SEQ ID NO.	Description	Sequence
1	r-AMS0401	GCUCUUCAGCCCCGCGCCGGGUCCCACUCUGCGCCGCUC
2	r-AMS0402	GUGCUUCAUAUCCGUCCCCCCCUUGCUGCG
3	r-AMS0403	AUUCUGUCUUUUUAAAUAAUGUCCCCUUGUUGCACAUUACAGAA
4	r-AMS0404	UUCUGUCUUUUUAGAUAAUGUCCCCUUGUUGCACAUUACA
5	r-AMS0405	GCUCUUCAAUGUUAACUACCAAUCGUUGAUCGUUUUGCAGAAAAUUCC
6	r-AMS0406	GCUCUUCGGUGUUAACUACCAAUCGUUGAUCGUUUUGCAAAAAAUUCC
7	r-AMS0407	UCCGCUUGCGGAGCAGCUUCUGUAUUCUGC
8	r-AMS0408	UCCGCUUGCGGAGCAUCUUCUGUAUUCUGC
9	r-AMS0409	UCCGCUUGCGGAGCACCUUCUGUAUUCUGC
10	r-AMS0410	GGAAAACAGUAUAAUGUCUUGUUUCUUCUC
11	r-AMS0411	GGUUUAUCUUAACUUUCUGCUACGAUACAGUUUUUGUUGA
12	r-AMS0416	CCAACCGUGUAUCUGUCUUGCUUGCGGCGGUUUUGUUGUG
13	r-AMS0419	AGUUAUUUGGUGGAGAGGAUCCAUUUAGCUUAUACAAUUC
14	r-AMS0420	CCGGCCAUUGGUGGAGAUUAUCCUUUGAGUACGGUUAUUC
15	r-AMS0421	UUUCGCUAGAUGGCUAACACGUAUAUUUGUGGAGAUGCACUAGCG
16	r-AMS0425	CGUGGAGAUGCAUUCUGUCGAGGAUUGGCUAACACGCGAA
17	r-AMS0421.1	UCCUUAGAUGGCUAACACGUAUAUUUGUGGAGAUGCACUA
18	r-AMS0427	CGCUAGAUGGCUAACACUAUUGUGGAGAUGCACUAGCG
19	r-AMS0421.3	GCGUAGAUGGCUAACACUAUUGUGGAGAUGCACUACGC
20	r-AMS0421.4	CGCUAGUGGCGAACACGUAUAUUUGUGGCGAUGCACUAGCG
21	r-AMS0421.5	CGCUAGAUGGCCUAACACGUAUAUUUGUGGAGAUGGCACUAGCG
22	r-AMS0425.1	CGUGGAGAUGCAUUCUGUCGAGGAUUGGCUAACACG
23	r-AMS0426	CGUGGAGAUGCAUUCUGUCGAAGAUUGGCUAACACG
24	r-AMS0425.3	CGUGGAGAUGCAUUCUUCGAGAUUGGCUAACACG
25	r-AMS0425.8	CGUGGAGAUGCGUUCCGUCGAGGAUCGGCUAACACG
26	r-AMS0425.10	CAGGUGGAGAUGCAUUCUGUCGAAGAUUGGCUAACACCUG
27	r-AMS0425.11	GCCGUGGAGAUGCGUUCCGUCGGGAUCGGCUAACACGGC
28	r-AMS0425.12	GCCGUGGAGAUGCGUUCCGUCGAGGAUCGGCUAACACGGC
29	r-AMS0425.16	CGUGGCGAUGCGUUCCGUCGAGGAUCGGCGAACACG
30	r-AMS0430	GCGGAUUGGCUAACACGGUCGACGUGGAGAUGCAUUCCGC
31	AMS0401	fGfCfUfCfUfUfCfAfGfCfCfCfCfGfCfGfCfCfGfGfGfUfCfCfCfAfCfUfCfUfGfCfG
		fCfCfGfCfUfC
32	AMS0402	fGfUfGfCfUfUfCfAfUfAfUfCfCfGfUfCfCfCfCfCfCfCfUfUfGfCfUfGfCfG
33	AMS0403	fAfUfUfCfUfGfUfCfUfUfUfUfUfAfAfAfUfAfAfUfGfUfCfCfCfCfUfUfGfUfUfG
		fCfAfCfAfUfUfAfCfAfGfAfA
34	AMS0404	fUfUfCfUfGfUfCfUfUfUfUfUfAfGfAfUfAfAfUfGfUfCfCfCfCfUfUfGfUfUfGfC
		fAfCfAfUfUfAfCfA
35	AMS0405	fGfCfUfCfUfUfCfAfAfUfGfUfUfAfAfCfUfAfCfCfAfAfUfCfGfUfUfGfAfUfCfGf
		UfUfUfUfGfCfAfGfAfAfAfAfUfUfCfC
36	AMS0406	fGfCfUfCfUfUfCfGfGfUfGfUfUfAfAfCfUfAfCfCfAfAfUfCfGfUfUfGfAfUfCfGf
		UfUfUfUfGfCfAfAfAfAfAfAfUfUfCfC
37	AMS0407	fUfCfCfGfCfUfUfGfCfGfGfAfGfCfAfGfCfUfUfCfUfGfUfAfUfUfCfUfGfC
38	AMS0408	fUfCfCfGfCfUfUfGfCfGfGfAfGfCfAfUfCfUfUfCfUfGfUfAfUfUfCfUfGfC
39	AMS0409	fUfCfCfGfCfUfUfGfCfGfGfAfGfCfAfCfCfUfUfCfUfGfUfAfUfUfCfUfGfC
40	AMS0410	fGfGfAfAfAfAfCfAfGfUfAfUfAfAfUfGfUfCfUfUfGfUfUfUfCfUfUfCfUfC
41	AMS0411	fGfGfUfUfUfAfUfCfUfUfAfAfCfUfUfUfCfUfGfCfUfAfCfGfAfUfAfCfAfGfUfU
		fUfUfUfGfUfUfGfA
42	AMS0416	fCfCfAfAfCfCfGfUfGfUfAfUfCfUfGfUfCfUfUfGfCfUfUfGfCfGfGfCfGfGfUfU
		fUfUfGfUfUfGfUfG
43	AMS0419	fAfGfUfUfAfUfUfUfGfGfUfGfGfAfGfAfGfGfAfUfCfCfAfUfUfUfAfGfCfUfUf
		AfUfAfCfAfAfUfUfC
44	AMS0420	fCfCfGfGfCfCfAfUfUfGfGfUfGfGfAfGfAfUfUfAfUfCfCfUfUfUfGfAfGfUfAfC
		fGfGfUfUfAfUfUfC
45	AMS0421	fUfUfUfCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfGfUfAfUfAfUfUfUfGfUfGfG
		fAfGfAfUfGfCfAfCfUfAfGfCfG
46	AMS0425	fCfGfUfGfGfAfGfAfUfGfCfAfUfUfCfUfGfUfCfGfAfGfGfAfUfUfGfGfCfUfAfA
		fCfAfCfGfCfGfAfA
47	AMS0421.1	fUfCfCfUfUfAfGfAfUfGfGfCfUfAfAfCfAfCfGfUfAfUfAfUfUfUfGfUfGfGfAfG
		fAfUfGfCfAfCfUfA
48	AMS0427	fCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfUfAfUfUfGfUfGfGfAfGfAfUfGfCfAf
		CfUfAfGfCfG
49	AMS0427.1	mCmGmCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfUfAfUfUfGfUfGfGfAfGfAfUfGf
		CfAfCfUfAmGmCmG
50	AMS0427.2	LCLGLCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfUfAfUfUfGfUfGfGfAfGfAfUfGfCf
		AfCfUfALGLCLG
51	AMS0421.3	fGfCfGfUfAfGfAfUfGfGfCfUfAfAfCfAfCfUfAfUfUfGfUfGfGfAfGfAfUfGfCfA
		fCfUfAfCfGfC
52	AMS0421.4	fCfGfCfUfAfGfUfGfGfCfGfAfAfCfAfCfGfUfAfUfAfUfUfUfGfUfGfGfCfGfAfU
		fGfCfAfCfUfAfGfCfG
53	AMS0421.5	fCfGfCfUfAfGfAfUfGfGfCfCfUfAfAfCfAfCfGfUfAfUfAfUfUfUfGfUfGfGfAfG
		fAfUfGfGfCfAfCfUfAfGfCfG
54	AMS0425.1	fCfGfUfGfGfAfGfAfUfGfCfAfUfUfCfUfGfUfCfGfAfGfGfAfUfUfGfGfCfUfAfA
		fCfAfCfG
55	AMS0426	fCfGfUfGfGfAfGfAfUfGfCfAfUfUfCfUfGfUfCfGfAfAfGfAfUfUfGfGfCfUfAfA
		fCfAfCfG
56	AMS0425.3	fCfGfUfGfGfAfGfAfUfGfCfAfUfUfCfUfUfCfGfAfGfAfUfUfGfGfCfUfAfAfCfA
		fCfG
57	AMS0425.8	fCfGfUfGfGfAfGfAfUfGfCfGfUfUfCfCfGfUfCfGfAfGfGfAfUfCfGfGfCfUfAfA
		fCfAfCfG
58	AMS0425.10	fCfAfGfGfUfGfGfAfGfAfUfGfCfAfUfUfCfUfGfUfCfGfAfAfGfAfUfUfGfGfCfU
		fAfAfCfAfCfCfUfG
59	AMS0425.11	fGfCfCfGfUfGfGfAfGfAfUfGfCfGfUfUfCfCfGfUfCfGfGfGfAfUfCfGfGfCfUfA
		fAfCfAfCfGfGfC
60	AMS0425.12	fGfCfCfGfUfGfGfAfGfAfUfGfCfGfUfUfCfCfGfUfCfGfAfGfGfAfUfCfGfGfCfU
		fAfAfCfAfCfGfGfC
61	AMS0430	fGfCfGfGfAfUfUfGfGfCfUfAfAfCfAfCfGfGfUfCfGfAfCfGfUfGfGfAfGfAfUfG
		fCfAfUfUfCfCfGfC
62	AMS0425.16	fCfGfUfGfGfCfGfAfUfGfCfGfUfUfCfCfGfUfCfGfAfGfGfAfUfCfGfGfCfGfAfAf
		CfAfCfG
63	r-AMS0525	GCAUAGAUAAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCACA
		CCGUCGCUUAGU
64	r-AMS0526	CGCAUAGAUAAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCG
65	r-AMS0525.3	GCCUUAGAUAAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCAGGC
66	r-AMS0525.4	CCGAUAAGCCAACAAACGUACAUUCUCUGUUAGUAUUCGG
67	AMS0525	fGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCfAfUfUfCfUfCfUfGf
		UfUfAfGfUfAfUfUfUfUfCfUfGfCfAfCfAfCfCfGfUfCfGfCfUfUfAfGfU
68	AMS0526	fCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCfAfUfUfCfUfCfUf
		GfUfUfAfGfUfAfUfUfUfUfCfUfGfCfG
69	AMS0525.3	fGfCfCfUfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCfAfUfUfCfUfCfUf
		GfUfUfAfGfUfAfUfUfUfUfCfAfGfGfC
70	AMS0525.4	fCfCfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCfAfUfUfCfUfCfUfGfUfUfAf
		GfUfAfUfUfCfGfG
71	AMS0525.5	mCmGmCmAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCfAfUfUfCfUf
		CfUfGfUfUfAfGfUfAfUfUfUfUfCmUmGmCmG
72	AMS0525.6	LCLGLCLAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCfAfUfUfCfUfCfUf
		GfUfUfAfGfUfAfUfUfUfUfCLULGLCLG
73	AMS0525.7	fCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAHfUfUfCfUfCfUfGfUfUfAfGf
		UfAfUfUfUfUfCfUfGfCfG
74	AMS0525.8	fCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCHfAfUfUfCfUfCfUfGfUfUfA
		fGfUfAfUfUfUfUfCfUfGfCfG
75	r-AMSB103	CGCUAGAUGGCUAACACUAUUGUGGAGAUGCACUAGCGCGCAUAGAUAA
		GCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCG
76	r-AMSB106	CGCAUAGAUAAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCGC
		GCUAGAUGGCUAACACUAUUGUGGAGAUGCACUAGCG
77	AMSB103	fCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfUfAfUfUfGfUfGfGfAfGfAfUfGfCfAf
		CfUfAfGfCfGfCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCfAfU
		fUfCfUfCfUfGfUfUfAfGfUfAfUfUfUfUfCfUfGfCfG
78	AMSB106	fCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCfAfUfUfCfUfCfUf
		GfUfUfAfGfUfAfUfUfUfUfCfUfGfCfGfCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAf
		CfUfAfUfUfGfUfGfGfAfGfAfUfGfCfAfCfUfAfGfCfG
79	VEGF 1st	fUfGfGfCfUfAfAfCfAfC
	consensus
	sequence f1
80	VEGF 1st	fUfGfGfCfU
	consensus
	sequence f2
81	VEGF 1st	fUfGfGfCfUfA
	consensus
	sequence f3
82	VEGF 1st	fUfGfGfCfUfAfA
	consensus
	sequence f4
83	VEGF 1st	fUfGfGfCfUfAfAfC
	consensus
	sequence f5
84	VEGF 1st	fUfGfGfCfUfAfAfCfA
	consensus
	sequence f6
85	VEGF 1st	UGGCUAACAC
	consensus
	sequence 1
86	VEGF 1st	UGGCU
	consensus
	sequence 2
87	VEGF 1st	UGGCUA
	consensus
	sequence 3
88	VEGF 1st	UGGCUAA
	consensus
	sequence 4
89	VEGF 1st	UGGCUAAC
	consensus
	sequence 5
90	VEGF 1st	UGGCUAACA
	consensus
	sequence 6
91	VEGF 2nd	fGfUfGfGfAfGfAfUfGfCfA
	consensus
	sequence f1
92	VEGF 2nd	fAfGfAfUfGfCfA
	consensus
	sequence f2
93	VEGF 2nd	fGfAfGfAfUfGfCfA
	consensus
	sequence f3
94	VEGF 2nd	fGfGfAfGfAfUfGfCfA
	consensus
	sequence f4
95	VEGF 2nd	fUfGfGfAfGfAfUfGfCfA
	consensus
	sequence f5
96	VEGF 2nd	GUGGAGAUGCA
	consensus
	sequence 1
97	VEGF 2nd	AGAUGCA
	consensus
	sequence 2
98	VEGF 2nd	GAGAUGCA
	consensus
	sequence 3
99	VEGF 2nd	GGAGAUGCA
	consensus
	sequence 4
100	VEGF 2nd	UGGAGAUGCA
	consensus
	sequence 5
101	Ang2 1st	fAfGfAfUfAfAfGfCfCfAfAfCfA
	consensus
	sequence f1
102	Ang2 1st	fUfAfGfAfUfAfAfGfCfCfAfAfCfA
	consensus
	sequence f2
103	Ang2 1st	fAfUfAfGfAfUfAfAfGfCfCfAfAfCfA
	consensus
	sequence f3
104	Ang2 1st	fCfUfAfGfAfUfAfAfGfCfCfAfAfCfA
	consensus
	sequence f4
105	Ang2 1st	AGAUAAGCCAACA
	consensus
	sequence 1
106	Ang2 1st	UAGAUAAGCCAACA
	consensus
	sequence 2
107	Ang2 1st	AUAGAUAAGCCAACA
	consensus
	sequence 3
108	Ang2 1st	CUAGAUAAGCCAACA
	consensus
	sequence 4
109	Ang2 2nd	fUfGfUfUfAfGfUfAfUfUfUfU
	consensus
	sequence f1
110	Ang2 2nd	fUfGfUfUfAfGfUfAfUfUfUfUfC
	consensus
	sequence f2
111	Ang2 2nd	fUfGfUfUfAfGfUfAfUfUfUfUfCfU
	consensus
	sequence f3
112	Ang2 2nd	fUfGfUfUfAfGfUfAfUfUfUfUfCfG
	consensus
	sequence f4
113	Ang2 2nd	UGUUAGUAUUUU
	consensus
	sequence 1
114	Ang2 2nd	UGUUAGUAUUUUC
	consensus
	sequence 2
115	Ang2 2nd	UGUUAGUAUUUUCU
	consensus
	sequence 3
116	Ang2 2nd	UGUUAGUAUUUUCG
	consensus
	sequence 4
117	human VEGF121	APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPL
		MRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLOHNKCECRP
		KKDRARQEKCDKPRR
118	VEGF Receptor	FMDVYQRSY
	Binding Domain1
119	VEGF Receptor	DIFQEYPDEIEYIFK
	Binding Domain2
120	VEGF Receptor	CNDEGL
	Binding Domain3
121	VEGF Receptor	QIMRIKPHQGQHI
	Binding Domain4
122	VEGF Receptor	KCECRPKK
	Binding Domain5
123	human	APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPL
	VEGF165,Monkey	MRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLOHNKCECRP
	VEGF-165	KKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRC
		DKPRR
124	Rabbit VEGF-189	APMAEEGDNKPHEVVKFMEVYRRSYCQPIETLVDIFQEYPDEIEYIFKPSCVPLVR
		CGGCCNDESLECVPTEEFNVTMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKD
		RARQEKKSVRGKGKGQKRKRKKSRYKSWSVPCGPCSERRKHLFVQDPQTCKCS
		CKNTDSRCKARQLELNERTCRCDKPRR
125	Rabbit VEGF-189	FMEVYRRSY
	receptor binding
	domain 1
126	Rabbit VEGF-189	CNDESL
	receptor binding
	domain 3
127	Rat VEGF 164	APTTEGEQKAHEVVKFMDVYQRSYCRPIETLVDIFQEYPDEIEYIFKPSCVPLMRC
		AGCCNDEALECVPTSESNVTMQIMRIKPHQSQHIGEMSFLQHSRCECRPKKDR
		TKPENHCEPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPR
		R
128	VEGF Receptor	CNDEAL
	Binding Domain3′
129	VEGF Receptor	RCECRPKK
	Binding Domain5′
130	Rat VEGF 120	APTTEGEQKAHEVVKFMDVYQRSYCRPIETLVDIFQEYPDEIEYIFKPSCVPLMRC
		AGCCNDEALECVPTSESNVTMQIMRIKPHQSQHIGEMSFLQHSRCECRPKKDR
		TKPEKCDKPRR
131	Mouse VEGF 164	APTTEGEQKSHEVIKFMDVYQRSYCRPIETLVDIFQEYPDEIEYIFKPSCVPLMRC
		AGCCNDEALECVPTSESNITMQIMRIKPHQSQHIGEMSFLQHSRCECRPKKDRT
		KPENHCEPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR
132	Mouse VEGF 120	APTTEGEQKSHEVIKFMDVYQRSYCRPIETLVDIFQEYPDEIEYIFKPSCVPLMRC
		AGCCNDEALECVPTSESNITMQIMRIKPHQSQHIGEMSFLQHSRCECRPKKDRT
		KPEKCDKPRR
133	Mouse VEGF 120	QIMRIKPHQSQHI
	receptor binding
	domain 4
134	VEGF forward	5′-CGCCCTCGTCCCATCTC-3′
	primer
135	VEGF reverse	5′-CGTTCTCGGTTGGTGTTC-3′
	primer
136	Ang2 forward	5′-ATCCAGAGTGACG-CTCTTCAGCA-3′
	primer
137	Ang2 reverse	5′-TGAAGAGCGTCACTCTGGAT-3′
	primer
138	r-AMSB101	UUUCGCUAGAUGGCUAACACGUAUAUUUGUGGAGAUGCACUAGCGCGC
		AUAGAUAAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCG
139	r-AMSB102	CGUGGAGAUGCAUUCUGUCGAAGAUUGGCUAACACGCGCAUAGAUAAG
		CCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCG
140	r-AMSB104	CGCAUAGAUAAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCGU
		UUCGCUAGAUGGCUAACACGUAUAUUUGUGGAGAUGCACUAGCG
141	r-AMSB105	CGCAUAGAUAAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCGC
		GUGGAGAUGCAUUCUGUCGAAGAUUGGCUAACACG
142	r-AMSB103.1	CGCUAGAUGGCUAACACUAUUGUGGAGAUGCACUAGCGHCGCAUAGAU
		AAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCG
143	r-AMSB103.2	CGCUAGAUGGCUAACACUAUUGUGGAGAUGCACUAGCGHHCGCAUAGA
		UAAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCG
144	r-AMSB103.3	CGCUAGAUGGCUAACACUAUUGUGGAGAUGCACUAGCGHHHCGCAUAG
		AUAAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCG
145	r-AMSB103.4	CGCUAGAUGGCUAACACUAUUGUGGAGAUGCACUAGCGHHHHCGCAUA
		GAUAAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCG
146	r-AMSB103.5	CGCUAGAUGGCUAACACUAUUGUGGAGAUGCACUAGCGHHHHHCGCAU
		AGAUAAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCG
147	r-AMSB103.6	CGCUAGAUGGCUAACACUAUUGUGGAGAUGCACUAGCGHHHHHHCGCA
		UAGAUAAGCCAACAAACGUACAUUCUCUGUUAGUAUUUUCUGCG
148	AMSB101	fUfUfUfCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfGfUfAfUfAfUfUfUfGfUfGfG
		fAfGfAfUfGfCfAfCfUfAfGfCfGfCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAf
		AfCfGfUfAfCfAfUfUfCfUfCfUfGfUfUfAfGfUfAfUfUfUfUfCfUfGfCfG
149	AMSB102	fCfGfUfGfGfAfGfAfUfGfCfAfUfUfCfUfGfUfCfGfAfAfGfAfUfUfGfGfCfUfAfA
		fCfAfCfGfCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCfAfUfUfC
		fUfCfUfGfUfUfAfGfUfAfUfUfUfUfCfUfGfCfG
150	AMSB104	fCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCfAfUfUfCfUfCfUf
		GfUfUfAfGfUfAfUfUfUfUfCfUfGfCfGfUfUfUfCfGfCfUfAfGfAfUfGfGfCfUfA
		fAfCfAfCfGfUfAfUfAfUfUfUfGfUfGfGfAfGfAfUfGfCfAfCfUfAfGfCfG
151	AMSB105	fCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCfAfUfUfCfUfCfUf
		GfUfUfAfGfUfAfUfUfUfUfCfUfGfCfGfCfGfUfGfGfAfGfAfUfGfCfAfUfUfCfU
		fGfUfCfGfAfAfGfAfUfUfGfGfCfUfAfAfCfAfCfG
152	AMSB103.1	fCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfUfAfUfUfGfUfGfGfAfGfAfUfGfCfAf
		CfUfAfGfCfGHfCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCfAf
		UfUfCfUfCfUfGfUfUfAfGfUfAfUfUfUfUfCfUfGfCfG
153	AMSB103.2	fCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfUfAfUfUfGfUfGfGfAfGfAfUfGfCfAf
		CfUfAfGfCfGHHfCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfCf
		AfUfUfCfUfCfUfGfUfUfAfGfUfAfUfUfUfUfCfUfGfCfG
154	AMSB103.3	fCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfUfAfUfUfGfUfGfGfAfGfAfUfGfCfAf
		CfUfAfGfCfGHHHfCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAfC
		fAfUfUfCfUfCfUfGfUfUfAfGfUfAfUfUfUfUfCfUfGfCfG
155	AMSB103.4	fCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfUfAfUfUfGfUfGfGfAfGfAfUfGfCfAf
		CfUfAfGfCfGHHHHfCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUfAf
		CfAfUfUfCfUfCfUfGfUfUfAfGfUfAfUfUfUfUfCfUfGfCfG
156	AMSB103.5	fCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfUfAfUfUfGfUfGfGfAfGfAfUfGfCfAf
		CfUfAfGfCfGHHHHHfCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfUf
		AfCfAfUfUfCfUfCfUfGfUfUfAfGfUfAfUfUfUfUfCfUfGfCfG
157	AMSB103.6	fCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfUfAfUfUfGfUfGfGfAfGfAfUfGfCfAf
		CfUfAfGfCfGHHHHHHfCfGfCfAfUfAfGfAfUfAfAfGfCfCfAfAfCfAfAfAfCfGfU
		fAfCfAfUfUfCfUfCfUfGfUfUfAfGfUfAfUfUfUfUfCfUfGfCfG

[0630] The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt, nucleotide(s); i.m., intramuscular (ly); i.p., intraperitoneal (ly); s.c., subcutaneous (ly); and the like.

[0631] The anti-VEGF aptamers of the present disclosure were identified using Chemically-Augmented Particle Display (CAPD), a platform as described (please refers to Wang et al. WO 2018064086A1; Wang et al. WO2018102115A1; Wang et al. PCT/US2013/071318). Detailed methods and steps were as described in e.g., WO 2018064086A1, which is incorporated herein by reference in its entirety.

[0632] Briefly, recombinant human VEGF-121 and VEGF-165 protein from various vendors (listed in Table 1 were used as the target protein for aptamer discovery. A typical initial selection library contains a 40-50 nucleotide random region in which all nucleotides containing a 2′-fluoro (2′-F) modification. For example, for the selection against human VEGF-121 (R&D systems, 4644-vs-CF), the forward primer used was 5′-CGCCCTCGTCCCATCTC-3′ (SEQ ID NO: 134) and the reverse primer used was 5′-CGTTCTCGGTTGGTGTTC-3′ (SEQ ID NO: 135).

[0633]

TABLE 1
Recombinant VEGFA Proteins for Aptamer Selections
		Catalog
Protein	Vendor	Number
Human VEGF-121	R&D systems	4644-vs-CF
Human VEGF-121	Novoprotein	C699
(C-6His)
Human VEGF-121	ACROBiosystems	VE1-H5246
(N-6His)
Biotinylated Human	ACROBiosystems	VE1-H82E7
VEGF-121 (Avitag ™)
Human VEGF-121	ProSpec	CYT-200
Human VEGF-121	Shenandoah Biotechnology	100-116
Human VEGF-165	R&D systems	293-VE-500
Human VEGF-165	ACROBiosystems	VE5-H5248
(N-6His)
Biotinylated Human	ACROBiosystems	VE5-H82Q0
VEGF-165 (Avitag ™)
Human VEGF-165	Shenandoah Biotechnology	100-44
Mouse VEGF-120	Shenandoah Biotechnology	200-52
Rat VEGF-164	R&D systems	564-RV/CF
Mouse VEGF-164	Shenandoah Biotechnology	200-34

[0634] The selection was started by synthesizing of a library of monoclonal modified aptamer particles (MAPs) each displaying multiple copies of fully 2′-fluoro modified aptamer candidate sequence on its surface. Then the emulsion was broken and the reverse strands were de-hybridized, leaving single strand 2′-fluoro modified aptamers (~90%) and natural DNA template sequences (~10%) on the particles, completing the formation of MAPs, which were now ready for screening. Like the template particles, MAPs are monoclonal wherein each particle displays ~10⁵ copies of a single modified aptamer sequence and ~10⁴ copies of amplifiable natural DNA sequences.

[0635] Then the MAPs were incubated with the one or more of the selection VEGF targets from Table 1 at 37° C. The MAPs were subsequently labeled using fluorescently labeled (Alexa Fluor 488 or 647) anti-VEGF antibody (for non-tagged VEGF), anti-His antibody (for His-tag VEGF), or streptavidin (for biotinylated VEGF). The fluorescence intensity of each MAP reflects the relative affinity of the 2′-fluoro modified aptamer for the VEGF target, which allows quantitatively isolating of aptamers with high affinity to target via a Fluorescence Activated Cell Sorter (FACS). To perform iterative rounds of CAPD to converge the pool on the best binders, PCR was used to amplify the natural DNA encoding strands on the enriched MAPS via high-fidelity polymerase and subsequent rounds of screening or sequencing were performed. Preferential selection of the anti-VEGF aptamers with higher affinity was achieved by shortening the incubation times, decreasing VEGF concentrations, and increasing the wash time in successive rounds. To avoid generating anti-VEGF aptamers that bind to VEGF mainly via the less specific polar interactions, increasing concentrations of polyanion competitors were introduced in successive rounds, such as salmon sperm DNA (Thermo Fisher Scientific, 15632011), herring sperm DNA (Sigma-Aldrich, D3159), and dextran sulfate (Sigma-Aldrich, 31404).

[0636] For ensuring binding specificity to the selection target, VEGF, increasing concentrations of non-target protein such as bovine serum albumin (BSA) or casein or non-target protein mixture such as diluted human serum (0%, 0.1%, 1% and 10%) can be introduced in the selections to ensure that the resulting anti-VEGF aptamers can specifically recognize VEGF with great affinity even when excessive amount (up to 109 fold concentration compared to VEGF) of interfering proteins are present.

[0637] Methods of enriching slow off-rate anti-VEGF aptamers were also incorporated in some selections. To do so, the enriched high-affinity MAPs were incubated with saturating concentration of biotinylated VEGF (ACROBiosystems, VE1-H82E7 or VE5-H82Q0) targets, and subsequently washed with an excess amount of non-labeled VEGF (ACROBiosystems, VE1-H5246 or VE5-H5248) for increasing duration of time (from 1 minute to 24 hours) in successive rounds. The non-labeled VEGF could replace the biotinylated VEGF that dissociated from the MAPs, and would not exhibit any fluorescent signal after the MAPs were labeled with fluorescent streptavidin. MAPs displaying slower off-rate anti-VEGF aptamers can retain more biotinylated VEGF targets after the wash procedure and exhibit higher fluorescence intensities, which allows quantitatively isolate anti-VEGF aptamers for VEGF with slow off-rate via a Fluorescence Activated Cell Sorter (FACS).

[0638] For some selections against VEGF-165, an excess amount of heparin (Sigma-Aldrich H3393) was introduced as competitors in the selections to ensure preferential selection of anti-VEGF aptamers with a higher affinity towards the receptor-binding domain of VEGF-165 and avoid yielding anti-VEGF aptamers that target the heparin-binding domain of VEGF-165. In this way, the resulting anti-VEGF aptamers are likely to recognize both VEGF-165 and VEGF-121.

[0639] To generate anti-VEGF aptamers that can recognize both VEGF-121 and VEGF-165 with similarly high affinity, some selections were done by toggling the protein target between human VEGF-121 and human VEGF-165 during alternating rounds of selection. The “toggle” selection process yielded a family of anti-VEGF aptamers, all of which bound both VEGF-121 and VEGF-165 with high affinity. Species cross-reactivity facilitates the pre-clinical evaluation of potentially therapeutic anti-VEGF aptamers in animal models. In order to generate aptamers that bind both human VEGF and rat/mouse VEGF, some selections were also done by toggling the protein target between human VEGF-121 and mouse VEGF-120 (Shenandoah Biotechnology, 200-52) or rat VEGF 164 (R&D Systems, 564-RV/CF) during alternating rounds of selection.

[0640] Truncated and modified variants of the anti-VEGF aptamers of the present disclosure were prepared by LGC Biosearch Technologies (Novato, CA), and their binding affinity to corresponding targets were examined. Briefly, the equilibrium binding constants of the enriched libraries were measured using magnetic bead partitioning. Biotinylated anti-VEGF aptamers were conjugated to the 1 μm streptavidin-coated MyOne Streptavidin C1 magnetic beads (Thermo Fisher Scientific, 65002). Binding assays were performed by incubating fixed concentration of anti-VEGF aptamer (0.01 nM) and target protein at concentrations ranging from 10⁻¹² to 10⁻⁸ M in PBSMCT buffer (DPBS, pH 7.2 with 2.5 mM MgCl₂, 1 mM CaCl₂) and 0.01% TWEEN-20) at 37° C. for 90 minutes. Bound target protein was partitioned by magnetic separation and subsequently labeled via fluorescent anti-VEGF or anti-His-tag antibody. The fraction of anti-VEGF aptamer bound was quantified by flow cytometry (BD Accuri C6 Plus). Raw binding data were corrected for nonspecific background binding of target protein to empty magnetic beads. The fraction of bound anti-VEGF aptamer was plotted as a function of protein concentration and a non-linear curve-fitting algorithm (Langmuir one-site binding model) was used to extract equilibrium binding constants (K_D values) from the data (FIG. 1).

[0641] Truncated variants of representative anti-aptamer sequences from selections for recombinant human VEGF-165 are shown in Table 2, with their K_D values for VEGF-121 and VEGF-165 indicated. Most of the anti-VEGF aptamers shown in Table 2 exhibit great affinity for VEGF-165, however, they fail to recognize VEGF-121 with decent affinity. These results demonstrated that the resulting anti-aptamers generated using VEGF-165 as targets (similar to Macugen®) most likely bind to the heparin-binding domain of VEGF-165, thus failed to recognize the VEGF-121 that lacks the heparin-binding domain.

[0642]

TABLE 2
Truncations of Aptamers Selected for Human VEGF-165
	Aptamer ID	KD (nM) for VEGF-121	KD (nM) for VEGF-165
	AMS0401	>100	0.087
	AMS0402	>100	0.098
	AMS0403	>100	0.111
	AMS0404	>100	0.419
	AMS0405	>100	1.012
	AMS0406	>100	0.324

[0643] Truncated variants of representative anti-VEGF aptamer sequences from selections for recombinant human VEGF-121 are shown in Table 3 and Table 4, with their K_D values for VEGF-121 and VEGF-165. The aptamers shown in Table 3 exhibit high affinity for VEGF-121; however, they exhibit inferior affinity for VEGF-165 (K_D>10 nM). These results suggest that the anti-VEGF aptamers in Table 3 do not bind to the VEGF receptor-binding domain on the VEGF-121, because VEGF-121 and VEGF-165 share the same VEGF receptor-binding domain. The truncated aptamer sequences listed in Table 4 were generated using the advanced selection methods as described above, such as including polyanion and/or protein competitors, improving off-rate, toggling between VEGF-121 and VEGF-165, and the resulting anti-VEGF aptamers can recognize both VEGF-121 and VEGF-165 with high affinity.

[0644]

TABLE 3
Aptamers for Human VEGF-121
	Aptamer ID	KD (nM) for VEGF-121	KD (nM) for VEGF-165
	AMS0407	0.761	>100
	AMS0408	10.1	>100
	AMS0409	14.2	>100
	AMS0410	11.8	>100
	AMS0411	1.19	>100

[0645]

TABLE 4
Aptamers for Both Human VEGF-121 & Human VEGF-165
	Aptamer ID	KD (nM) for VEGF-121	KD (nM) for VEGF-165
	AMS0416	0.654	29.4
	AMS0419	0.095	0.99
	AMS0420	0.082	0.87
	AMS0421	0.045	0.096
	AMS0425	0.092	0.102

[0646] Various mutations and truncated variants of two high-affinity anti-VEGF aptamers (AMS0421 and AMS0425) and their K_D values for VEGF-121 and VEGF-165 were shown in Table 5 and Table 6, respectively. AMS0421 has been truncated to a 38-mer (AMS0427) in a series of mutation and deletion experiments (Table 5), and AMS0425 has been truncated to a 36-mer (AMS0426) in a series of mutation and deletion experiments (Table 6).

[0647]

TABLE 5
Variants of anti-VEGF aptamer AMS0421
	Aptamer ID	KD (nM) for VEGF-121	KD (nM) for VEGF-165
	AMS0421	0.045	0.096
	AMS0421.1	0.049	0.101
	AMS0427	0.045	0.091
	AMS0421.3	0.051	0.098
	AMS0421.4	0.471	1.92
	AMS0421.5	0.511	0.879
	AMS0427.1	0.047	0.092
	AMS0427.2	0.043	0.087

[0648]

TABLE 6
Variants of anti-VEGF aptamer AMS0425
	Aptamer ID	KD (nM) for VEGF-121	KD (nM) for VEGF-165
	AMS0425	0.092	0.102
	AMS0425.1	0.294	0.356
	AMS0426	0.214	0.349
	AMS0425.3	0.245	0.981
	AMS0425.8	0.287	0.876
	AMS0425.10	0.107	0.652
	AMS0425.11	0.124	0.349
	AMS0425.12	0.126	0.678
	AMS0430	0.104	0.176
	AMS0425.16	>100	>100

[0649] FIG. 1A is a determination of K_D values to human VEGF-121 for two representative fully modified anti-VEGF aptamers of the present disclosure, Macugen®, and Lucentis®; FIG. 1B is a determination of K_D values to human VEGF-165 for two representative fully modified anti-VEGF aptamers of the present disclosure, Macugen®, and Lucentis®. FIG. 1C is a determination of K_D values to various VEGFA isoforms for AMS0427, a 38-nucleotide fully 2′-fluoro modified anti-VEGF aptamer of the present disclosure.

[0650] As can been seen from FIG. 1, compared to the only FDA-approved aptamer-based anti-VEGF nucleic acid therapeutic (Macugen®) that binds only to VEGF-165, both AMS0426 and AMS0427 bind to human VEGF-121 and human VEGF-165 with comparable and superior affinities of less than 150 pM (FIG. 1A and FIG. 1B). Compared to FDA-approved protein-based anti-VEGF therapeutic (Lucentis®) that binds to both VEGF-121 and VEGF-165 with similar affinities, both AMS0426 and AMS0427 showed more than 10-fold improvement of binding affinity. The truncated variant that encompasses the shortest sequence with the highest affinity binding, the 38-mer AMS0427, binds to human VEGF-121 (from five different vendors), human VEGF-165 (from three different vendors), mouse VEGF 120 and rat VEGF-164 with comparable affinities, ranging from 0.01-0.15 nM (FIG. 1C).

[0651] To demonstrate that the anti-VEGF aptamers of the present disclosure are potent inhibitors of vascular endothelial growth factor-A (VEGF) signaling, in vitro competition assays were performed to verify that selected anti-VEGF aptamers can block the interaction between VEGFA and VEGF-R (such as VEGF-R1 and VEGF-R2). Three different recombinant VEGF receptors (two VEGF-R1 and one VEGF-R2) were tested (R&D Systems, 3516-FL, 321-FL, and 357-KD). For determination of the receptor competition IC₅₀, 0.1 nM of VEGF-121 or VEGF-165 with His tag was conjugated to Dynabeads His-Tag Isolation and Pulldown beads (Thermo Fisher Scientific, 10104D), and then incubated with 1 nM of VEGF receptor and aptamers at concentrations ranging from 10⁻¹² to 10⁻⁸ M in PBSMCT buffer (DPBS, pH 7.2 with 2.5 mM MgCl₂, 1 mM CaCl₂) and 0.01% TWEEN-20) at 37° C. for 90 minutes. VEGF-bound receptors and anti-VEGF aptamers were partitioned by magnetic separation, and the VEGF-bound VEGF receptors were subsequently labeled via a fluorescent anti-human antibody. The fraction of VEGF-bound receptor was quantified by flow cytometry (BD Accuri C6 Plus). Raw binding data were corrected for nonspecific background binding and normalized to the fluorescence intensity from the sample with 1 nM VEGF receptor but without anti-VEGF aptamer competition. The fraction of bound receptor was plotted as a function of anti-VEGF aptamer concentration and a non-linear curve-fitting algorithm (dose-response inhibition) was used to extract IC₅₀ values from the data (FIG. 2).

[0652] FIG. 2A demonstrates a determination of IC₅₀ values to human VEGF-121 and human VEGF Receptor 1 for two representative fully modified anti-VEGF aptamers AMS0427 and AMS0426, a non-binding fully modified anti-VEGF aptamer AMS0421.12, and Lucentis®; FIG. 2B demonstrates a determination of IC₅₀ values to human VEGF-165 and human VEGF Receptor 1 for two representative fully modified anti-VEGF aptamers AMS0427 and AMS0426, a non-binding fully modified anti-VEGF aptamer AMS0421.12, and Lucentis®; FIG. 2C demonstrates a determination of IC₅₀ values to human VEGF-121 and human VEGF Receptor 2 for two representative fully modified anti-VEGF aptamers AMS0427 and AMS0426, a non-binding fully modified anti-VEGF aptamer AMS0421.12, and Lucentis®; FIG. 2D demonstrates a determination of IC₅₀ values to human VEGF-165 and human VEGF Receptor 2 for two representative fully modified anti-VEGF aptamers AMS0427 and AMS0426, a non-binding fully modified anti-VEGF aptamer AMS0421.12, and Lucentis®.

[0653] It can be seen that AMS0427 and AMS0426 potently inhibited the interaction between VEGF-121 and VEGF-R1, VEGF-165 and VEGF-R1, VEGF-121 and VEGF-R2, and VEGF-165 and VEGF-R2 (IC₅₀ values around 100 μM), indicating that the anti-VEGF aptamers of the present disclosure bind to and block the receptor-binding domain on both VEGF isoforms.

[0654] 2-dimensional structures analysis for the selected anti-VEGF aptamers was performed using the NUPACK software suite. The results are illustrated in FIG. 3. FIG. 3A demonstrates the secondary structural predictions of AMS0421 and AMS0427. Conserved nucleotide sequences were highlighted by the dashed box; FIG. 3B demonstrates the secondary structural predictions of AMS0426, AMS0427 and AMS0430. Conserved nucleotide sequences were highlighted by the dashed box.

[0655] From the various mutations and truncated variants of AMS0421 and their K_D values for VEGF-121 and VEGF-165 (shown in Table 5), it is demonstrated that the sequences highlighted in the dashed box containing 3 bulge areas (i.e., first bulge, second bulge and third bulge) are highly conserved (FIG. 3A).

[0656] The nucleotide composition of the top loop region (i.e., first loop) is not essential and can tolerate modifications or be replaced by a hexa-ethylene glycol (Heg) linker. A similar analysis was also performed for the various mutations and truncated variants of AMS0426. Although the sequence AMS0426 and AMS0427 appear quite different, their predicted structures are very similar. The sequences indicated in the dashed box are highly conserved in both AMS0426 and AMS0427, AMS0426 contains the identical bulge area (the first bulge and the second bulge) as the ones in AMS0427 (FIG. 3B), but the position of the non-essential top loop and bottom stem regions are the opposite for AMS0426 and AMS0427. To validate this finding, AMS0426 to AMS0430 were mutated by switching their top loop (i.e., first loop) and bottom stem (i.e., first stem) regions, making it more similar to AMS0427, and confirmed the performance of AMS0426 and AMS0430 are unchanged (FIG. 3B). It is demonstrated the conserved nucleotide sequence (i.e., consensus sequence 1 and consensus sequence 2) in the selected anti-VEGF aptamers are the ones shown in the dashed box in FIG. 3, and these conserved nucleotide sequences were underlined in Table 7.

[0657]

TABLE 7
Conserved Sequence of anti-VEGF aptamers
		KD (nM) for	KD (nM) for
Aptamer ID	Sequence (5′ → 3′)	VEGF-121	VEGF-165
AMS0421	fUfUfUfCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfG	0.045	0.096
	fUfAfUfAfUfUfUfGfUfGfGfAfGfAfUfGfCfAfCfUf
	AfGfCfG (SEQ ID NO: 45)
AMS0427	fCfGfCfUfAfGfAfUfGfGfCfUfAfAfCfAfCfUfAfUfU	0.045	0.091
	fGfUfGfGfAfGfAfUfGfCfAfCfUfAfGfCfG (SEQ ID
	NO: 48)
AMS0425	fCfGfUfGfGfAfGfAfUfGfCfAfUfUfCfUfGfUfCfGf	0.092	0.102
	AfGfGfAfUfUfGfGfCfUfAfAfCfAfCfGfCfGfAfA
	(SEQ ID NO: 46)
AMS0426	fCfGfUfGfGfAfGfAfUfGfCfAfUfUfCfUfGfUfCfGf	0.086	0.151
	AfAfGfAfUfUfGfGfCfUfAfAfCfAfCfG (SEQ ID
	NO: 55)
AMS0430	fGfCfGfGfAfUfUfGfGfCfUfAfAfCfAfCfGfGfUfCfG	0.104	0.176
	fAfCfGfUfGfGfAfGfAfUfGfCfAfUfUfCfCfGfC
	(SEQ ID NO: 61)

[0658] The study was performed by largely following previously established procedure. 6-8 weeks old Male Brown Norway (BN) rats were used for the study. Rats were anesthetized with sodium pentobarbital (30 mg/kg, injected via tail vein), pupils were dilated, and CNV was induced in the rats by laser rupture of the Bruch's membrane (6 burns/eye. Laser setting: 150 mW, 100 μm diameter, 100 ms duration). Only the laser spots where a bubble was formed without hemorrhage were considered effective CNV lesions and included in the study. The following agents were then administered via intravitreal injection on the same day (D1): Group 1:5 μl of drug vehicle as the negative control; Group 2:5 μl of 40 mg/ml Aflibercept; Group 3:5 μl of 40 mg/ml AMS0427. Fluorescein angiography (FA) was performed to visualize the CNV lesions: 10% sodium fluorescein was injected at 0.5 ml/kg of body weight; following the injection, a rapid series of red-free images were taken in the first minute (early-phase images) and after 5 minutes (late-phase images). The leakage severity of each lesion was scored based on the late-phase images according to a scale of grade 0-3 (grade 3 is the most severe, representing clinically meaningful CNV). The percentage of lesions with grade 3 leakage and the mean leakage score was then calculated for each group.

[0659] The previous examples have proven high in vitro potency of the anti-VEGF aptamers against both VEGF-165 and VEGF-121, and suggested that they may work as potent therapeutic agents for retinal diseases such as wet AMD, DME, and DR. In this example, the anti-VEGF aptamer of the present disclosure has been tested in a laser-induced CNV model, which is a commonly used animal model for wet AMD. It is also worth noting that previously Macugen® has been tested in this model (please refer to Mark H et al., Comparing Pegaptanib and Triamcinolone Efficacy in the Rat Choroidal Neovascularization Model, (REPRINTED) ARCH OPHTHALMOL/VOL 126 (NO. 7), July 2008), and reported negligible benefits compared to the drug vehicle control (PBS solution).

[0660] The results are shown in FIG. 8. These results demonstrated that comparing to the negative control, the anti-VEGF aptamer of the present disclosure significantly reduced the number of grade 3 lesions as well as the mean leakage score to a comparable extent as Aflibercept. This proves the in vivo efficacy of the anti-VEGF aptamer.

[0661] The selection method is similar to that of Example 1.

[0662] Briefly, recombinant human Angiopoietin-2 protein (R&D Systems, 623-AN) was used as the target protein for anti-Ang2 aptamer discovery. A typical initial selection library contains a 40 nucleotide random region in which all nucleotides containing a 2′-fluoro (2′-F) modification. The forward primer was 5′-ATCCAGAGTGACG-CTCTTCAGCA-3′ (SEQ ID NO: 136) and the reverse primer was 5′-TGAAGAGCGTCACTCTGGAT-3′ (SEQ ID NO: 137). The following example describes the selection and production process of an aptamer that specifically binds recombinant human Ang2.

[0663] Each selection was started by synthesizing of a library of monoclonal modified aptamer particles (MAPs) each displaying multiple copies of fully 2′-fluoro modified aptamer candidate sequence on its surface. Then the MAPs were incubated with the Ang2 targets at 37° C. The MAPs were subsequently labeled using fluorescently labeled (Alexa Fluor 488 or 647) anti-Ang2 antibody or anti-His antibody (for His-tag Ang2), or streptavidin (for biotinylated Ang2). The fluorescence intensity of each MAP reflects the relative affinity of the 2′-fluoro modified aptamer for the Ang2 target, which allows quantitatively isolating aptamers with high affinity to target via a Fluorescence Activated Cell Sorter (FACS). To perform iterative rounds of CAPD to converge the pool on the best binders, PCR was conducted to amplify the natural DNA encoding strands on the enriched MAPs via high-fidelity polymerase and perform subsequent rounds of screening or sequencing. Preferential selection of anti-Ang2 aptamers with higher affinity was achieved by shortening the incubation times, decreasing Ang2 concentrations, and increasing the wash time in successive rounds. To avoid generating anti-Ang2 aptamers that bind to Ang2 mainly via the less specific polar interactions, increasing concentrations of polyanion competitors were introduced in successive rounds, such as salmon sperm DNA (Thermo Fisher Scientific, 15632011), herring sperm DNA (Sigma-Aldrich, D3159), and dextran sulfate (Sigma-Aldrich, 31404).

[0664] During some selections, advanced techniques were adopted to ensure the exquisite binding specificity requirement for the anti-Ang2 aptamer—an ideal anti-Ang2 aptamer should not bind to its homolog, Ang1, which plays the opposite role of Ang2. For example, increasing concentrations of non-target protein such as bovine serum albumin (BSA) or casein or Ang1 (R&D Systems, 923-AN) or non-target protein mixture such as diluted human serum (0%, 0.1%, 1% and 10%) can be introduced in the selections to ensure that the resulting anti-Ang2 aptamers can specifically recognize Ang2 with great affinity even when excessive amount (up to 109-fold concentration compared to Ang2) of interfering proteins are present.

[0665] Methods of enriching slow off-rate anti-Ang2 aptamers were also incorporated in some selections. To do so, the enriched high-affinity MAPs were incubated with saturating concentration of biotinylated Ang2 targets, and subsequently wash with excessive amount of non-labeled Ang2 for increasing duration of time (from 1 minute to 24 hours) in successive rounds. The non-labeled Ang2 could replace the biotinylated Ang2 that dissociated from the MAPs, and would not exhibit any fluorescent signal after the MAPs were labeled with fluorescent streptavidin. MAPs displaying slower off-rate aptamers can retain more biotinylated Ang2 targets after the wash procedure and exhibit higher fluorescence intensities, which lets us quantitatively isolate anti-Ang2 aptamers for Ang2 with slow off-rate via a Fluorescence Activated Cell Sorter (FACS).

[0666] Species cross-reactivity facilitates the pre-clinical evaluation of potentially therapeutic anti-Ang2 aptamers in animal models. In order to generate anti-Ang2 aptamers that bind both human Ang2 and mouse Ang2, some selections were also done by toggling the protein target between human Ang2 and mouse Ang2 (R&D Systems, 7186-AN) during alternating rounds of selection. The toggle selection process yielded a family of anti-Ang2 aptamers, all of which bound human and mouse Ang2 with high affinity.

[0667] Various mutations and truncated variants of a representative anti-Ang2 aptamer (AMS0525) were obtained and their binding affinity to Ang2 were measured using magnetic bead partitioning. Biotinylated anti-Ang2 aptamers were conjugated to the 1 μm streptavidin-coated MyOne Streptavidin C1 magnetic beads (Thermo Fisher Scientific, 65002). Binding assays were performed by incubating fixed concentration of aptamer (0.01 nM) and Ang2 protein at concentrations ranging from 10⁻¹² to 10⁻⁸ M in PBSMCT buffer (DPBS, pH 7.2 with 2.5 mM MgCl₂, 1 mM CaCl₂) and 0.01% TWEEN-20) at 37° C. for 90 minutes. Bound target protein was partitioned by magnetic separation and subsequently labeled via fluorescent anti-His-tag antibody. The fraction of anti-Ang2 aptamer bound was quantified by flow cytometry (BD Accuri C6 Plus). Raw binding data were corrected for nonspecific background binding of target protein to empty magnetic beads. The fraction of bound anti-Ang2 aptamer was plotted as a function of protein concentration and a non-linear curve-fitting algorithm (Langmuir one-site binding model) was used to extract equilibrium binding constants (K_D values) from the data (FIG. 4A).

[0668] The obtained K_D values for Ang2 were shown in Table 8. AMS0525 has been truncated to a 48-mer (AMS0526) in a series of mutation and deletion experiments. Compared to previously reported partially-modified anti-Ang2 aptamer that binds to human Ang2 with K_D of 3.1 nM, AMS0526 is a fully modified aptamer that binds to both human and mouse Ang2 with superior affinities (K_D<100 pM), and does not recognize human Ang1 (FIG. 4A).

[0669]

TABLE 8
Variants of anti-Ang2 aptamer AMS0525
	Aptamer ID	KD (nM) for Ang2
	AMS0525	0.404
	AMS0526	0.032
	AMS0525.3	0.033
	AMS0525.4	>1
	AMS0525.5	0.043
	AMS0525.6	0.029
	AMS0525.7	0.093
	AMS0525.8	0.085

[0670] Interestingly, it was noted that replacing the 2′F modified nucleotides to corresponding nucleotides modified with 2′OMe or LNA did not significantly affect the binding of the resulted anti-Ang2 aptamers to the target Ang2 (as demonstrated by the K_D values of AMS0525.5 and AMS0525.6).

[0671] In addition, 2-dimensional structure analysis for the anti-Ang2 aptamer AMS0526 was performed using the NUPACK software suite, and the predicted structure is shown in FIG. 4B.

[0672] To demonstrate that the anti-Ang2 aptamer of the present disclosure are potent inhibitors of Ang2 signaling, in vitro competition assays was performed to verify that selected anti-Ang2aptamers can block the interaction between Ang2 and Tie-2. For determination of the receptor competition IC₅₀, 0.01 nM of Ang2 with His tag was conjugated to Dynabeads His-Tag Isolation and Pulldown beads (Thermo Fisher Scientific, 10104D), and then incubated with 1 nM of Tie2 (R&D Systems, 313-TI) and anti-Ang2 aptamers at concentrations ranging from 10⁻¹² to 10⁻⁸ M in PBSMCT buffer (DPBS, pH 7.2 with 2.5 mM MgCl₂, 1 mM CaCl₂) and 0.01% TWEEN-20) at 37° C. for 90 minutes. Ang2-bound Tie2 and anti-Ang2 aptamers were partitioned by magnetic separation, and the Ang2-bound Tie2 were subsequently labeled via a fluorescent anti-human antibody. The fraction of Ang2-bound receptor was quantified by flow cytometry (BD Accuri C6 Plus). Raw binding data were corrected for nonspecific background binding and normalized to the fluorescence intensity from the sample with 1 nM Tie2 but without anti-Ang2 aptamer competition. The fraction of bound receptor was plotted as a function of anti-Ang2 aptamer concentration and a non-linear curve-fitting algorithm (dose-response inhibition) was used to extract IC₅₀ values from the data (FIG. 4C). It can be seen that AMS0526 could potently inhibit the interaction between Ang2 and Tie2 with IC₅₀ of 83.9 μM, which is 30-fold better compared to the previously reported anti-Ang2 aptamer (FIG. 4C).

[0673] The study was performed by largely following previously established procedure to test the effect on both angiogenesis and vascular permeability. OIR was induced by placing litters of C57BL/6 mice in 75% O₂ on postnatal day 7 (P7). On P12, the mice were returned to room air. To test the effect on angiogenesis, the mice received intravitreal injections on the same day with the following agents: Group 1 (G1): PBS (negative control); G2: 20 μg of Aflibercept; G3: 20 μg of AMS0525; G4: 20 μg of Aflibercept+20 μg of AMS0525. On P17, the mice were sacrificed, fixed in 10% phosphate-buffered formalin for 4 hours at 22° C., the eyes of the mice were dissected and retina flat mounts were prepared. The retina was then stained with FITC-labeled Griffonia simplicifolia (GSA) lectin (Vector Laboratories) for 45 min to visualize NV. Digital photographs were obtained with a Zeiss fluorescent microscope at 5× magnification, and images were merged into a single image to show the entire retina using photomerge option of Photoshop CS5.4. The area of retinal NV was measured by Image-Pro Plus (Media Cybernetics) by a separate researcher blinded with respect to sample identity. To test the effect on vascular permeability, OIR was induced via the same procedure as described above, but mice received the injection on P16-when the neovascularization (NV) was already developed. This way, only the drug effect on vascular permeability was tested, not NV growth. On P17, vitreous samples from the mice was obtained and the amount of albumin leaked from the blood vessels was measured by ELISA.

[0674] The results are shown in FIG. 11. While inhibiting Ang2 alone has very modest effect (consistent with previous studies), combining the anti-Ang2 aptamer of the present disclosure with Aflibercept almost completely eliminated NV, resulting in significantly better efficacy than Aflibercept alone (FIG. 11B). As shown in FIG. 11C, despite the established NV, the combination of the anti-Ang2 aptamer of the present disclosure and Aflibercept reduced leakage to a superior extent over Aflibercept alone.

[0675] With the aim of developing bispecific aptamer constructs with specificity for VEGF and Ang2, a variety of aptamer constructs comprising an anti-VEGF aptamer linked to an anti-Ang2 aptamer were designed and tested. The bispecific aptamer constructs were tested combining one of three anti-VEGF aptamers (AMS0421, AMS0426, or AMS0427) and an anti-Ang2 aptamer AMS0526. Bispecific aptamer constructs were synthesized head-to-tail, connected directly without any linker, in both orientations (either with the anti-VEGF aptamer at the 5′ end or anti-Ang2 aptamer at the 5′ end). The results are shown in Table 9.

[0676]

TABLE 9
VEGF/Ang2 Bispecific Aptamers
		KD	KD
		(nM)	(nM)
Aptamer	Sequence (5’->3’)	for	for
ID	Aptamer 1	Aptamer 2	VEGF-121	Ang2
AMSB101	anti-VEGF aptamer	anti-Ang2 aptamer	0.125	0.138
	AMS0421	AMS0526
AMSB102	anti-VEGF aptamer	anti-Ang2 aptamer	0.092	0.056
	AMS0426	AMS0526
AMSB103	anti-VEGF aptamer	anti-Ang2 aptamer	0.039	0.029
	AMS0427	AMS0526
AMSB104	anti-Ang2 aptamer	anti-VEGF aptamer	0.087	0.159
	AMS0526	AMS0421
AMSB105	anti-Ang2 aptamer	anti-VEGF aptamer	0.123	0.052
	AMS0526	AMS0426
AMSB106	anti-Ang2 aptamer	anti-VEGF aptamer	0.043	0.031
	AMS0526	AMS0427

[0677] Based on binding affinity for VEGF-121 and Ang2, bispecific aptamer AMSB103 and AMSB106 appeared to give the best results in this experiment. Both of these bispecific aptamers (AMSB103 and AMSB106) were synthesized by combining anti-VEGF aptamer AMS0427 and anti-Ang2 aptamer AMS0526. Both AMSB103 and AMSB106 showed binding affinity for VEGF-121 and Ang2 that was equivalent to or better than the binding affinity of its precursor aptamers (Table 9). The non-essential bottom stem region of both anti-VEGF aptamer AMS0427 and anti-Ang2 aptamer AMS0526 were carefully selected (CGC for both aptamers) in order to minimize the chance of misfolding and interference between the two individual aptamers. As shown in the predicted secondary structures of AMSB103 and AMSB106 (FIG. 5), the structures of both the anti-VEGF aptamer AMS0427 and the anti-Ang2 aptamer AMS0526 were well retained in the both of the bispecific constructs.

[0678] Another set of bispecific aptamers comprising aptamers anti-VEGF aptamer AMS0427 and anti-Ang2 aptamer AMS0526 (with the anti-VEGF aptamer at the 5′ end) was tested to determine the effect of overall linker length. Aptamer constructs were synthesized head-to-tail, connected by zero (AMSB103) to six hexa-ethyleneglycol (Heg) linkers (AMSB103.1-AMSB103.6). The results are shown in Table 10. All of the bispecific aptamer constructs performed well, demonstrating that the Heg linker lengths have little impacts on the binding affinity of AMSB103 for both VEGF and Ang2. AMSB103 without Heg linker was used for future experiments.

[0679]

TABLE 10
VEGF/Ang2 Bispecific Aptamers AMSB103 with Various Linkers
	Sequence (5’->3’)	KD (nM) for	KD (nM)
Aptamer ID	Aptamer 1	Linker	Aptamer 2	VEGF-121	for Ang2
AMSB103	VEGF AMS0427	N/A	Ang2 AMS0526	0.039	0.029
AMSB103.1	VEGF AMS0427	H	Ang2 AMS0526	0.041	0.026
AMSB103.2	VEGF AMS0427	HH	Ang2 AMS0526	0.038	0.034
AMSB103.3	VEGF AMS0427	HHH	Ang2 AMS0526	0.036	0.031
AMSB103.4	VEGF AMS0427	HHHH	Ang2 AMS0526	0.041	0.032
AMSB103.5	VEGF AMS0427	HHHHH	Ang2 AMS0526	0.038	0.027
AMSB103.6	VEGF AMS0427	HHHHHH	Ang2 AMS0526	0.039	0.034

[0680] “H” denotes hexa-ethyleneglycol (Heg) linker.

[0681] The bispecific aptamer AMSB103 was also tested in the VEGF and Ang2 functional receptor competition assays. Based on the functional competition assay data, AMSB103 inhibited both the VEGF/VEGFR2 and the Ang2/Tie2 interactions with IC₅₀ values of <100 μM (FIGS. 6A and 6B).

[0682] To demonstrate the ability of the bispecific aptamers of the present disclosure to bind VEGF and Ang2 simultaneously, a modified magnetic bead based binding affinity assay was developed. Briefly, 5′-biotinylated bispecific aptamer AMSB103 was synthesized by LGC Biosearch Technologies (Novato, CA) and conjugated to the 2.8 μm streptavidin-coated M-270 Streptavidin magnetic beads (Thermo Fisher Scientific, 65305). Binding affinity of AMSB103 to VEGF-121 and VEGF-165 were performed by first saturating fixed concentration of aptamer (0.01 nM) with excessive amount of Ang2 (1 μM) in PBSMCT buffer (DPBS, pH 7.2 with 2.5 mM MgCl₂, 1 mM CaCl₂) and 0.01% TWEEN-20) at 37° C. for 30 minutes. VEGF concentrations ranging from 10⁻¹² to 10⁻⁸ M in PBSMCT buffer were then added to the bispecific aptamer & Ang2 mixture and incubated at 37° C. for 90 minutes. Bound VEGF and Ang2 protein were partitioned by magnetic separation and subsequently labeled via fluorescent anti-VEGF and anti-Ang2 antibody (different fluorescent label for each antibody). The bispecific aptamer-bound Ang2 and VEGF was quantified by flow cytometry (BD Accuri C6 Plus). Raw binding data were corrected for nonspecific background binding of target protein to empty magnetic beads. The fraction of VEGF-bound bispecific aptamer was plotted as a function of VEGF concentration and a non-linear curve-fitting algorithm (Langmuir one-site binding model) was used to extract equilibrium binding constants (K_D values) of AMSB103 for VEGF proteins from the data (FIGS. 7A and 7B).

[0683] Interestingly, the presence of high concentration of Ang2 did not affect AMSB103's binding to VEGF-165.

[0684] Binding affinity of AMSB103 to Ang2 was measured using a similar assay, i.e., by first saturating fixed concentration of the bispecific aptamer (0.01 nM) coated M-270 Streptavidin magnetic beads with an excess amount of VEGF-165 (1 μM) in PBSMCT buffer at 37° C. for 30 minutes. Ang2 concentrations ranging from 10⁻¹² to 10⁻⁸ M in PBSMCT buffer were then added to the bispecific aptamer-VEGF mixture and incubated at 37° C. for 90 minutes. Bound VEGF and Ang2 protein was partitioned by magnetic separation and subsequently labeled via fluorescent anti-VEGF and anti-Ang2 antibody (different fluorescent label for each antibody). The aptamer-bound VEGF and Ang2 was quantified by flow cytometry (BD Accuri C6 Plus). Raw binding data were corrected for nonspecific background binding of target protein to empty magnetic beads. The fraction of Ang2-bound specific aptamer was plotted as a function of Ang2 concentration and a non-linear curve-fitting algorithm (Langmuir one-site binding model) was used to extract equilibrium binding constants (K_D values) of AMSB103 for Ang2 from the data (FIG. 7C).

[0685] The presence of high concentration of VEGF-121 does not affect AMSB103's binding to Ang2.

[0686] AMSB103 showed <100 pM affinity for VEGF-121, VEGF-165, and Ang2 (FIG. 7). Because the VEGF-121 or VEGF-165 binding affinity test was performed in the presence of a saturating concentration (1 μM) of Ang2, and because the Ang2 binding test was performed in the presence of a saturating concentration (1 μM) of VEGF-121, it can be concluded that AMSB103 bind to both VEGF and Ang2 simultaneously.

[0687] The study was performed by largely following previously established procedure. 6-8 weeks old Male Brown Norway (BN) rats were used for the study. Rats were anesthetized with sodium pentobarbital (30 mg/kg, injected via tail vein), pupils were dilated, and CNV was induced in the rats by laser rupture of the Bruch's membrane (6 burns/eye. Laser setting: 150 mW, 100 μm diameter, 100 ms duration). Only the laser spots where a bubble was formed without hemorrhage were considered effective CNV lesions and included in the study. The following agents were then administered via intravitreal injection on the same day (D1): Group 1: 5 μl of drug vehicle as the negative control; Group 2: 5 μl of 40 mg/ml Aflibercept; Group 3: 5 μl of 40 mg/ml AMSB103. Fluorescein angiography (FA) was performed to visualize the CNV lesions: 10% sodium fluorescein was injected at 0.5 ml/kg of body weight; following the injection, a rapid series of red-free images were taken in the first minute (early-phase images), and additional images were taken after 5 minutes (late-phase images). The leakage severity of each lesion was scored based on the late-phase images according to a scale of grade 0-3 (grade 3 is the most severe, representing clinically meaningful CNV). The percentage of lesions with grade 3 leakage and the mean leakage score was then calculated for each group.

[0688] The results are shown in FIG. 9. From the result, it can be seen that compared to the negative control, AMSB103 significantly reduced the number of grade 3 lesions as well as the mean leakage score. Compared to Aflibercept, AMSB103 also showed a trend of better reduction of grade 3 lesions and better reduction of mean leakage score. This proves the in vivo efficacy of the bispecific aptamer of the present disclosure.

[0689] Accordingly, the bispecific aptamers of the present disclosure (e.g. AMSB103) may work as potent therapeutic agents for various diseases, such as wet Age-related Macular Degeneration (AMD), Diabetic Macular Edema (DME), and Diabetic Retinopathy (DR), as the laser-induced CNV model is a commonly used animal model for wet AMD.

[0690] The study was performed by largely following previously established procedure. Adult cynomolgus monkeys (2-4 kg) were used for the study. Monkeys were anesthetized with ketamine hydrochloride (10 mg/kg, intramuscular injection) and sodium pentobarbital (12 mg/kg, intravenous injection), pupils were dilated, and CNV was induced in the monkeys by laser rupture of the Bruch's membrane around the macula (6-8 burns per eye. Laser setting: 532 nm wavelength, 400-500 mW, 50 μm diameter, 100 ms duration). Only the laser spots where a bubble was formed without hemorrhage were considered effective CNV lesions and included in the study. Two weeks after the laser treatment (D15), fluorescein angiography (FA) was performed to visualize the CNV lesions: 100 mg/ml sodium fluorescein was injected at 10 mg/kg of body weight; following the injection, a rapid series of images were taken in the first minute (early-phase images) and after 5 minutes (late-phase images). The leakage of each lesion was visualized by comparing the late-phase images against the early-phase images, and the leakage severity was scored according to a scale of Grade I to IV: Grade I—no hyperfluorescence; Grade II—hyperfluorescence without leakage; Grade III—hyperfluorescence in the early or mid-transit images and late leakage; Grade IV—bright hyperfluorescence in the transit and late leakage beyond the treated areas. Grade IV lesions are considered as clinically significant. The monkeys were then assigned to different experimental groups so that each group has a comparable percentage of lesions with Grade IV leakage. Optical coherence tomography (OCT) was also performed to measure the height of the subretinal hyperreflective material (SHRM) at the laser burns, which is analogous to human pathologic CNV. The following agents were then administered via intravitreal injection on the same day (D15): Group 1: 50 μl of PBS as the negative control; Group 2: 50 μl of 2 mg/ml AMSB103. The CNV lesions were measured again on D29 by FA imaging following the procedure described above, and the effect of each agent was measured by comparing the leakage area of the Grade IV lesions before and after injection. Moreover, OCT was also performed and the height of the SHRM before and after injection was compared.

[0691] The results are shown in FIG. 10. From the result, it can be seen that the injection of the PBS (Group 1) resulted in little reduction in SHRM thickness and no reduction in leakage area. In contrast, the injection of AMSB103 resulted in a significant reduction in both SHRM thickness and leakage area, confirming its potent efficacy in inhibiting CNV in NHPs.

[0692] Interestingly, Macugen® was also tested in this model, but having been reported to have no effect in inhibiting CNV, likely due to the fact that it does not inhibit some of the VEGF isoforms such as VEGF-121 (please refer to R. T. Tzekov et al., Evaluation of intravitreal application of Pegaptanib Sodium on Laser-induced CNV in Cynomolgus Monkeys, ARVO Annual Meeting Abstract, May 2006). However, the bispecific aptamers of the present disclosure exhibited a potent efficacy in this model.

[0693] 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. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. 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.