Aptamers and uses thereof

Abstract

The present disclosure provides an anti-VEGF aptamer, a medicament or composition comprising the anti-VEGF aptamer, and uses thereof.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of February 25, 2021, application number 202180015992.3, and invention name “Apptamers and Their Uses”. Background Art

[0002] Neovascularization (NV) and vascular leakage are hallmarks of various retinal neovascular diseases, such as diabetic macular edema (DME), wet age-related macular degeneration (AMD), and diabetic retinopathy (DR).

[0003] VEGF plays a key role in regulating angiogenesis and vascular permeability. Angiogenesis is the main mechanism of NV in the eye, and vascular permeability determines the degree of leakage. Therefore, VEGF is an effective target for a variety of diseases.

[0004] Extensive studies have demonstrated that the Ang2 / Tie2 pathway is an important player in supplementing VEGF and has great potential as a new target for treating a variety of diseases such as retinal diseases. It is reported that 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 supplements the function of VEGF by promoting vascular permeability and stimulating the secretion of pro-angiogenic cytokines, which are required for VEGF-induced ocular NV and vascular leakage.

[0005] Despite the success of current anti-VEGF drugs, their efficacy and duration are still limited. Increasing the dose of anti-VEGF agents can improve the efficacy and duration of the drug. Nucleic acid aptamers have high solubility and small molecular weight (15kDa), so much higher molar doses can be achieved.

[0006] However, previous anti-VEGF aptamers, e.g. This potential cannot be realized. It cannot recognize all VEGFA isoforms and therefore has low efficacy. Therefore, there is an urgent need to develop aptamers that are more effective than those in the art.

[0007] There is an ongoing need to develop aptamers targeting VEGF and / or Ang2 that are more specific, have higher affinity, and / or are more potent. Summary of the invention

[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 aptamers provided in the present disclosure are capable of at least one of the following: 1) D The invention relates to an anti-VEGF that has the following properties: 1) binds to all VEGFA isoforms (e.g., VEGF-121, VEGF-165, and VEGF 189); 2) forms a specific secondary structure comprising, from the 5' to the 3' direction, a first stem, a first loop, a second stem, and a second loop; 3) is formulated at a concentration of up to 200 mg / ml or higher; 4) is resistant to nucleases; 5) binds to VEGFA isoforms from multiple species, such as human VEGF, mouse VEGF, rat VEGF, rabbit VEGF, and monkey VEGF; 6) inhibits the interaction between VEGF and VEGF receptors; and 7) prevents, treats, and / or ameliorates neovascular diseases, disorders, or conditions.

[0010] The anti-Ang2 aptamers provided in the present disclosure are capable of at least one of the following: 1) activating the Ang2 aptamer with a K of about 500 picomolar (pM) or less; D 1) binds to Ang2; 2) does not substantially bind to human Ang1; 3) forms a specific secondary structure, which comprises a first stem, a first ridge, a second stem, a second ridge, a third stem, a third ridge, a fourth stem and a first loop from 5' to 3' direction; 4) binds to both human Ang2 and mouse Ang2; 5) is resistant to nucleases; 6) inhibits the interaction between Ang2 and Tie2; and 7) prevents, treats and / or improves neovascular diseases, disorders or conditions.

[0011] The bispecific aptamers and / or compositions provided in the present disclosure are capable of at least one of the following: 1) performing a K of about 20 nanomolar (nM) or less; D The invention relates to an anti-VEGF agent that binds to all VEGFA isoforms (e.g., VEGF-121, VEGF-165, and VEGF189) with a KD value of about 500 picomolar (pM) or less; 2) binds to Ang2 with a KD value of about 500 picomolar (pM) or less; 3) is resistant to nucleases; 4) inhibits the interaction between Ang2 and Tie2; 5) inhibits the interaction between VEGF and VEGF receptors; and 6) prevents, treats, and / or ameliorates neovascular diseases, disorders, or conditions.

[0012] Anti-VEGF Aptamer

[0013] In one aspect, the present disclosure provides an anti-VEGF aptamer that has a K of about 20 nanomolar (nM) or less. D The value binds to VEGF-121 with a K of about 20 nanomolar (nM) or less. D Values ​​are bound to VEGF-165.

[0014] In some embodiments, the anti-VEGF aptamer has a K of about 2 nanomolar (nM) or less. D In some embodiments, the anti-VEGF aptamer binds to VEGF-121 with a KD value of about 2 nanomolar (nM) or less.

[0015] In some embodiments, VEGF-121 is human VEGF-121, mouse VEGF-120, monkey VEGF-121, rabbit VEGF-121, and / or rat VEGF-120. In some embodiments, VEGF-165 is human VEGF-165, mouse VEGF-164, monkey VEGF-165, rabbit VEGF-165, and / or rat VEGF-164. In some embodiments, the anti-VEGF aptamer binds to VEGF-189, such as rabbit VEGF-189.

[0016] In some embodiments, the anti-VEGF aptamer specifically binds to both the VEGF receptor binding domain of VEGF-121 or a fragment thereof and the VEGF receptor binding domain of VEGF-165 or a fragment thereof.

[0017] In some embodiments, the VEGF receptor binding domain of VEGF-121 comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 30-34, 40, 41, and 45. In some embodiments, the VEGF receptor binding domain of VEGF-165 comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 30-34, 40, and 41.

[0018] In some embodiments, the anti-VEGF aptamer inhibits the interaction between the VEGF-121 and VEGF-R1. In some embodiments, the anti-VEGF aptamer inhibits the interaction between the VEGF-121 and VEGF-R2.

[0019] In some embodiments, the anti-VEGF aptamer inhibits the interaction between the VEGF-165 and VEGF-R1. In some embodiments, the anti-VEGF aptamer inhibits the interaction between the VEGF-165 and VEGF-R2.

[0020] In some embodiments, the anti-VEGF aptamer is capable of reducing and / or ameliorating damage and / or leakage in a laser-induced choroidal neovascularization (CNV) rat model.

[0021] In some embodiments, anti-VEGF aptamers are capable of reducing and / or ameliorating damage and / or leakage in a non-human primate (NHP) laser-induced CNV model.

[0022] In some embodiments, the anti-VEGF aptamer is highly soluble.

[0023] 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 nucleotides. In some embodiments, the anti-VEGF aptamer is resistant to nucleases. In some embodiments, all nucleotides of the anti-VEGF aptamer are modified nucleotides. In some embodiments, such modified nucleotides comprise chemical substitutions or modifications 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 the following: 2'-position sugar modification, 2'-amino (2'-NH2) modification, 2'-fluoro (2'-F) modification, 2'-O-methyl (2'-OMe) modification, 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, 5-position modified pyrimidine, modification at the amine outside the cytosine ring, substitution with 5'-bromouracil, substitution with 5'-bromodeoxyuridine, substitution with 5'-bromodeoxycytidine, backbone modification, locked nucleic acid (LNA), methylation, 3' cap and 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-benzylcarboxamide)-2'-deoxyuridine, 5-(N-isobutylcarboxamide)-2'-deoxyuridine, 5-(N-naphthylmethylcarboxamide)-2'-deoxyuridine and 5-(N-tryptaminecarboxamide)-2'-deoxyuridine.

[0024] In some embodiments, the anti-VEGF aptamer comprises at least one 2'-modified nucleotide. In some embodiments, all nucleotides of the anti-VEGF aptamer are 2'-modified nucleotides. In some embodiments, the 2'-modified nucleotides are selected from 2'-amino-modified nucleotides, 2'-fluoro-modified nucleotides, and 2'-O-methyl-modified nucleotides and 2'-O-(2-methoxyethyl)-modified nucleotides. 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.

[0025] In some embodiments, all 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 cytidines of the anti-VEGF aptamer are 2'-deoxy-2'-fluoro cytidines. In some embodiments, all uridines of the anti-VEGF aptamer are 2'-modified uridines. In some embodiments, all uridines of the anti-VEGF aptamer are 2'-fluoro-modified uridines and / or 2'-O-methyl-modified uridines. In some embodiments, all uridines of the anti-VEGF aptamer are 2'-deoxy-2'-fluoro uridines. In some embodiments, all adenosines of the anti-VEGF aptamer are 2'-modified adenosines. In some embodiments, all adenosines of the anti-VEGF aptamer are 2'-fluoro-modified adenosines and / or 2'-O-methyl-modified adenosines. In some embodiments, all adenosines of the anti-VEGF aptamer are 2'-deoxy-2'-fluoroadenosine. In some embodiments, all guanosines of the anti-VEGF aptamer are 2'-modified guanosine. In some embodiments, all guanosines of the anti-VEGF aptamer are 2'-fluoro-modified guanosine and / or 2'-O-methyl-modified guanosine. In some embodiments, all guanosines of the anti-VEGF aptamer are 2'-deoxy-2'-fluoroguanosine.

[0026] In some embodiments, all nucleotides of the anti-VEGF aptamer are 2'-fluoro-modified nucleotides and / or 2'-O-methyl-modified nucleotides.

[0027] In some embodiments, all nucleotides of the anti-VEGF aptamer are 2'-fluoro-modified nucleotides.

[0028] In some embodiments, the anti-VEGF aptamer does not comprise any naturally occurring nucleotides.

[0029] In some embodiments, the anti-VEGF aptamer comprises about 10 to about 150 nucleotides.

[0030] In some embodiments, the anti-VEGF aptamer competes for binding to VEGF-165 and / or VEGF-121 with an anti-VEGF reference aptamer, wherein the anti-VEGF reference aptamer comprises a secondary structure comprising (e.g., from the 5' to 3' direction) a first stem, a first loop, a second stem, and a second loop.

[0031] In some embodiments, an anti-VEGF reference aptamer comprises a secondary structure consisting of (eg, from the 5' to 3' direction) a first stem, a first loop, a second stem, and a second loop.

[0032] In some embodiments, the anti-VEGF reference aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 136-137. In some embodiments, the anti-VEGF reference aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 138-139.

[0033] In some embodiments, the first consensus sequence comprises nucleotide sequences that form a first stem and a first loop of an anti-VEGF reference aptamer. In some embodiments, the first consensus sequence is not contained in a nucleotide sequence that forms a second stem or a second loop of an anti-VEGF reference aptamer. In some embodiments, the second consensus sequence comprises nucleotide sequences that form a second stem and a second loop of an anti-VEGF reference aptamer. In some embodiments, the second consensus sequence is not contained in a nucleotide sequence that forms a first stem or a first loop of an anti-VEGF reference aptamer.

[0034] In some embodiments, the anti-VEGF reference aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, or a variant or truncated form thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201. In some embodiments, the variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, with one or more nucleotide additions, deletions and / or substitutions. In some embodiments, the anti-VEGF reference aptamer comprises about 10 to about 150 nucleotides.

[0035] In some embodiments, the anti-VEGF reference aptamer comprises a first consensus sequence and a second consensus sequence and the first consensus sequence is located 5' relative to the second consensus sequence.

[0036] In embodiments, the anti-VEGF reference aptamer comprises a first consensus sequence and a second consensus sequence and the second consensus sequence is located 5' relative to the first consensus sequence.

[0037] In some embodiments, the anti-VEGF aptamer comprises a secondary structure, and the secondary structure (e.g., from 5' to 3' direction) comprises a first stem, a first loop, a second stem, and a second loop. In some embodiments, the anti-VEGF aptamer comprises a secondary structure, and the secondary structure (e.g., from 5' to 3' direction) consists of a first stem, a first loop, a second stem, and a second loop.

[0038] In some embodiments, the anti-VEGF aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 136-137. In some embodiments, the anti-VEGF aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 138-139.

[0039] In some embodiments, the first consensus sequence comprises nucleotide sequences that form a first stem and a first loop of an anti-VEGF aptamer. In some embodiments, the first consensus sequence is not contained in a nucleotide sequence that forms a second stem or a second loop of an anti-VEGF aptamer. In some embodiments, the second consensus sequence comprises nucleotide sequences that form a second stem and a second loop of an anti-VEGF aptamer. In some embodiments, the second consensus sequence is not contained in a nucleotide sequence that forms a first stem or a first loop of an anti-VEGF aptamer.

[0040] In some embodiments, the anti-VEGF aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, or a variant or truncated form thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201. In some embodiments, the variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, with one or more nucleotide additions, deletions and / or substitutions.

[0041] In some embodiments, the anti-VEGF aptamer comprises a first consensus sequence and a second consensus sequence, and the first consensus sequence is located 5' relative to the second consensus sequence. In some embodiments, the anti-VEGF aptamer comprises a first consensus sequence and a second consensus sequence, and the second consensus sequence is located 5' relative to the first consensus sequence.

[0042] In some embodiments, anti-VEGF aptamers are used to modulate the biological activity of VEGF or a VEGF receptor.

[0043] In some embodiments, the anti-VEGF aptamer is used to prevent, treat and / or improve VEGF-related diseases, disorders or conditions. 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.

[0044] In one aspect, the present disclosure provides an anti-VEGF agent comprising an 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' end of the aptamer.

[0045] In one aspect, the present disclosure provides a composition comprising an anti-VEGF aptamer or an anti-VEGF agent of the present disclosure.

[0046] 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 a therapeutically effective amount of an anti-VEGF aptamer or anti-VEGF agent.

[0047] In one aspect, the present disclosure provides a method for modulating the biological activity of VEGF and / or a VEGF receptor, comprising administering an effective amount of an anti-VEGF aptamer or an anti-VEGF agent of the present disclosure to a subject in need thereof.

[0048] In one aspect, the present disclosure provides a method for preventing, treating and / or ameliorating VEGF-related diseases, disorders or conditions, comprising administering an effective amount of an anti-VEGF aptamer or anti-VEGF agent of the present disclosure to a subject in need thereof. In some embodiments, the VEGF-related disease, disorder or condition is a neovascular disease, disorder or condition. In some embodiments, the anti-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.

[0049] In one aspect, the present disclosure provides the use of the anti-VEGF aptamer or anti-VEGF agent of the present disclosure for the manufacture of a medicament for modulating the biological activity of VEGF or a VEGF receptor.

[0050] In one aspect, the present disclosure provides the use of an anti-VEGF aptamer or anti-VEGF agent of the present disclosure for the manufacture of a medicament for preventing, treating and / or ameliorating a VEGF-related disease, disorder or condition. In some embodiments, a VEGF-related disease, disorder or condition is a neovascular disease, disorder or condition. In some embodiments, a VEGF-related disease, disorder or condition is an ocular neovascular disease, disorder or condition. In some embodiments, a 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, a VEGF-related disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization and proliferative diabetic retinopathy.

[0051] Anti-Ang2 Aptamer

[0052] In one aspect, the present disclosure provides an anti-Ang2 aptamer having a K of about 500 picomolar (pM) or less. D The value specifically binds to Ang2.

[0053] In some embodiments, the anti-Ang2 aptamer has a K of about 100 picomolar (pM) or less. D Values ​​are bound to Ang2.

[0054] In some embodiments, Ang2 is human Ang2, mouse Ang2, monkey Ang2, rabbit Ang2, and / or rat Ang2.

[0055] In some embodiments, the anti-Ang2 aptamer does not substantially bind to human Ang1.

[0056] In some embodiments, the anti-Ang2 aptamer inhibits the interaction between Ang2 and Tie2.

[0057] In some embodiments, the anti-Ang2 aptamer is capable of reducing neovascularization and / or decreasing vascular permeability in a mouse oxygen-induced ischemic retinopathy (OIR) model.

[0058] In some embodiments, the anti-Ang2 aptamer is highly soluble.

[0059] 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 resistant to nucleases. In some embodiments, all 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 the following: 2'-position sugar modification, 2'-amino (2'-NH2) modification, 2'-fluoro (2'-F) modification, 2'-O-methyl (2'-OMe) modification, 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, 5-position modified pyrimidine, modification at the amine outside the cytosine ring, substitution with 5'-bromouracil, substitution with 5'-bromodeoxyuridine, substitution with 5'-bromodeoxycytidine, backbone modification, locked nucleic acid (LNA), methylation, 3' cap and 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-benzylcarboxamide)-2'-deoxyuridine, 5-(N-isobutylcarboxamide)-2'-deoxyuridine, 5-(N-naphthylmethylcarboxamide)-2'-deoxyuridine and 5-(N-tryptaminecarboxamide)-2'-deoxyuridine.

[0060] In some embodiments, the anti-Ang2 aptamer comprises at least one 2'-modified nucleotide. In some embodiments, all nucleotides of the anti-Ang2 aptamer are 2'-modified nucleotides. In some embodiments, the 2'-modified nucleotides are selected from 2'-amino-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, and 2'-O-methoxy-modified nucleotides.

[0061] 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 cytidines of the anti-Ang2 aptamer are 2'-modified cytidines. In some embodiments, all cytidines of the anti-Ang2 aptamer are 2'-fluoro-modified cytidines and / or 2'-O-methyl-modified cytidines. In some embodiments, all cytidines of the anti-Ang2 aptamer are 2'-deoxy-2'-fluoro cytidines. In some embodiments, all uridines of the anti-Ang2 aptamer are 2'-modified uridines. In some embodiments, all uridines of the anti-Ang2 aptamer are 2'-fluoro-modified uridines and / or 2'-O-methyl-modified uridines. In some embodiments, all uridines of the anti-Ang2 aptamer are 2'-deoxy-2'-fluoro uridines. In some embodiments, all adenosines of the anti-Ang2 aptamer are 2'-modified adenosines. In some embodiments, all adenosines of the anti-Ang2 aptamer are 2'-fluoro-modified adenosines and / or 2'-O-methyl-modified adenosines. In some embodiments, all adenosines of the anti-Ang2 aptamer are 2'-deoxy-2'-fluoro adenosines. In some embodiments, all guanosines of the anti-Ang2 aptamer are 2'-modified guanosines. In some embodiments, all guanosines of the anti-Ang2 aptamer are 2'-fluoro-modified guanosines and / or 2'-O-methyl-modified guanosines. In some embodiments, all guanosines of the anti-Ang2 aptamer are 2'-deoxy-2'-fluoro guanosines. In some embodiments, all nucleotides of the anti-Ang2 aptamer are 2'-fluoro-modified nucleotides and / or 2'-O-methyl-modified nucleotides. In some embodiments, all nucleotides of the anti-Ang2 aptamer are 2'-fluoro-modified nucleotides.

[0062] In some embodiments, the anti-Ang2 aptamer does not comprise any natural nucleotides. In some embodiments, the anti-Ang2 aptamer comprises about 10 to about 150 nucleotides.

[0063] In some embodiments, the anti-Ang2 aptamer competes for binding to Ang2 with an anti-Ang2 reference aptamer, 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.

[0064] In some embodiments, the anti-Ang2 reference aptamer comprises a secondary structure consisting of, from 5' to 3' direction, a first stem, a first knob, a second stem, a second knob, a third stem, a third knob, a fourth stem, and a first loop.

[0065] In some embodiments, the anti-Ang2 reference aptamer comprises a first consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 13-20. In some embodiments, the anti-Ang2 reference aptamer comprises a second consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 21-28.

[0066] In some embodiments, the first consensus sequence is comprised in the nucleotide sequence forming the first knob, the second stem, the second knob, the third stem, the third knob 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 the first loop of the anti-Ang2 reference aptamer.

[0067] In some embodiments, the second consensus sequence is comprised in the nucleotide sequence forming the first knob, the second stem, the second knob, the third stem, the third knob 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 the first loop of the anti-Ang2 reference aptamer.

[0068] In some embodiments, the anti-Ang2 reference aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12 or a variant or truncated form thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as shown in any one of SEQ ID NOs: 1-12. In some embodiments, the variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, with one or more nucleotide additions, deletions and / or substitutions. In some embodiments, the anti-Ang2 reference aptamer comprises about 10 to about 150 nucleotides.

[0069] In some embodiments, the anti-Ang2 reference aptamer comprises a first consensus sequence and a second consensus sequence, and the first consensus sequence is located 5′ relative to the second consensus sequence.

[0070] In some embodiments, the anti-Ang2 aptamer comprises a secondary structure, which comprises, from 5' to 3' direction, a first stem, a first carina, a second stem, a second carina, a third stem, a third carina, a fourth stem, and a first loop. In some embodiments, the anti-Ang2 aptamer comprises a secondary structure, which consists, from 5' to 3' direction, of a first stem, a first carina, a second stem, a second carina, a third stem, a third carina, a fourth stem, and a first loop.

[0071] In some embodiments, the anti-Ang2 aptamer comprises a first consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 13-20. In some embodiments, the anti-Ang2 aptamer comprises a second consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 21-28.

[0072] In some embodiments, the first consensus sequence is included in the nucleotide sequence forming the first carina, the second stem, the second carina, the third stem, the third carina, and / or the fourth stem of the anti-Ang2 aptamer. In some embodiments, the first consensus sequence is not included in the nucleotide sequence forming the first loop of the anti-Ang2 aptamer. In some embodiments, the second consensus sequence is included in the nucleotide sequence forming the first carina, the second stem, the second carina, the third stem, the third carina, and / or the fourth stem of the anti-Ang2 aptamer. In some embodiments, the second consensus sequence is not included in the nucleotide sequence forming the first loop of the anti-Ang2 aptamer.

[0073] The first loop of the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise 2-30 nucleotides.

[0074] In some embodiments, the anti-Ang2 aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, or a variant or truncated form thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 1-12. In some embodiments, the variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, with one or more nucleotide additions, deletions and / or substitutions.

[0075] In some embodiments, the anti-Ang2 aptamer comprises a first consensus sequence and a second consensus sequence, and the first consensus sequence is located 5′ relative to the second consensus sequence.

[0076] In some embodiments, the anti-Ang2 aptamer comprises a first consensus sequence and a second consensus sequence, and the second consensus sequence is located 5′ relative to the first consensus sequence.

[0077] In some embodiments, anti-Ang2 aptamers are used to modulate the biological activity of Ang2 or Tie2.

[0078] In some embodiments, anti-Ang2 aptamers are used to prevent, treat and / or improve Ang2-related diseases, disorders or conditions. In some embodiments, Ang2-related diseases, disorders or conditions are neovascular diseases, disorders or conditions. In some embodiments, Ang2-related diseases, disorders or conditions are ocular neovascular diseases, disorders or conditions. In some embodiments, Ang2-related diseases, disorders or conditions are selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion and diabetic retinopathy. In some embodiments, Ang2-related diseases, disorders or conditions are selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion and diabetic retinopathy.

[0079] In one aspect, the present disclosure provides an anti-Ang2 agent comprising an 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' end of the anti-Ang2 aptamer.

[0080] In one aspect, the present disclosure provides a composition comprising an anti-Ang2 aptamer or an anti-Ang2 agent of the present disclosure.

[0081] 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 an anti-Ang2 aptamer or anti-Ang2 agent.

[0082] In some embodiments, the composition further comprises an anti-VEGF agent. In some embodiments, the anti-VEGF agent comprises Aflibercept.

[0083] In one aspect, the present disclosure provides a method for modulating the biological activity of Ang2 or Tie2, comprising administering an effective amount of an anti-Ang2 aptamer or anti-Ang2 agent of the present disclosure to a subject in need thereof.

[0084] In one aspect, the present disclosure provides a method for preventing, treating and / or ameliorating an Ang2-related disease, disorder or condition, comprising administering an effective amount of an anti-Ang2 aptamer or anti-Ang2 agent of the present disclosure to a subject in need thereof.

[0085] 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 an anti-VEGF agent to the subject. In some embodiments, the anti-VEGF agent comprises aflibercept.

[0086] In one aspect, the present disclosure provides use of the anti-Ang2 aptamer or anti-Ang2 agent of the present disclosure for the manufacture of a medicament for modulating the biological activity of Ang2 or Tie2.

[0087] In one aspect, the present disclosure provides use of the anti-Ang2 aptamer or anti-Ang2 agent of the present disclosure for the manufacture of a medicament for preventing, treating and / or ameliorating an Ang2-related disease, disorder or condition.

[0088] 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 wet age-related macular degeneration, myopic choroidal neovascularization and proliferative diabetic retinopathy.

[0089] In one aspect, the present disclosure provides 1) the use of an anti-Ang2 aptamer or anti-Ang2 agent of the present disclosure in combination with 2) an anti-VEGF agent for 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.

[0090] Bispecific Aptamers

[0091] In another aspect, the present disclosure provides a bispecific aptamer, which comprises an anti-Ang2 aptamer and an anti-VEGF aptamer. The bispecific aptamer may be included in the composition of the present disclosure.

[0092] In some embodiments, the anti-VEGF aptamer has a K of about 20 nanomolar (nM) or less. D The value binds to VEGF-121 with a K of about 20 nanomolar (nM) or less. D Values ​​are bound to VEGF-165.

[0093] In some embodiments, the anti-Ang2 aptamer has a K of about 500 picomolar (pM) or less. D The value specifically binds to Ang2.

[0094] In some embodiments, the anti-VEGF aptamer has a K of about 2 nanomolar (nM) or less. D In some embodiments, the anti-VEGF aptamer binds to VEGF-121 with a K of about 2 nanomolar (nM) or less. D Values ​​are bound to VEGF-165.

[0095] In some embodiments, VEGF-121 is human VEGF-121, mouse VEGF-120, monkey VEGF-121, rabbit VEGF-121, and or rat VEGF-120. In some embodiments, VEGF-165 is human VEGF-165, mouse VEGF-164, monkey VEGF-165, rabbit VEGF-165, or rat VEGF-164.

[0096] In some embodiments, the anti-VEGF aptamer specifically binds to both the VEGF receptor binding domain of VEGF-121 and the VEGF receptor binding domain of VEGF-165.

[0097] In some embodiments, the VEGF receptor binding domain of VEGF-121 comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 30-34, 40, 41, and 45. In some embodiments, the VEGF receptor binding domain of VEGF-165 comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 30-34, 40, and 41.

[0098] In some embodiments, the anti-VEGF aptamer inhibits the interaction between VEGF-121 and VEGF-R1. In some embodiments, the anti-VEGF aptamer inhibits the interaction between VEGF-121 and VEGF-R2. In some embodiments, the anti-VEGF aptamer inhibits the interaction between VEGF-165 and VEGF-R1. In some embodiments, the anti-VEGF aptamer inhibits the interaction between VEGF-165 and VEGF-R2.

[0099] In some embodiments, the anti-VEGF aptamer is capable of reducing and / or ameliorating damage and / or leakage in a laser-induced choroidal neovascularization (CNV) rat model.

[0100] In some embodiments, anti-VEGF aptamers are capable of reducing and / or ameliorating damage and / or leakage in a non-human primate (NHP) laser-induced CNV model.

[0101] In some embodiments, the anti-VEGF aptamer is highly soluble.

[0102] 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 resistant to nucleases. In some embodiments, the anti-VEGF aptamer comprises one or more modified nucleotides. In some embodiments, all 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 the following: 2'-position sugar modification, 2'-amino (2'-NH2) modification, 2'-fluoro (2'-F) modification, 2'-O-methyl (2'-OMe) modification, 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, 5-position modified pyrimidine, modification at the amine outside the cytosine ring, substitution with 5'-bromouracil, substitution with 5'-bromodeoxyuridine, substitution with 5'-bromodeoxycytidine, backbone modification, locked nucleic acid (LNA), methylation, 3' cap and 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-benzylcarboxamide)-2'-deoxyuridine, 5-(N-isobutylcarboxamide)-2'-deoxyuridine, 5-(N-naphthylmethylcarboxamide)-2'-deoxyuridine and 5-(N-tryptaminecarboxamide)-2'-deoxyuridine.

[0103] In some embodiments, the anti-VEGF aptamer comprises at least one 2'-modified nucleotide. In some embodiments, all nucleotides of the anti-VEGF aptamer are 2'-modified nucleotides. In some embodiments, the 2'-modified nucleotides are selected from 2'-amino-modified nucleotides, 2'-fluoro-modified nucleotides, and 2'-O-methyl-modified nucleotides and 2'-O-(2-methoxyethyl)-modified nucleotides. 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.

[0104] In some embodiments, all 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 cytidines of the anti-VEGF aptamer are 2'-deoxy-2'-fluoro cytidines. In some embodiments, all uridines of the anti-VEGF aptamer are 2'-modified uridines. In some embodiments, all uridines of the anti-VEGF aptamer are 2'-fluoro-modified uridines and / or 2'-O-methyl-modified uridines. In some embodiments, all uridines of the anti-VEGF aptamer are 2'-deoxy-2'-fluoro uridines. In some embodiments, all adenosines of the anti-VEGF aptamer are 2'-modified adenosines. In some embodiments, all adenosines of the anti-VEGF aptamer are 2'-fluoro-modified adenosines and / or 2'-O-methyl-modified adenosines. In some embodiments, all adenosines of the anti-VEGF aptamer are 2'-deoxy-2'-fluoroadenosine. In some embodiments, all guanosines are 2'-modified guanosine. In some embodiments, all guanosines of the anti-VEGF aptamer are 2'-fluoro-modified guanosine and / or 2'-O-methyl-modified guanosine. In some embodiments, all guanosines of the anti-VEGF aptamer are 2'-deoxy-2'-fluoroguanosine.

[0105] In some embodiments, all nucleotides of the anti-VEGF aptamer are 2'-fluoro-modified nucleotides and / or 2'-O-methyl-modified nucleotides.

[0106] In some embodiments, all nucleotides of the anti-VEGF aptamer are 2'-fluoro-modified nucleotides.

[0107] In some embodiments, the anti-VEGF aptamer does not comprise any naturally occurring nucleotides.

[0108] In some embodiments, the anti-VEGF aptamer comprises about 10 to about 150 nucleotides.

[0109] In some embodiments, the anti-VEGF aptamer competes with an anti-VEGF reference aptamer for binding to VEGF-165 and / or VEGF-121, and the anti-VEGF reference aptamer comprises a secondary structure comprising, from the 5' to 3' direction, a first stem, a first loop, a second stem, and a second loop.

[0110] In some embodiments, the anti-VEGF reference aptamer comprises a secondary structure consisting of, from the 5' to the 3' direction, a first stem, a first loop, a second stem, and a second loop.

[0111] In some embodiments, the anti-VEGF reference aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 136-137. In some embodiments, the anti-VEGF reference aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 138-139.

[0112] In some embodiments, the first consensus sequence comprises nucleotide sequences that form a first stem and a first loop of an anti-VEGF reference aptamer. In some embodiments, the first consensus sequence is not contained in a nucleotide sequence that forms a second stem or a second loop of an anti-VEGF reference aptamer. In some embodiments, the second consensus sequence comprises nucleotide sequences that form a second stem and a second loop of an anti-VEGF reference aptamer. In some embodiments, the second consensus sequence is not contained in a nucleotide sequence that forms a first stem or a first loop of an anti-VEGF reference aptamer.

[0113] In some embodiments, the anti-VEGF reference aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, or a variant or truncated form thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201. In some embodiments, the variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, with one or more nucleotide additions, deletions and / or substitutions. In some embodiments, the anti-VEGF reference aptamer comprises about 10 to about 150 nucleotides.

[0114] In some embodiments, the anti-VEGF reference aptamer comprises a first consensus sequence and a second consensus sequence, and the first consensus sequence is located 5' relative to the second consensus sequence.

[0115] In some embodiments, the anti-VEGF reference aptamer comprises a first consensus sequence and a second consensus sequence, wherein the second consensus sequence is located 5' relative to the first consensus sequence.

[0116] In some embodiments, the anti-VEGF aptamer comprises a secondary structure, which comprises, from the 5' to the 3' direction, a first stem, a first loop, a second stem, and a second loop. In some embodiments, the anti-VEGF aptamer comprises a secondary structure, which consists, from the 5' to the 3' direction, of a first stem, a first loop, a second stem, and a second loop.

[0117] In some embodiments, the anti-VEGF aptamer comprises a first consensus sequence comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 136-137. In some embodiments, the anti-VEGF aptamer comprises a second consensus sequence comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 138-139.

[0118] In some embodiments, the first consensus sequence comprises nucleotide sequences that form a first stem and a first loop of an anti-VEGF aptamer. In some embodiments, the first consensus sequence is not contained in a nucleotide sequence that forms a second stem or a second loop of an anti-VEGF aptamer. In some embodiments, the second consensus sequence comprises nucleotide sequences that form a second stem and a second loop of an anti-VEGF aptamer. In some embodiments, the second consensus sequence is not contained in a nucleotide sequence that forms a first stem or a first loop of an anti-VEGF aptamer.

[0119] In some embodiments, the anti-VEGF reference aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, or a variant or truncated form thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201. In some embodiments, the variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, with one or more nucleotide additions, deletions and / or substitutions.

[0120] In some embodiments, the anti-VEGF aptamer comprises a first consensus sequence and a second consensus sequence, and the first consensus sequence is located 5' relative to the second consensus sequence.

[0121] In some embodiments, the anti-VEGF aptamer comprises a first consensus sequence and a second consensus sequence, and the second consensus sequence is located 5' relative to the first consensus sequence.

[0122] In some embodiments, the anti-Ang2 aptamer has a K of about 100 picomolar (pM) or less. D Values ​​are bound to Ang2.

[0123] In some embodiments, Ang2 is human Ang2, mouse Ang2, monkey Ang2, rabbit Ang2, and / or rat Ang2.

[0124] In some embodiments, the anti-Ang2 aptamer does not substantially bind to human Ang1.

[0125] In some embodiments, the anti-Ang2 aptamer inhibits the interaction between Ang2 and Tie2.

[0126] In some embodiments, the anti-Ang2 aptamer is capable of reducing neovascularization and / or decreasing vascular permeability in a mouse oxygen-induced ischemic retinopathy (OIR) model.

[0127] In some embodiments, the anti-Ang2 aptamer is highly soluble.

[0128] 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 resistant to nucleases. In some embodiments, all 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 the following: 2'-position sugar modification, 2'-amino (2'-NH2) modification, 2'-fluoro (2'-F) modification, 2'-O-methyl (2'-OMe) modification, 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, 5-position modified pyrimidine, modification at the amine outside the cytosine ring, substitution with 5'-bromouracil, substitution with 5'-bromodeoxyuridine, substitution with 5'-bromodeoxycytidine, backbone modification, locked nucleic acid (LNA), methylation, 3' cap and 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-benzylcarboxamide)-2′-deoxyuridine, 5-(N-isobutylcarboxamide)-2′-deoxyuridine, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine and 5-(N-tryptaminecarboxamide)-2′-deoxyuridine.

[0129] In some embodiments, the anti-Ang2 aptamer comprises at least one 2'-modified nucleotide. In some embodiments, all nucleotides of the anti-Ang2 aptamer are 2'-modified nucleotides. In some embodiments, the 2'-modified nucleotides are selected from 2'-amino-modified nucleotides, 2'-fluoro-modified nucleotides and 2'-O-methyl-modified nucleotides and 2'-O-(2-methoxyethyl)-modified nucleotides. 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.

[0130] In some embodiments, the anti-Ang2 aptamer comprises at least one 2'-fluoro-modified nucleotide. In some embodiments, all cytidines of the anti-Ang2 aptamer are 2'-modified cytidines. In some embodiments, all cytidines of the anti-Ang2 aptamer are 2'-fluoro-modified cytidines and / or 2'-O-methyl-modified cytidines. In some embodiments, all cytidines of the anti-Ang2 aptamer are 2'-deoxy-2'-fluoro cytidines. In some embodiments, all uridines of the anti-Ang2 aptamer are 2'-modified uridines. In some embodiments, all uridines of the anti-Ang2 aptamer are 2'-fluoro-modified uridines and / or 2'-O-methyl-modified uridines. In some embodiments, all uridines of the anti-Ang2 aptamer are 2'-deoxy-2'-fluoro uridines. In some embodiments, all adenosines of the anti-Ang2 aptamer are 2'-modified adenosines. In some embodiments, all adenosines of the anti-Ang2 aptamer are 2'-fluoro-modified adenosines and / or 2'-O-methyl-modified adenosines. In some embodiments, all adenosines of the anti-Ang2 aptamer are 2'-deoxy-2'-fluoroadenosine. In some embodiments, all guanosines of the anti-Ang2 aptamer are 2'-modified guanosines. In some embodiments, all guanosines of the anti-Ang2 aptamer are 2'-fluoro-modified guanosines and / or 2'-O-methyl-modified guanosines. In some embodiments, all guanosines of the anti-Ang2 aptamer are 2'-deoxy-2'-fluoroguanosines. In some embodiments, all nucleotides of the anti-Ang2 aptamer are 2'-fluoro-modified nucleotides. In some embodiments, all nucleotides of the anti-Ang2 aptamer are 2'-fluoro-modified nucleotides and / or 2'-O-methyl-modified nucleotides.

[0131] In some embodiments, the anti-Ang2 aptamer does not comprise any natural nucleotides. In some embodiments, the anti-Ang2 aptamer comprises about 10 to about 150 nucleotides.

[0132] In some embodiments, the anti-Ang2 aptamer competes for binding to Ang2 with an anti-Ang2 reference aptamer, 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.

[0133] In some embodiments, the anti-Ang2 reference aptamer comprises a secondary structure consisting of, from 5' to 3' direction, a first stem, a first knob, a second stem, a second knob, a third stem, a third knob, a fourth stem, and a first loop.

[0134] In some embodiments, the anti-Ang2 reference aptamer comprises a first consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 13-20. In some embodiments, the anti-Ang2 reference aptamer comprises a second consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 21-28.

[0135] In some embodiments, the first consensus sequence is comprised in the nucleotide sequence forming the first knob, the second stem, the second knob, the third stem, the third knob 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 the first loop of the anti-Ang2 reference aptamer.

[0136] In some embodiments, the second consensus sequence is comprised in the nucleotide sequence forming the first knob, the second stem, the second knob, the third stem, the third knob 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 the first loop of the anti-Ang2 reference aptamer.

[0137] In some embodiments, the anti-Ang2 reference aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12 or a variant or truncated form thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence as shown in any one of SEQ ID NOs: 1-12. In some embodiments, the variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, with one or more nucleotide additions, deletions and / or substitutions. In some embodiments, the anti-Ang2 reference aptamer comprises about 10 to about 150 nucleotides.

[0138] In some embodiments, the anti-Ang2 reference aptamer comprises a first consensus sequence and a second consensus sequence, and the first consensus sequence is located 5′ relative to the second consensus sequence.

[0139] In some embodiments, the anti-Ang2 reference aptamer comprises a first consensus sequence and a second consensus sequence, and the second consensus sequence is located 5′ relative to the first consensus sequence.

[0140] In some embodiments, the anti-Ang2 aptamer comprises a secondary structure, which comprises, from 5' to 3' direction, a first stem, a first carina, a second stem, a second carina, a third stem, a third carina, a fourth stem, and a first loop. In some embodiments, the anti-Ang2 aptamer comprises a secondary structure, which consists, from 5' to 3' direction, of a first stem, a first carina, a second stem, a second carina, a third stem, a third carina, a fourth stem, and a first loop.

[0141] In some embodiments, the anti-Ang2 aptamer comprises a first consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 13-20. In some embodiments, the anti-Ang2 aptamer comprises a second consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 21-28.

[0142] In some embodiments, the first consensus sequence is included in the nucleotide sequence forming the first carina, the second stem, the second carina, the third stem, the third carina, and / or the fourth stem of the anti-Ang2 aptamer. In some embodiments, the first consensus sequence is not included in the nucleotide sequence forming the first loop of the anti-Ang2 aptamer. In some embodiments, the second consensus sequence is included in the nucleotide sequence forming the first carina, the second stem, the second carina, the third stem, the third carina, and / or the fourth stem of the anti-Ang2 aptamer. In some embodiments, the second consensus sequence is not included in the nucleotide sequence forming the first loop of the anti-Ang2 aptamer.

[0143] In some embodiments, the anti-Ang2 aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, or a variant or truncated form thereof. In some embodiments, the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 1-12. In some embodiments, the variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, with one or more nucleotide additions, deletions and / or substitutions.

[0144] In some embodiments, the anti-Ang2 aptamer comprises a first consensus sequence and a second consensus sequence, and the first consensus sequence is located 5′ relative to the second consensus sequence.

[0145] In some embodiments, the anti-Ang2 aptamer comprises a first consensus sequence and a second consensus sequence, and the second consensus sequence is located 5′ relative to the first consensus sequence.

[0146] 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' end of the anti-VEGF aptamer. In some embodiments, the PEG moiety is conjugated to the 3' end 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' end of the anti-Ang2 aptamer. In some embodiments, the PEG moiety is conjugated to the 3' end of the anti-Ang2 aptamer.

[0147] In some embodiments, the bispecific aptamer has the formula A1-(L)n-A2, wherein A1 is an anti-VEGF aptamer and A2 is an anti-Ang2 aptamer, or A1 is an anti-Ang2 aptamer and A2 is an 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.

[0148] In some embodiments, the bispecific aptamer is capable of specifically binding to both VEGF and Ang2.

[0149] In some embodiments, the bispecific aptamer has a K of about 500 pM or less. D values ​​bind to VEGF-121 with a K of about 500 pM or less D Values ​​are bound to Ang2.

[0150] In some embodiments, the bispecific aptamer has a K of about 200 pM or less. D values ​​bind to VEGF-121 with a K of about 200 pM or less D Values ​​are bound to Ang2.

[0151] In some embodiments, the bispecific aptamer has a K of about 100 pM or less. D values ​​bind to VEGF-121 with a K of about 100 pM or less D Values ​​are bound to Ang2.

[0152] 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.

[0153] In some embodiments of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, or a variant or truncated form thereof; and A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, or a variant or truncated form thereof.

[0154] In some embodiments of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, or a variant or truncated form thereof; and A2 is an anti-VEGF aptamer comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, or a variant or truncated form thereof.

[0155] In some embodiments of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising the nucleotide sequence as shown in SEQ ID NO:83 or a truncated form thereof, A2 is an anti-Ang2 aptamer comprising the nucleotide sequence as shown in SEQ ID NO:2 or a truncated form 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-VEGF aptamer comprising the nucleotide sequence as shown in SEQ ID NO:128 or a truncated form thereof, A2 is an anti-Ang2 aptamer comprising the nucleotide sequence as shown in SEQ ID NO:6 or a truncated form thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0157] In some embodiments of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising the nucleotide sequence as shown in SEQ ID NO:2 or a truncated form thereof, A2 is an anti-VEGF aptamer comprising the nucleotide sequence as shown in SEQ ID NO:83 or a truncated form thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0158] In some embodiments of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising the nucleotide sequence as shown in SEQ ID NO:6 or a truncated form thereof, A2 is an anti-VEGF aptamer comprising the nucleotide sequence as shown in SEQ ID NO:128 or a truncated form thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0159] In some embodiments, the bispecific aptamer is selected from r-AMSB107.0, r-AMSB108.0, r-AMSB107.1, r-AMSB107.2, r-AMSB107, r-AMSB107.4, r-AMSB107.5, r-AMSB107.6, r-AMSB107.7, r-AMSB107.8, r-AMSB107.9, r-AMSB107.10, r-AMSB107.11, r-AMSB107.12, r-AMSB107.13, r-AMSB107.14, r-AMSB107.15, r-AMSB107.16, r-AMSB107.17, r-AMSB107 .18, r-AMSB107.19, r-AMSB107.22, r-AMSB107.23, r-AMSB107.24, r-AMSB107.25, r-AMSB107.26, r-AMS B107.27, AMSB107.0, AMSB108.0, AMSB107.1, AMSB107.2, AMSB107, AMSB107.4, AMSB107.5, AMSB107.6, AMSB107.7, AMSB107.8, AMSB107.9, AMSB107.10, AMSB107.11, AMSB107.12, AMSB107.13, AMSB107.14, AM SB107.15, AMSB107.16, AMSB107.17, AMSB107.18, AMSB107.19, AMSB107.20, AMSB107.21, AMSB107.22, AMSB107.23, AMSB107.24, AMSB107.25, AMSB107.26, AMSB107.P6, AMSB107.P40K, AMSB107P and AMSB107.27.

[0160] In some embodiments, the bispecific aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 140-195, SEQ ID NOs: 202-204.

[0161] Composition

[0162] In one aspect, the present disclosure provides a composition comprising an anti-Ang2 aptamer of the present disclosure, an anti-VEGF aptamer of the present disclosure and / or a bispecific aptamer of the present disclosure.

[0163] In some embodiments, the composition is a pharmaceutical composition.

[0164] In some embodiments, the composition comprises a pharmaceutically acceptable excipient or carrier.

[0165] In some embodiments, the composition is used to prevent, treat and / or improve neovascular diseases, disorders or conditions. The neovascular diseases, disorders or conditions may be ocular neovascular diseases, disorders or conditions. In some embodiments, the neovascular diseases, disorders or conditions are selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion and diabetic retinopathy. In some embodiments, the neovascular diseases, disorders or conditions are selected from wet age-related macular degeneration, myopic choroidal neovascularization and proliferative diabetic retinopathy.

[0166] Medical uses and methods

[0167] 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 an anti-Ang2 aptamer of the present disclosure, an effective amount of an anti-VEGF aptamer of the present disclosure, an effective amount of a bispecific aptamer of the present disclosure and / or an effective amount of a composition of the present disclosure.

[0168] 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.

[0169] 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 for the manufacture of a medicament for preventing, treating and / or ameliorating a neovascular disease, disorder or condition.

[0170] 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.

[0171] Other aspects and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description, wherein only exemplary embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details can be modified in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and descriptions are to be regarded as illustrative, not restrictive.

[0172] Incorporated by Reference

[0173] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0174] The novel features of the present invention are set forth in detail in the appended claims. The features and advantages of the present invention will be better understood with reference to the following detailed description and the accompanying drawings (also referred to herein as "figures" and "FIG."), which set forth exemplary embodiments employing the principles of the present invention, wherein:

[0175] Figure 1A-1B The binding affinity of the anti-VEGF aptamers of the present disclosure is shown.

[0176] Figure 2 An in vitro VEGF receptor competition assay for the anti-VEGF aptamers of the present disclosure is shown.

[0177] Figure 3A shows the binding affinity of the anti-Ang2 aptamers of the present disclosure; Figure 3B Inhibition of binding of the anti-Ang2 aptamers of the present disclosure to human Ang2 and Tie2 is shown.

[0178] Figure 4A-4B The binding affinities of the bispecific aptamers of the present disclosure to VEGF and Ang2 are shown. Figure 4C-4D Shown is an in vitro VEGF and Ang2 receptor competition assay for the bispecific aptamers of the present disclosure.

[0179] Figures 5A-5C The secondary structures of the anti-VEGF aptamer, anti-Ang2 aptamer and bispecific aptamer of the present disclosure are shown.

[0180] Figures 6A-6C The effects of the anti-Ang2 aptamers of the present disclosure on neovascularization and vascular permeability are shown.

[0181] Figure 7A-7BShown are the results of the anti-VEGF aptamers of the present disclosure in an in vivo efficacy test in a non-human primate (NHP) laser-induced CNV model.

[0182] Figure 8 Representative monochrome imaging photographs of rabbit DL-AAA study results of the anti-VEGF aptamers of the present disclosure are shown.

[0183] Fig. 9 Representative FA photographs showing the results of rabbit DL-AAA studies of the anti-VEGF aptamers of the present disclosure. DETAILED DESCRIPTION

[0184] Although multiple embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many changes, modifications and substitutions may be made by those skilled in the art without departing from the present invention. It should be understood that a variety of alternatives to the embodiments of the present invention described herein may be employed.

[0185] As used herein, the term "aptamer" refers generally to a non-naturally occurring nucleic acid that has a desired effect on a target molecule. The desired effect may include, but is not limited to, binding to a target, catalytically altering a target, reacting with a target in a manner that modifies or changes a target or a functional activity of a target, covalently attaching to a target (such as in a suicide inhibitor), and promoting a reaction between a target and another molecule. In some embodiments, the effect may be a specific binding affinity to a target molecule, which is a three-dimensional chemical structure other than a polynucleotide that binds to a nucleic acid ligand by a mechanism unrelated to Watson / Crick base pairing or triple helix formation, wherein an aptamer is not a nucleic acid with a known physiological function of being bound by a target molecule. An aptamer for a given target includes a nucleic acid identified from a candidate nucleic acid mixture by a method in which the aptamer can be a ligand for the target, the method comprising: (a) contacting the candidate mixture with the target, wherein nucleic acids having increased affinity for the target relative to other nucleic acids in the candidate mixture can be separated from the remaining nucleic acids in the candidate mixture; (b) separating the nucleic acids with increased affinity from the remaining nucleic acids in the candidate mixture; and (c) amplifying the nucleic acids with increased affinity to produce a ligand-enriched nucleic acid mixture, thereby identifying an aptamer for the target molecule. It should be recognized that affinity interactions are a matter of degree; however, in this case, the "specific binding affinity" of an aptamer for its target means that the affinity with which the aptamer binds to its target is generally much higher than the affinity with which it binds to other non-target components in the mixture or sample. An "aptamer" or "nucleic acid ligand" is a set of copies of a type or species of nucleic acid molecules having a specific nucleotide sequence. An aptamer can include any suitable number of nucleotides. The aptamer can be DNA and / or RNA (or a combination of DNA and RNA), can be single-stranded, double-stranded or contain double-stranded or triple-stranded regions. In some cases, the aptamer can also include 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 can be replaced by 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 can be a hexaethylene glycol (H or PEG6) linker. Such substitutions may occur in any nucleotide of the aptamer, as long as such substitutions do not substantially affect the activity (e.g., binding activity or inhibitory activity) and / or function of the aptamer. In some cases, the substitutions do not substantially affect the secondary structure of the aptamer, for example, the substitutions may occur in nucleotides contained in the knob region or loop region of the secondary structure of the aptamer.In some cases, the substitution does not occur for any nucleotides included in the stem region of the secondary structure of the aptamer.

[0186] As used herein, the term "anti-VEGF aptamer" refers broadly to a nucleic acid molecule that has specific binding affinity for VEGF. In some cases, as defined herein, an 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. For example, one or more nucleotides of an anti-VEGF aptamer may be substituted with a polyethylene glycol moiety or a linker comprising a plurality (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) of polyethylene glycol moieties. In some cases, the linker can be a hexaethylene glycol (H or PEG6) linker. Such substitutions can occur in any nucleotide of the anti-VEGF aptamer, as long as such substitutions do not substantially affect the activity (e.g., binding activity or inhibitory activity) and / or function of the anti-VEGF aptamer. In some cases, the substitutions do not substantially affect the secondary structure of the anti-VEGF aptamer, for example, the substitutions can occur in nucleotides included in the bulge region or loop region of the secondary structure of the anti-VEGF aptamer. In some cases, the substitutions do not occur in any nucleotides included in the stem region of the secondary structure of the anti-VEGF aptamer.

[0187] As used herein, the term "VEGF" refers to vascular endothelial growth factor A (VEGFA or VEGF-A) and its splice variants and isoforms. Four different splice variants of VEGF can be found, referred to 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 in the form of disulfide-linked homodimers. In a variety of cell types, the secretion pattern of the isoforms is different, but VEGF-165 is observed to be the most common isoform. Although these isoforms bind to two receptors Flt-1 and Flt-1 / KDR with high affinity, they have different binding affinities for heparin and the extracellular matrix. The accession number of human VEGF in the NCBI database may be NP_001273973.1, NP_001028928.1, NP_001020540.2, NP_001020539.2, NP_001020538.2, NP_003367.4 or NP_001020537.2.

[0188] As used herein, the term "K D The term "value" generally refers to a dissociation constant, which is measured by a technique appropriate for the aptamer and target pair, such as by using a ligand binding assay, using radioactive or fluorescent measurements, surface plasmon resonance (SPR), biomembrane interferometry (BLI, e.g. System), SRU Biological System Isothermal titration calorimetry (ITC) or Microscale Thermophoresis (MST) measurement. In some embodiments, K D The values ​​are determined using standard fluorescence-based ligand binding assays and saturation analysis. In one example, multiple concentrations of fluorescently labeled target molecules are incubated with the particles of the present disclosure at room temperature for at least 3 hours under gentle rotation. Each sample is then washed, and the target bound is quantitatively maintained by measuring the fluorescence of each particle using a flow cytometer. Then, for example, by using an equilibrium binding model (e.g., according to the law of mass action), the background-subtracted fluorescence values ​​are fitted into a saturation binding curve.

[0189] The terms "specific binding" or "binds specifically to" or "specifically for" are used interchangeably herein and refer generally 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 portion thereof) that is measurably and / or statistically distinct from nonspecific interactions (e.g., nonspecific interactions may be binding to a reference molecule or a random molecule). Specific binding can be measured, for example, by competition with a control molecule similar to the target, such as an excess of unlabeled target, in which case specific binding is indicated if binding of the labeled target to the candidate agent is competitively inhibited by an excess of unlabeled target. K for the target D Molecules of at least about 1 nM, at least about 900 pM, at least about 800 pM, at least about 700 pM, at least about 600 pM, at least about 500 pM, at least about 400 pM, at least about 300 pM, at least about 200 pM, at least about 100 pM, at least about 90 pM, at least about 80 pM, at least about 70 pM, at least about 60 pM, at least about 50 pM, at least about 40 pM, at least about 30 pM, at least about 20 pM, at least about 10 pM, at least about 5 pM, at least about 1 pM or more can exhibit specific binding.

[0190] As used herein, the term "VEGF receptor binding domain" generally refers to a domain of VEGF that binds to or is recognized by a corresponding VEGF receptor.

[0191] As used herein, the term "VEGF-R" or "VEGF receptor" refers broadly to receptors that bind VEGF or VEGF family members, including their splice variants and isoforms. VEGFRs may include: (i) Flt-1 (fin-like tyrosine kinase), 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), a 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, 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 on vascular endothelium (Pajusola et al., Cancer Res (1992), 52:5738-43).

[0192] As used herein, the term "RNA aptamer" refers broadly to an aptamer comprising ribonucleotide units."RNA aptamer" is also meant to encompass RNA analogs as defined herein.

[0193] As used herein, the term "DNA aptamer" refers broadly to an aptamer comprising deoxyribonucleotide units."DNA aptamer" is also meant to encompass DNA analogs as defined herein.

[0194] As used herein, the term "highly soluble" refers broadly to the characteristic that an agent is soluble in a solvent (e.g., water or an appropriate buffer) to a relatively high degree (e.g., 80% or more by weight, 85% or more by weight, 90% or more by weight, 95% or more by weight, 99% or more by weight).

[0195] As used herein, the term "nuclease-resistant" refers generally to the characteristic of an agent that it can resist cleavage by a nuclease. In some cases, exonucleases acting on the 5' or 3' ends of nucleic acids can destroy nucleic acid molecules. In addition, endonucleases can cleave DNA or RNA at internal phosphodiester bonds between individual nucleotides. However, when a molecule (e.g., DNA or RNA) is resistant to nucleases (e.g., after certain modifications), exonucleases or endonucleases can no longer destroy or digest it.

[0196] As used herein, the term "modified nucleotide" refers to a nucleotide that is not naturally occurring, but is an analog or ester of a naturally occurring nucleotide. When used in the context of an oligonucleotide or nucleic acid molecule (e.g., an aptamer), it generally means that at least one of the four constituent nucleotides of the oligonucleotide (i.e., A, G, T / U, and C) is an analog or ester of a naturally occurring nucleotide. In some embodiments, modified nucleotides can make the oligonucleotide resistant to nucleases. In some embodiments, the modified nucleotides mainly cause hydrophobic interactions between the aptamer and its protein target, resulting in high binding efficiency and a stable co-crystallized complex. A pyrimidine having a substitution at the C-5 position is an example of a modified nucleotide. Modifications may include backbone modifications, methylation, rare base pairing combinations (e.g., isobases, isocytidine, and isoguanidine), etc. Modifications may also include 3' and 5' modifications, such as capping. Other modifications may include substitution of one or more natural nucleotides with analogs, internucleotide modifications, such as those with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and those with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), modifications with intercalators (e.g., acridine, psoralen, etc.), modifications comprising chelators (e.g., metals, radioactive metals, boron, oxidized metals, etc.), modifications comprising alkylating agents, and modifications with modified bonds (e.g., alpha anomeric nucleic acids, etc.). In addition, any of the hydroxyl groups typically present on the sugar of a nucleotide may be replaced by a phosphonate or phosphate group; protected with a standard protecting group; or activated to form additional bonds with another nucleotide or solid support. The 5' and 3' terminal OH groups can be phosphorylated or substituted with an amine, an organic end-capping group moiety having about 1 to about 20 carbon atoms, a polyethylene glycol (PEG) polymer in the range of about 10 kDa to about 80 kDa in some embodiments, a PEG polymer in the range of about 20 kDa to about 60 kDa in some embodiments, or other hydrophilic or hydrophobic biological or synthetic polymers. In some embodiments, the modification can be at the C-5 position of the pyrimidine. These modifications can be generated by amide linkages directly at the C-5 position or by other types of linkages.

[0197] As used herein, the term "2'-modified nucleotide" refers generally to a nucleotide having a modification of the 2' carbon atom. For example, cytidine, uridine, adenosine, guanosine can have a modification of the 2' carbon atom.

[0198] As used herein, the term "2'-fluoro-modified nucleotide" refers generally to a nucleotide having a 2'-fluoro substituent. 2'-deoxy-2'-fluoro modified oligonucleotides can be prepared by the method described in US Pat. No. 6,531,584.

[0199] As used herein, the term "cytidine" refers generally to a nucleoside molecule formed when cytosine is linked to a ribose ring (also known as furanose) via a β-N1-glycosidic bond. Cytidine may have the following structure:

[0200]

[0201] As used herein, the term "2'-modified cytidine" generally refers to a modified cytidine having a 2' carbon atom.

[0202] As used herein, the term "2'-deoxy-2'-fluorocytidine" refers generally to a chemical substance having the following structure:

[0203] As used herein, the term "2'-modified uridine" refers generally to a modified uridine having a 2' carbon atom. Uridine is a glycosylated pyrimidine analog containing uracil linked to a ribose ring (or more specifically, ribofuranose) via a β-N1-glycosidic bond. Uridine may have the following structure:

[0204] As used herein, the term "2'-deoxy-2'-fluorouridine" refers generally to a chemical substance having the following structure:

[0205] As used herein, the term "2'-modified adenosine" refers generally to a modified adenosine having a 2' carbon atom. Adenosine is a purine nucleoside composed of an adenine molecule linked to a ribose molecule (ribofuranose) moiety via a β-N9-glycosidic bond. Adenosine may have the following structure:

[0206] As used herein, the term "2'-deoxy-2'-fluoroadenosine" refers generally to a chemical substance having the following structure:

[0207] As used herein, the term "2'-modified guanosine" refers generally to modified guanosine having a 2' carbon atom. Guanosine is a purine nucleoside comprising guanine linked to a ribose (ribofuranose) ring via a β-N9-glycosidic bond. Guanosine may have the following structure:

[0208] As used herein, the term "2'-deoxy-2'-fluoroguanosine" refers generally to a chemical substance having the following structure:

[0209] As used herein, the term "nucleotide" refers generally to an organic molecule used as a monomeric unit to form the nucleic acid polymer deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleotides are composed of three subunit molecules: a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. Nucleosides are composed of a nitrogenous base and a 5-carbon sugar. Therefore, a nucleoside plus a phosphate group produces a nucleotide.

[0210] As used herein, the term "natural nucleotides" refers generally to naturally occurring nucleotides. For example, natural nucleotides may include cytidine, adenosine, guanosine, uridine. In some embodiments, "natural nucleic acids" also include synthetic or modified nucleotides that do not interfere with amplification and / or sequencing.

[0211] As used herein, the term "reference aptamer" broadly refers to an aptamer that competitively binds to a target with an aptamer of the present disclosure. In some embodiments, a reference aptamer may bind to the same target as an aptamer of the present disclosure.

[0212] As used herein, the term "competition" generally refers to the action of one chemical substance inhibiting another chemical substance by competing with it for binding or bonding. For example, a reference aptamer can compete with an aptamer of the present disclosure for binding to a target. As another example, an aptamer of the present disclosure can compete with a receptor or ligand of the target for binding to the target.

[0213] As used herein, the term "secondary structure" refers generally to the high-order structure of a nucleic acid molecule. The secondary structure can be formed by base pairing interactions within a single nucleic acid polymer or between two polymers. It can be expressed as a series of bases paired in a nucleic acid molecule. The secondary structure can generally be divided into a spiral (adjacent base pairs) and a variety of loops (unpaired nucleotides surrounded by a spiral). Typically, these elements or their combinations can be further divided into additional categories comprising stems, protuberances, loops and spirals. In the present disclosure, the secondary structure is determined by using the NUPACK software suite.

[0214] As used herein, the term "5'-3'" refers generally to the order of a DNA or RNA chain from the 5'-terminus to the 3'-terminus. The 5'-terminus may refer to the end of a DNA or RNA chain having the fifth carbon in the sugar ring of deoxyribose or ribose at the end. The phosphate group attached to the 5'-terminus allows the connection of two nucleotides, that is, the 5'-phosphate is covalently bound to the 3'-hydroxyl of another nucleotide, forming a phosphodiester bond. The 3'-terminus may refer to the third carbon in the sugar ring terminating at the hydroxyl group, also known as the tail end. The 3'-hydroxyl group is often required in the synthesis of new nucleic acid molecules because it binds (joins) the 5'-phosphate of a separate nucleotide, allowing the formation of a chain of connected nucleotides.

[0215] As used herein, the term "stem" broadly refers to a region of nucleic acid secondary structure, and the stem comprises a base-paired helix.

[0216] As used herein, the term "knob" broadly refers to a region of nucleic acid secondary structure where one strand of the helix has "extra" intervening bases that have no counterpart in the opposing strand in the secondary structure of the nucleic acid molecule.

[0217] As used herein, the term "loop" broadly refers to a region of nucleic acid secondary structure in which one or more bases of one strand of the helix do not have a complementary counterpart in the opposite strand in the secondary structure of the nucleic acid molecule.

[0218] As used herein, the term "consensus sequence" refers generally to certain positions (not necessarily adjacent) of an oligonucleotide being specified. "Specified" means that the composition of the position is not completely random. Not all oligonucleotides in a mixture will have the same nucleotide at that position; for example, a consensus sequence may contain specific nucleotides at a known ratio.

[0219] As used herein, the term "truncated form" refers generally to an aptamer that is truncated by the deletion of nucleotides but still has the desired or even improved binding characteristics. The length of the truncated form may vary depending on the length of the starting aptamer and is as defined herein with respect to the term "aptamer". The truncation in the truncated form may occur in the fixed region or the variable region of the starting aptamer, or in both the fixed region and the variable region.

[0220] As used herein, the term "modulate biological activity" generally refers to adjusting (eg, increasing or decreasing) the biological activity of a target molecule (eg, protein, such as a receptor or ligand). For example, the anti-VEGF aptamers of the present disclosure can be used to modulate the biological activity of VEGF and / or VEGF receptors.

[0221] As used herein, the term "neovascular disease or disorder" refers broadly to a disease or disorder associated with new blood vessel formation. Neovascular diseases or disorders may include ocular neovascular diseases (e.g., retinal and choroidal neovascular diseases), rubroiridis glaucoma, pterygium, solid tumor cancers, osteoarthritis, rheumatoid arthritis, vascular abnormalities and malformations (e.g., hemangiomas, lymphangiomas, etc.), and psoriasis.

[0222] As used herein, the term "ocular neovascular disease" broadly refers to a disease characterized by ocular neovascularization, ie, abnormal blood vessel development in the eye of a subject.

[0223] As used herein, the term "VEGF-related diseases, disorders or conditions" refers generally to diseases, disorders or conditions associated with abnormal activity or loss of activity of VEGF. For example, VEGF-related diseases, disorders or conditions may include abnormal angiogenesis. For example, VEGF-related diseases, disorders or conditions may include cancer, rheumatoid arthritis, bullous diseases (including bullous pemphigoid, dermatitis herpetiformis and erythema multiforme) and psoriasis.

[0224] As used herein, the term "age-related macular degeneration" or "AMD" refers generally to macular degeneration associated with aging. Macular degeneration can affect the central area of ​​the retina called the macula, and can cause gradual or rapid loss of vision to a level of 20 / 200 or lower. Moreover, AMD can also affect central vision, which is used for reading and watching television, for example, resulting in loss of central vision, but peripheral vision remains relatively intact.

[0225] As used herein, the term "diabetic retinopathy" or "DR" refers broadly to late microvascular complications of diabetes, such as type II diabetes. Diabetic retinopathy is a retinal microvascular disease that can present as a series of stages of increasing severity and worsening visual prognosis. DR can be broadly divided into 2 major clinical stages: non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR), where the term "proliferative" refers to the presence of retinal neovascularization.

[0226] As used herein, the term "diabetic macular edema" refers broadly to symptoms associated with any stage of diabetic retinopathy that cause a significant decrease in central visual acuity.

[0227] As used herein, the term "retinal vein occlusion" refers generally to a complete or partial blockage of a retinal blood vessel that restricts blood flow through the retinal tissue. The site of blockage is usually on the venous side and may be in a branch vessel (branch retinal vein occlusion-BRVO) or the central retinal vein (central retinal vein occlusion-CRVO).

[0228] As used herein, the term "myopic choroidal neovascularization" refers broadly to a disease that causes visual impairment in people with pathological myopia. When new blood vessels form in the macula, a pigmented fibrous scar often forms, which can create a dark spot in the center of vision. Myopic choroidal neovascularization is a type of choroidal neovascularization.

[0229] As used herein, the term "anti-VEGF agent" broadly refers to compounds that inhibit the activity or production of vascular endothelial growth factor ("VEGF").

[0230] As used herein, the term "composition" refers broadly to the distribution of individual substances that make up a mixture. The individual substances can be named as components.

[0231] As used herein, the term "pharmaceutical composition" refers generally to a preparation prepared in a form suitable for pharmaceutical administration. A pharmaceutical composition can generally be 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, such as intravenous, intradermal, subcutaneous, inhalation, topical, transdermal, transmucosal, and rectal administration.

[0232] As used herein, the term "pharmaceutically acceptable excipient or carrier" refers broadly to any excipient or carrier approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" may refer to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered, including, but not limited to, sterile liquids such as water and oils.

[0233] As used herein, the term "effective amount" refers broadly to an amount required 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" when referring to an anti-VEGF aptamer, anti-Ang2 aptamer or bispecific aptamer, composition or agent of the present disclosure means that amount of the aptamer will provide the desired specific pharmacological response after administration to a large number of individuals in need of such treatment. It is emphasized that a therapeutically effective amount of an aptamer administered to a particular individual under a particular situation will not always be effective in treating the condition / disease described herein, even if such a dosage is considered by one skilled in the art to be a therapeutically effective amount.

[0234] As used herein, the term "Ang2" refers generally to angiopoietin-2, which is an angiogenic factor belonging to the E2F family. Ang2 is considered to be a naturally occurring antagonist of Tie2. Unless otherwise expressly stated, the term should be considered to cover not only human Ang2, but also Ang2 from multiple species. The term "Ang2" also covers functional fragments and components of full-length Ang2. Human Ang2 is also known as Ensembl: ENSG00000091879 or MIM: 601922.

[0235] As used herein, the term "the interaction between Ang2 and Tie2" generally refers to the activity of Ang2 binding to Tie2. Tie2 is an endothelial receptor tyrosine kinase (RTK) that is associated with both embryonic vascular development and pathological angiogenesis.

[0236] As used herein, the term "substantially does not bind" refers generally to almost no binding to a specific substance. For example, the aptamers of the present disclosure can hardly (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%) bind to their target.

[0237] As used herein, the term "anti-Ang2 aptamer" refers generally to a nucleic acid molecule that has specific binding affinity for Ang2. In some cases, as defined herein, an 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. For example, one or more nucleotides of an anti-Ang2 aptamer may be substituted with a polyethylene glycol moiety or a linker comprising a plurality (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) of polyethylene glycol moieties. In some cases, the linker can be a hexaethylene glycol (H or PEG6) linker. Such substitutions can occur in any nucleotide of the anti-VEGF aptamer, as long as it does not substantially affect the activity (e.g., binding activity or inhibitory activity) and / or function of the anti-Ang2 aptamer. In some cases, the substitutions do not substantially affect the secondary structure of the anti-Ang2 aptamer, for example, the substitutions can occur in nucleotides included in the protuberance region or loop region of the secondary structure of the anti-Ang2 aptamer. In some cases, the substitutions do not occur in any nucleotides included in the stem region of the secondary structure of the anti-Ang2 aptamer.

[0238] As used herein, the term "anti-Ang2 agent" broadly refers to an agent that inhibits the activity or production of Ang2.

[0239] As used herein, the term "aflibercept" refers generally to a recombinant fusion protein consisting of the vascular endothelial growth factor (VEGF) binding portion from the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1 immunoglobulin.

[0240] As used herein, the term "linker" generally refers to an agent designed to facilitate the functional connection of two parts into one connected part. For example, a linker can be used to make a single strand to achieve a desired biological activity. For example, a linker can be used to make a single strand to impart the stability required to produce connected nucleotides.

[0241] As used herein, the term "nucleotide linker" refers broadly to a linker made of nucleotides. The length of a nucleotide linker connecting adjacent nucleotides can vary.

[0242] As used herein, the term "modified nucleotide linker" generally refers to a nucleotide linker having one or more modifications.

[0243] As used herein, the term "hydrocarbon joint" refers generally to a joint made of hydrocarbons. Hydrocarbons are organic compounds composed entirely of hydrogen and carbon. Examples of hydrocarbons include, but are not limited to, for example, methane, ethane, propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane.

[0244] As used herein, the term "polyethylene glycol linker" broadly refers to a linker made of one or more polyethylene glycol (PEG) subunits.

[0245] As used herein, the term "fragment thereof" generally refers to a partial region that can be obtained from a complete parent molecule (e.g., an aptamer of the present disclosure). A fragment can still retain the properties of the parent molecule from which it is derived. In some cases, a fragment can provide many advantages over a complete parent molecule for manufacturing and / or medical use.

[0246] As used herein, the term "variant" refers generally to an aptamer molecule that differs from a parent molecule (e.g., an aptamer) by at least one nucleotide. A variant may refer to the molecule itself, a composition comprising the molecule. When such a molecule is DNA or RNA, it also refers to the nucleotide sequence of the molecule. In some cases, a variant is different from its parent molecule (e.g., an aptamer) by adding, deleting, or replacing one or more nucleotides, e.g., 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 nucleotides. In some cases, a variant may have 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 more) sequence homology / identity with the nucleotide sequence of its parent molecule.

[0247] As used herein, the term "reducing neovascularization" refers broadly to reducing the extent of neovascularization. Neovascularization is the natural formation of new human blood vessels, which are usually in the form of a functional microvascular network that is able to be filled with red blood cells and serve as collateral circulation in response to local hypoperfusion or ischemia. In this disclosure, it is used interchangeably with "angiogenesis".

[0248] As used herein, the term "reducing vascular permeability" refers generally to the degree of reducing vascular permeability in a tissue. It is believed that changes in vascular permeability play a role in 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 the disease conditions associated with angiogenesis and / or increased vascular permeability can be cancer, diabetes, psoriasis, rheumatoid arthritis, Kaposi's sarcoma, hemangioma, acute and chronic kidney disease, atherosclerosis, arterial restenosis, autoimmune diseases, acute inflammation, excessive scarring and adhesions, endometriosis, functional menstrual disorders, and eye diseases with retinal vascular proliferation.

[0249] As used herein, the term "mouse oxygen-induced ischemic retinopathy (OIR) model" refers generally to an animal model of oxygen-induced retinopathy in mice with reproducible and quantifiable proliferative retinal neovascularization. This model is suitable for examining the pathogenesis and therapeutic intervention of retinal neovascularization in retinopathy of prematurity (ROP) and other vascular diseases. The preparation of this model is described, for example, in Smith LE et al., Oxygen-induced retinopathy in the mouse. Invest. Opthalmol. Vis. Sci. 1994; 35: 101-111.

[0250] As used herein, the term "Ang2-related disease, disorder or condition" refers generally to a disease, disorder or condition associated with abnormal activity or loss of activity of Ang2. For example, an Ang2-related disease, disorder or condition has an effect on angiogenesis. Examples of such diseases, disorders or conditions may be cancer or carcinoma, eye diseases (e.g., age-related macular degeneration and diabetic retinopathy) and / or chronic kidney disease (e.g., diabetic nephropathy, post-renal failure, pre-renal azotemia and internal renal failure).

[0251] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a particle" includes a plurality of such particles, reference to "the sequence" includes reference to one or more of the sequences and equivalents thereof known to those skilled in the art, and so forth.

[0252] As used herein, the term "about" refers to an approximation of a given value based on reasonable inferences made by one of ordinary skill in the art, including equivalents and approximations resulting from experimental and / or measurement conditions of such given values. For example, it may refer to a value that is up to 10% higher or lower than the value modified by the term.

[0253] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can also be used to practice or test the present invention, 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 to which the cited publications are related.

[0254] As will be apparent to those skilled in the art upon reading this disclosure, each of the various embodiments described and illustrated herein has discrete components and features that are readily separated or combined with features of any of the other several embodiments without departing from the scope or spirit of the invention. Any narrated method may be performed in the order of events narrated or in any other order that is logically possible. It is intended to provide support for all such combinations.

[0255] Anti-VEGF Aptamer

[0256] In one aspect, the present disclosure provides an anti-VEGF aptamer that has a K of about 20 nanomolar (nM) or less. D The value binds to VEGF-121 with a K of about 20 nanomolar (nM) or less. D Values ​​are bound to VEGF-165.

[0257] For example, the anti-VEGF aptamer can be 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, or about 1 nanomolar (nM) or less. 0.05 nanomolar (nM) or less, 0.04 nanomolar (nM) or less, 0.04 nanomolar (nM) or less, 0.05 nanomolar (nM) or less, 0.06 nanomolar (nM) or less, 0.01 nanomolar (nM) or less, 0.01 nanomolar (nM) or less, 0.02 nanomolar (nM) or less, 0.03 nanomolar (nM) or less, 0.06 nanomolar (nM) or less, 0.07 nanomolar (nM) or less, 0.08 nanomolar (nM) or less, 0.07 nanomolar (nM) or less, 0.06 nanomolar (nM) or less, 0.08 nanomolar (nM) or less, 0.09 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. D Values ​​are bound to VEGF-121.

[0258] For example, the anti-VEGF aptamer can be 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. D Values ​​are bound to VEGF-165.

[0259] VEGF-121 may be human VEGF-121, mouse VEGF-120, monkey VEGF-121, rabbit VEGF-121 and / or rat VEGF- 120. VEGF-165 may be human VEGF-165, mouse VEGF-164, monkey VEGF-165, rabbit VEGF-165 and / or rat VEGF-164.

[0260] Human VEGF-121 (NCBI No. P15692-9) may comprise the amino acid sequence as shown in SEQ ID NO: 29. Mouse VEGF-120 (NCBI No. Q00731-3) may comprise the amino acid sequence as shown in SEQ ID NO: 44. Rat VEGF-120 (NCBI No. P16612-4) may comprise the amino acid sequence as shown in SEQ ID NO: 42.

[0261] Human VEGF-165 (NCBI No. P15692-4) may comprise the amino acid sequence as shown in SEQ ID NO: 35. Mouse VEGF-164 (NCBI No. Q00731-2) may comprise the amino acid sequence as shown in SEQ ID NO: 43. Monkey VEGF-165 (NCBI No. H9EQI6) may comprise the amino acid sequence as shown in SEQ ID NO: 35. Rat VEGF-164 (NCBI No. P16612-2) may comprise the amino acid sequence as shown in SEQ ID NO: 39.

[0262] Rabbit VEGF-189 (NCBI No. G1T1L9) may comprise the amino acid sequence as shown in SEQ ID NO:36.

[0263] The anti-VEGF aptamer can specifically bind to both: 1) the VEGF receptor binding domain of VEGF-121 or a fragment thereof; and 2) the VEGF receptor binding domain of VEGF-165 or a fragment thereof.

[0264] The VEGF receptor binding domain of VEGF-121 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 30-34, 40, 41, and 45. The VEGF receptor binding domain of VEGF-165 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 30-34, 40, and 41.

[0265] The receptor binding domain 1 of human VEGF-121 may comprise the amino acid sequence as shown in SEQ ID NO: 30. The receptor binding domain 2 of human VEGF-121 may comprise the amino acid sequence as shown in SEQ ID NO: 31. The receptor binding domain 3 of human VEGF-121 may comprise the amino acid sequence as shown in SEQ ID NO: 32. The receptor binding domain 4 of human VEGF-121 may comprise the amino acid sequence as shown in SEQ ID NO: 33. The receptor binding domain 5 of human VEGF-121 may comprise the amino acid sequence as shown in SEQ ID NO: 34.

[0266] The receptor binding domain 1 of human VEGF-165 may comprise the amino acid sequence as shown in SEQ ID NO: 30. The receptor binding domain 2 of human VEGF-165 may comprise the amino acid sequence as shown in SEQ ID NO: 31. The receptor binding domain 3 of human VEGF-165 may comprise the amino acid sequence as shown in SEQ ID NO: 32. The receptor binding domain 4 of human VEGF-165 may comprise the amino acid sequence as shown in SEQ ID NO: 33. The receptor binding domain 5 of human VEGF-165 may comprise the amino acid sequence as shown in SEQ ID NO: 34.

[0267] The receptor binding domain 1 of monkey VEGF-165 may comprise the amino acid sequence as shown in SEQ ID NO: 30. The receptor binding domain 2 of monkey VEGF-165 may comprise the amino acid sequence as shown in SEQ ID NO: 31. The receptor binding domain 3 of monkey VEGF-165 may comprise the amino acid sequence as shown in SEQ ID NO: 32. The receptor binding domain 4 of monkey VEGF-165 may comprise the amino acid sequence as shown in SEQ ID NO: 33. The receptor binding domain 5 of monkey VEGF-165 may comprise the amino acid sequence as shown in SEQ ID NO: 34.

[0268] The receptor binding domain 1 of rabbit VEGF-189 may comprise the amino acid sequence as shown in SEQ ID NO: 37. The receptor binding domain 2 of rabbit VEGF-189 may comprise the amino acid sequence as shown in SEQ ID NO: 31. The receptor binding domain 3 of rabbit VEGF-189 may comprise the amino acid sequence as shown in SEQ ID NO: 38. The receptor binding domain 4 of rabbit VEGF-189 may comprise the amino acid sequence as shown in SEQ ID NO: 33. The receptor binding domain 5 of rabbit VEGF-189 may comprise the amino acid sequence as shown in SEQ ID NO: 34.

[0269] The receptor binding domain 1 of rat VEGF-164 may comprise the amino acid sequence as shown in SEQ ID NO: 30. The receptor binding domain 2 of rat VEGF-164 may comprise the amino acid sequence as shown in SEQ ID NO: 31. The receptor binding domain 3 of rat VEGF-164 may comprise the amino acid sequence as shown in SEQ ID NO: 40. The receptor binding domain 4 of rat VEGF-164 may comprise the amino acid sequence as shown in SEQ ID NO: 33. The receptor binding domain 5 of rat VEGF-164 may comprise the amino acid sequence as shown in SEQ ID NO: 41.

[0270] The receptor binding domain 1 of rat VEGF-120 may comprise the amino acid sequence as shown in SEQ ID NO: 30. The receptor binding domain 2 of rat VEGF-120 may comprise the amino acid sequence as shown in SEQ ID NO: 31. The receptor binding domain 3 of rat VEGF-120 may comprise the amino acid sequence as shown in SEQ ID NO: 40. The receptor binding domain 4 of rat VEGF-120 may comprise the amino acid sequence as shown in SEQ ID NO: 33. The receptor binding domain 5 of rat VEGF-120 may comprise the amino acid sequence as shown in SEQ ID NO: 41.

[0271] The receptor binding domain 1 of mouse VEGF-164 may comprise the amino acid sequence as shown in SEQ ID NO: 30. The receptor binding domain 2 of mouse VEGF-164 may comprise the amino acid sequence as shown in SEQ ID NO: 31. The receptor binding domain 3 of mouse VEGF-164 may comprise the amino acid sequence as shown in SEQ ID NO: 40. The receptor binding domain 4 of mouse VEGF-164 may comprise the amino acid sequence as shown in SEQ ID NO: 33. The receptor binding domain 5 of mouse VEGF-164 may comprise the amino acid sequence as shown in SEQ ID NO: 41.

[0272] The receptor binding domain 1 of mouse VEGF-120 may comprise the amino acid sequence as shown in SEQ ID NO: 30. The receptor binding domain 2 of mouse VEGF-120 may comprise the amino acid sequence as shown in SEQ ID NO: 31. The receptor binding domain 3 of mouse VEGF-120 may comprise the amino acid sequence as shown in SEQ ID NO: 40. The receptor binding domain 4 of mouse VEGF-120 may comprise the amino acid sequence as shown in SEQ ID NO: 45. The receptor binding domain 5 of mouse VEGF-120 may comprise the amino acid sequence as shown in SEQ ID NO: 41.

[0273] The anti-VEGF aptamer may inhibit the interaction between VEGF-121 and VEGF-R1. The anti-VEGF aptamer may inhibit the interaction between VEGF-121 and VEGF-R2. The anti-VEGF aptamer may inhibit the interaction between VEGF-165 and VEGF-R1. The anti-VEGF aptamer may simultaneously or additionally inhibit the interaction between VEGF-165 and VEGF-R2.

[0274] The anti-VEGF aptamers of the present disclosure are capable of reducing and / or ameliorating neovascularization and / or leakage in animal models, such as ocular animal models.

[0275] The animal model can be a vertebrate animal model, and in some cases, the animal model can be a mammalian model. The animal can be a rat, mouse, rabbit, goat, sheep, non-human primate, or any other suitable animal.

[0276] In some embodiments, the anti-VEGF aptamers of the present disclosure are capable of reducing and / or ameliorating damage and / or leakage in a laser-induced choroidal neovascularization (CNV) model, such as a rat model or a non-human primate (NHP) model.

[0277] The anti-VEGF aptamers of the present disclosure are highly soluble. For example, the anti-VEGF aptamer has a relatively high degree of solubility in a solvent (e.g., 80% or more of the entire weight, 85% or more of the entire weight, 90% or more of the entire weight, 95% or more of the entire weight, 99% or more of the entire weight). For example, the anti-VEGF aptamer can be formulated to a concentration of 100 mg / ml or more, or 150 mg / ml or more, such as 200 mg / ml or more.

[0278] The anti-VEGF aptamer can be an RNA aptamer, a DNA aptamer, or a combination thereof. For example, the anti-VEGF aptamer can comprise primarily RNA, it can comprise primarily DNA, or it can comprise both RNA and DNA.

[0279] The anti-VEGF aptamer can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) modified nucleotides. In some cases, all nucleotides of the anti-VEGF aptamer can be modified nucleotides. For example, the anti-VEGF aptamer can have 20 nucleotides, all of which can be modified nucleotides.

[0280] In some cases, the anti-VEGF aptamers of the present disclosure may be resistant to nucleases.

[0281] The modified nucleotide may comprise a chemical substitution or modification at one or more positions independently selected from the ribose position, the deoxyribose position, the phosphate position and the base position. The modified nucleotide may comprise one or more modifications independently selected from the following: 2'-position sugar modification, 2'-amino (2'-NH2) modification, 2'-fluoro (2'-F) modification, 2'-O-methyl (2'-OMe) modification, 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, 5-position modified pyrimidine, modification at the amine outside the cytosine ring, substitution with 5'-bromouracil, substitution with 5'-bromodeoxyuridine, substitution with 5'-bromodeoxycytidine, backbone modification, locked nucleic acid (LNA), methylation, 3' cap and 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-benzylcarboxamide)-2'-deoxyuridine, 5-(N-isobutylcarboxamide)-2'-deoxyuridine, 5-(N-naphthylmethylcarboxamide)-2'-deoxyuridine and 5-(N-tryptaminecarboxamide)-2'-deoxyuridine.

[0282] 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 nucleotides may be 2'-modified nucleotides. The 2'-modified nucleotides may be selected from 2'-amino-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides and 2'-O-(2-methoxyethyl)-modified nucleotides.

[0283] In some cases, all cytidines may be 2'-modified cytidines. In some cases, all cytidines may be 2'-fluoro-modified cytidines and / or 2'-O-methyl-modified cytidines. In some cases, all cytidines may be 2'-deoxy-2'-fluoro cytidines. In some cases, all uridines may be 2'-modified uridines. In some cases, all uridines may be 2'-fluoro-modified uridines and / or 2'-O-methyl-modified uridines. In some cases, all uridines may be 2'-deoxy-2'-fluoro uridines. In some cases, all adenosines may be 2'-modified adenosines. In some cases, all adenosines may be 2'-fluoro-modified adenosines and / or 2'-O-methyl-modified adenosines. In some cases, all adenosines may be 2'-deoxy-2'-fluoro adenosines. In some cases, all guanosines may be 2'-modified guanosines. In some cases, all guanosines may be 2'-fluoro-modified guanosines and / or 2'-O-methyl-modified guanosines. In some cases, all guanosines may be 2'-deoxy-2'-fluoroguanosines.

[0284] For example, the anti-VEGF aptamer comprises at least one 2'-modified nucleotide. In some embodiments, all nucleotides of the anti-VEGF aptamer may be 2'-modified nucleotides. In some embodiments, all 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 nucleotide may be a 2'-fluoro (2'-F)-modified nucleotide or a 2'-fluoro-2'-deoxy-modified nucleotide. For example, the 2'-fluoro-modified nucleotide may comprise the following structure:

[0285] In some cases, the anti-VEGF aptamer does not comprise any native nucleotides (eg, unmodified A, C, T, U, or G).

[0286] 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.

[0287] The anti-VEGF aptamer can compete with the anti-VEGF reference aptamer for binding to VEGF-165 and / or VEGF-121.

[0288] The anti-VEGF reference aptamers or anti-VEGF aptamers disclosed herein may comprise a nucleotide sequence with or without modification. For example, an aptamer may comprise natural nucleotides and / or modified nucleotides. When an aptamer is defined as comprising or consisting of a nucleotide sequence as shown in a particular SEQ ID NO, the nucleotides within the sequence may be further modified.

[0289] According to the present disclosure, the anti-VEGF aptamer r-AMS0431.0 has natural nucleotides, and its nucleotide sequence is shown in SEQ ID NO: 46; the anti-VEGF aptamer AMS0431.0 has corresponding 2'-fluoro-modified nucleotides, and its nucleotide sequence is shown in SEQ ID NO: 91. Therefore, an aptamer comprising the nucleotide sequence shown in SEQ ID NO: 46 can cover both r-AMS0431.0 and AMS0431.0, but an aptamer comprising the nucleotide sequence shown in SEQ ID NO: 91 cannot cover r-AMS0431.0.

[0290] Similarly, in the present disclosure, generally, aptamers (e.g., aptamers of the present disclosure) designated as "r-AMSxxxx" (e.g., r-AMS0433) have natural nucleotides (but may include a linker, such as an H linker as described herein), while aptamers including corresponding 2′-fluoro-modified nucleotides are designated as "AMSxxxx" (e.g., AMS0433).

[0291] The anti-VEGF reference aptamer or anti-VEGF aptamer of the present disclosure may comprise a secondary structure, the secondary structure (e.g., from 5' to 3' direction) comprising a first stem, a first loop, a second stem, and a second loop. The anti-VEGF aptamer may comprise a secondary structure, the secondary structure (e.g., from 5' to 3' direction) consisting of a first stem, a first loop, a second stem, and a second loop.

[0292] An anti-VEGF reference aptamer or an anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 136-137.

[0293] Alternatively or additionally, an anti-VEGF reference aptamer or an anti-VEGF aptamer of the present disclosure may comprise a second consensus sequence comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 138-139.

[0294] For example, the anti-VEGF reference aptamer or anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as shown in SEQ ID NO: 136, and a second consensus sequence comprising a nucleotide sequence as shown in SEQ ID NO: 138; the anti-VEGF reference aptamer or anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence comprising a nucleotide sequence as shown in SEQ ID NO: 137, and a second consensus sequence comprising a nucleotide sequence as shown in SEQ ID NO: 139.

[0295] The first consensus sequence may include nucleotide sequences that form the first stem and first loop of an anti-VEGF reference aptamer or anti-VEGF aptamer of the present disclosure. In some cases, the first consensus sequence may not be included in the nucleotide sequences that form the second stem or second loop of an anti-VEGF reference aptamer or anti-VEGF aptamer of the present disclosure.

[0296] The second consensus sequence may include nucleotide sequences that form the second stem and the second loop of the anti-VEGF reference aptamer or anti-VEGF aptamer of the present disclosure. In some cases, the second consensus sequence may not be included in the nucleotide sequences that form the first stem or the first loop of the anti-VEGF reference aptamer or anti-VEGF aptamer of the present disclosure.

[0297] An anti-VEGF reference aptamer or an anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence and a second consensus sequence, wherein the first consensus sequence may be located 5′ relative to the second consensus sequence.

[0298] In some cases, an anti-VEGF reference aptamer or an anti-VEGF aptamer of the present disclosure may comprise a first consensus sequence and a second consensus sequence, wherein the second consensus sequence may be located 5′ relative to the first consensus sequence.

[0299] The anti-VEGF reference aptamer or anti-VEGF aptamer of the present disclosure may comprise a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, or a variant or truncated form thereof. The variant may comprise a nucleotide sequence that is at least 50% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201. The variant may comprise a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, having 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 additions, deletions and / or substitutions.

[0300] 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.

[0301] Anti-VEGF aptamers can be used to modulate the biological activity of VEGF or VEGF receptors.

[0302] Anti-VEGF aptamers can be used to prevent, treat and / or improve VEGF-related diseases, disorders or conditions. For example, VEGF-related diseases, disorders or conditions can be neovascular diseases, disorders or conditions. For example, VEGF-related diseases, disorders or conditions can be ocular neovascular diseases, disorders or conditions. For example, VEGF-related diseases, disorders or conditions can be selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion and diabetic retinopathy. For example, VEGF-related diseases, disorders or conditions can be selected from wet age-related macular degeneration, myopic choroidal neovascularization and proliferative diabetic retinopathy.

[0303] In one aspect, the present disclosure provides an anti-VEGF agent comprising an 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' end and / or the 3' end of the anti-VEGF aptamer.

[0304] In another aspect, the present disclosure provides a composition comprising the anti-VEGF aptamer and / or anti-VEGF agent of the present application.

[0305] In some cases, the composition can be a pharmaceutical composition. For example, the composition can include a pharmaceutically acceptable excipient or carrier.

[0306] In some cases, the composition may be presented in discrete dosage forms, each containing a predetermined amount of the active ingredient in powder form, or in the form of granules, a solution or suspension in an aqueous or non-aqueous liquid. Such dosage forms may be prepared by any method known to the skilled artisan, for example, it may include the step of bringing the active ingredient into association with a carrier that constitutes one or more other ingredients. In general, the composition is prepared by uniformly and intimately mixing the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired appearance.

[0307] The composition may include a therapeutically effective amount of an active composition (e.g., an anti-VEGF aptamer or anti-VEGF agent of the present disclosure). A therapeutically effective amount may be an amount of the subject composition that is capable of preventing and / or curing (at least in part) the condition or disorder and / or any complications thereof in a subject suffering from a neovascular disease or disorder or at risk of developing the disease or disorder. The specific amount / concentration of the active agent included may vary depending on the method of administration and the needs of the patient, and may be determined based on, for example, the patient's volume, viscosity and / or body weight, etc. For example, a suitable dosage may be from about 0.1 mg or 1 mg / kg per day to about 50 mg / kg per day; sometimes, the dosage may be higher. It should be understood that these specific dosages are preferably adjusted by a skilled person in the art (e.g., a physician or pharmacist) based on the condition, formulation and / or disease of the particular patient.

[0308] The composition may also contain an effective amount of additional therapeutically active components, such as additional therapeutically active components for preventing, treating and / or ameliorating neovascular diseases or conditions. Each active component may be present in the pharmaceutical composition in a pharmaceutically effective amount. The anti-VEGF aptamers of the present application may or may not be associated with additional active components.

[0309] Non-limiting exemplary compositions and methods for preparing such compositions are described below. The composition can be, for example, in a form suitable for oral administration, such as tablets, capsules, pills, powders, sustained release formulations, solutions, suspensions; suitable for parenteral injection, such as sterile solutions, suspensions or emulsions; suitable for topical administration, such as ointments or creams; or suitable for rectal administration, such as suppositories. The composition can be in a unit dosage form suitable for a single administration of a precise dose. In some embodiments, the composition can be a liquid pharmaceutical composition.

[0310] In the composition, the anti-VEGF aptamer can be combined with a pharmaceutical carrier in an intimate mixture according to conventional pharmaceutical compounding techniques. The carrier can take a variety of forms, depending on the form of preparation desired for administration.

[0311] The composition may also include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, but are not limited to, anti-adherents, defoaming compositions, buffer compositions, polymers, antioxidants, preservatives, chelating compositions, viscosity modifiers, tonicity agents, flavoring agents, coloring agents, flavor enhancers, opacifiers, suspending compositions, adhesives, fillers, plasticizers, lubricants and / or mixtures thereof.

[0312] In another aspect, the present disclosure provides a method for modulating the biological activity of VEGF or a VEGF receptor, comprising administering an effective amount of an anti-VEGF aptamer or an anti-VEGF agent of the present disclosure to a subject in need thereof.

[0313] In another aspect, the present disclosure provides a method for preventing, treating and / or ameliorating a VEGF-related disease, disorder or condition, comprising administering an effective amount of an anti-VEGF aptamer or anti-VEGF agent of the present disclosure to a subject in need thereof.

[0314] For example, a VEGF-related disease, disorder or condition can be a neovascular disease, disorder or condition. For example, a VEGF-related disease, disorder or condition can be an ocular neovascular disease, disorder or condition. For example, a VEGF-related disease, disorder or condition can be selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion, and diabetic retinopathy. For example, a VEGF-related disease, disorder or condition can be selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0315] In one aspect, the present disclosure provides the use of an anti-VEGF aptamer or an anti-VEGF agent of the present disclosure for the manufacture of a medicament for modulating the biological activity of VEGF or a VEGF receptor.

[0316] In one aspect, the present disclosure provides use of the aptamer of the present disclosure or the anti-VEGF agent of the present disclosure for the manufacture of a medicament for preventing, treating and / or ameliorating a VEGF-related disease, disorder or condition.

[0317] VEGF-related diseases, disorders or conditions can be neovascular diseases, disorders or conditions. For example, VEGF-related diseases, disorders or conditions can be ocular neovascular diseases, disorders or conditions. For example, VEGF-related diseases, disorders or conditions can be selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion and diabetic retinopathy. For example, VEGF-related diseases, disorders or conditions can be selected from wet age-related macular degeneration, myopic choroidal neovascularization and proliferative diabetic retinopathy.

[0318] Anti-Ang2 Aptamer

[0319] In one aspect, the present disclosure provides an aptamer that has a K of about 500 picomolar (pM) or less. D For example, the anti-Ang2 aptamer may have a K 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. D Values ​​are bound to Ang2.

[0320] Ang2 can be human Ang2, mouse Ang2, monkey Ang2, rabbit Ang2 and / or rat Ang2.

[0321] The anti-Ang2 aptamer is substantially unable to bind to human Ang1. For example, the affinity of the anti-Ang2 aptamer binding to Ang1 may be significantly lower than its affinity for Ang2 (at least 1-fold lower, at least 2-fold lower, at least 3-fold lower, at least 4-fold lower, or more). In some cases, the K of the anti-Ang2 aptamer binding to Ang1 is D Values ​​may not be determined using ligand binding assays, using radioactivity or fluorescence measurements, surface plasmon resonance (SPR), biomembrane interferometry (BLI, e.g. System), SRU Biological System Isothermal Titration Calorimetry (ITC) or Microscale Thermophoresis (MST) measured.

[0322] Anti-Ang2 aptamers can inhibit the interaction between Ang2 and Tie2.

[0323] The anti-Ang2 aptamers disclosed herein can reduce and / or improve neovascularization and / or vascular permeability in an animal model, such as an eye animal model. For example, the eye animal model can be an oxygen-induced ischemic retinopathy (OIR) model. The animal model can be a vertebrate animal model, and in some cases, the animal model can be a mammalian model. The animal can be a rat, a mouse, a rabbit, a goat, a sheep, a non-human primate, or any other suitable animal.

[0324] The anti-Ang2 aptamers of the present disclosure are capable of reducing neovascularization and / or decreasing vascular permeability in a mouse oxygen-induced ischemic retinopathy (OIR) model.

[0325] The anti-Ang2 aptamers of the present disclosure are highly soluble. For example, the solubility of the anti-Ang2 aptamer in the solvent is relatively high (e.g., 80% or more of the whole weight, 85% or more of the whole weight, 90% or more of the whole weight, 95% or more of the whole weight, 99% or more of the whole weight). For example, the anti-Ang2 aptamer can be formulated to a concentration of 100 mg / ml or more, or 150 mg / ml or more, such as a concentration of at least 200 mg / ml.

[0326] 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.

[0327] The anti-Ang2 aptamer may comprise one or more modified nucleotides. In some cases, all nucleotides of the anti-Ang2 aptamer may be modified nucleotides.

[0328] For example, a 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. A modified nucleotide may comprise one or more modifications independently selected from the following: 2'-position sugar modification, 2'-amino (2'-NH2) modification, 2'-fluoro (2'-F) modification, 2'-O-methyl (2'-OMe) modification, 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, 5-position modified pyrimidine, modification at the amine outside the cytosine ring, substitution with 5'-bromouracil, substitution with 5'-bromodeoxyuridine, substitution with 5'-bromodeoxycytidine, backbone modification, locked nucleic acid (LNA), methylation, 3' cap, and 5' cap. The 5-position modified pyrimidine can 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-benzylcarboxamide)-2′-deoxyuridine, 5-(N-isobutylcarboxamide)-2′-deoxyuridine, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine and 5-(N-tryptaminecarboxamide)-2′-deoxyuridine.

[0329] 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 nucleotides of the anti-Ang2 aptamer may be 2'-modified nucleotides. The 2'-modified nucleotides may be selected from 2'-amino-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, and 2'-O-(2-methoxyethyl)-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 structures:

[0330] 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.

[0331] In some cases, all cytidines of the anti-Ang2 aptamer may be 2'-modified cytidines. In some cases, all cytidines may be 2'-fluoro-modified cytidines and / or 2'-O-methyl-modified cytidines. In some cases, all cytidines may be 2'-deoxy-2'-fluoro cytidines. In some cases, all uridines may be 2'-modified uridines. In some cases, all uridines may be 2'-fluoro-modified uridines and / or 2'-O-methyl-modified uridines. In some cases, all uridines may be 2'-deoxy-2'-fluoro uridines. In some cases, all adenosines may be 2'-modified adenosines. In some cases, all adenosines may be 2'-fluoro-modified adenosines and / or 2'-O-methyl-modified adenosines. In some cases, all adenosines may be 2'-deoxy-2'-fluoro adenosines. In some cases, all guanosines may be 2'-modified guanosines. In some cases, all guanosines can be 2'-fluoro-modified guanosines and / or 2'-O-methyl-modified guanosines. In some cases, all guanosines can be 2'-deoxy-2'-fluoroguanosines. In some cases, all nucleotides can be 2'-fluoro-modified nucleotides and / or 2'-O-methyl-modified nucleotides. In some cases, all nucleotides can be 2'-fluoro-modified nucleotides.

[0332] In some cases, the anti-Ang2 aptamer does not comprise any natural nucleotides (eg, unmodified A, C, T, U, or G).

[0333] 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.

[0334] The anti-Ang2 aptamer can compete with the anti-Ang2 reference aptamer for binding to Ang2.

[0335] The anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a nucleotide sequence with or without modification. For example, the aptamer may comprise natural nucleotides and / or modified nucleotides. When an aptamer is defined as comprising or consisting of a nucleotide sequence as shown in a specific SEQ ID NO, the nucleotides within the sequence may be further modified.

[0336] According to the present disclosure, the anti-Ang2 aptamer r-AMS0526 has natural nucleotides, the nucleotide sequence of which is shown in SEQ ID NO: 2, and the anti-Ang2 aptamer AMS0526 has corresponding 2'-fluoro-modified nucleotides, the nucleotide sequence of which is shown in SEQ ID NO: 6. Therefore, an aptamer comprising the nucleotide sequence as shown in SEQ ID NO: 2 can encompass both r-AMS0526 and AMS0526, but an aptamer comprising the nucleotide sequence as shown in SEQ ID NO: 6 cannot encompass r-AMS0526.

[0337] Similarly, the anti-Ang2 aptamer r-AMS0525 has natural nucleotides, the nucleotide sequence of which is shown in SEQ ID NO: 1; the anti-Ang2 aptamer AMS0525 has corresponding 2'-fluoro-modified nucleotides, the nucleotide sequence of which is shown in SEQ ID NO: 5. The anti-Ang2 aptamer r-AMS0525.3 has natural nucleotides, the nucleotide sequence of which is shown in SEQ ID NO: 3; the anti-Ang2 aptamer AMS0525.3 has corresponding 2'-fluoro-modified nucleotides, the nucleotide sequence of which is shown in SEQ ID NO: 7. The anti-Ang2 aptamer r-AMS0525.4 has natural nucleotides, the nucleotide sequence of which is shown in SEQ ID NO: 4; the anti-Ang2 aptamer AMS0525.4 has corresponding 2'-fluoro-modified nucleotides, the nucleotide sequence of which is shown in SEQ ID NO: 8. The anti-Ang2 aptamer r-AMS0525 has natural nucleotides, and its nucleotide sequence is shown in SEQ ID NO: 1; the anti-Ang2 aptamers AMS0525.5, AMS0525.6, AMS0525.7 or AMS0525.8 have corresponding nucleotides with various modifications, and their nucleotide sequences are shown in SEQ ID NOs: 9-12.

[0338] 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 carina, a second stem, a second carina, a third stem, a third carina, 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 carina, a second stem, a second carina, a third stem, a third carina, a fourth stem, and a first loop.

[0339] 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 shown in any one of SEQ ID NOs: 13-20.

[0340] Alternatively or additionally, 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 shown in any one of SEQ ID NOs: 21-28.

[0341] For example, an anti-Ang2 reference aptamer or an anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence, the first consensus sequence comprising a nucleotide sequence as shown in SEQ ID NO: 13, and a second consensus sequence, the second consensus sequence comprising a nucleotide sequence as shown in SEQ ID NO: 21; an anti-Ang2 reference aptamer or an anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence, the first consensus sequence comprising a nucleotide sequence as shown in SEQ ID NO: 14, and a second consensus sequence, the second consensus sequence comprising a nucleotide sequence as shown in SEQ ID NO: 22; an anti-Ang2 reference aptamer or an anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence, the first consensus sequence comprising a nucleotide sequence as shown in SEQ ID NO: 15, and a second consensus sequence, the second consensus sequence comprising a nucleotide sequence as shown in SEQ ID NO: 23; an anti-Ang2 reference aptamer or an anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence, the first consensus sequence comprising a nucleotide sequence as shown in SEQ ID NO: 16, and a second consensus sequence, the second consensus sequence comprising a nucleotide sequence as shown in SEQ ID NO: NO:24; the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence, the first consensus sequence comprising the nucleotide sequence as shown in SEQ ID NO:17, and a second consensus sequence, the second consensus sequence comprising the nucleotide sequence as shown in SEQ ID NO:25; the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence, the first consensus sequence comprising the nucleotide sequence as shown in SEQ ID NO:18, and a second consensus sequence, the second consensus sequence comprising the nucleotide sequence as shown in SEQ ID NO:26; the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence, the first consensus sequence comprising the nucleotide sequence as shown in SEQ ID NO:19, and a second consensus sequence, the second consensus sequence comprising the nucleotide sequence as shown in SEQ ID NO:27; the anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence, the first consensus sequence comprising the nucleotide sequence as shown in SEQ ID NO:20, and a second consensus sequence, the second consensus sequence comprising the nucleotide sequence as shown in SEQ ID NO:28.

[0342] The first consensus sequence may be included in the nucleotide sequence of the first stem, the first carina, the second stem, the second carina, the third stem, the third carina, 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 included 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 for the region forming the first loop.

[0343] In some cases, the first consensus sequence may be included in the first stem, the first carina, and the second stem. In some cases, the first consensus sequence may be included in the first stem, the first carina, the second stem, the second carina, and the third stem. In some cases, the first consensus sequence may be included in the first stem, the first carina, the second stem, the second carina, and the third stem. In some cases, the first consensus sequence may be included in the first stem, the first carina, the second stem, the second carina, the third stem, and the third carina. In some cases, the first consensus sequence may be included in the first stem, the first carina, the second stem, the second carina, the third stem, the third carina, and the fourth stem. In some cases, the first consensus sequence may be included in the second stem, the second carina, and the third stem. In some cases, the first consensus sequence may be included in the second stem, the second carina, the third stem, and the third carina. In some cases, the first consensus sequence may be included in the second stem, the second carina, the third stem, and the third carina. In some cases, the first consensus sequence may be included in the second stem, the second carina, the third stem, the third carina, and the fourth stem. In some cases, the first consensus sequence may be included in the third stem, the third carina, and the fourth stem.

[0344] The second consensus sequence may be included in the nucleotide sequence forming the first stem, the first carina, the second stem, the second carina, the third stem, the third carina, 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 included 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 for the region forming the first loop.

[0345] In some cases, the second consensus sequence may be included in the first stem, the first carina, the second stem, and the second stem. In some cases, the second consensus sequence may be included in the first stem, the first carina, the second stem, the second carina, and the third stem. In some cases, the second consensus sequence may be included in the first stem, the first carina, the second stem, the second carina, the third stem, and the third carina. In some cases, the second consensus sequence may be included in the first stem, the first carina, the second stem, the second carina, the third stem, and the third carina. In some cases, the second consensus sequence may be included in the first stem, the first carina, the second stem, the second carina, the third stem, the third carina, and the fourth stem. In some cases, the second consensus sequence may be included in the second stem, the second carina, the third stem, and the third carina. In some cases, the second consensus sequence may be included in the second stem, the second carina, the third stem, and the third carina. In some cases, the second consensus sequence may be included in the second stem, the second carina, the third stem, and the third carina. In some cases, the second consensus sequence may be included in the second stem, the second carina, the third stem, the third carina, and the fourth stem. In some cases, the second consensus sequence may be included in the third stem, the third carina, and the fourth stem.

[0346] The first consensus sequence and the second consensus sequence can be paired to ensure that the secondary structure of the aptamer is properly formed. For example, when selecting the first consensus sequence, those skilled in the art can adjust the nucleotide sequence of the second consensus sequence as long as the secondary structure of the aptamer can be properly formed.

[0347] An anti-Ang2 reference aptamer or an anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence and a second consensus sequence, wherein the first consensus sequence may be located 5′ relative to the second consensus sequence.

[0348] In some cases, an anti-Ang2 reference aptamer or an anti-Ang2 aptamer of the present disclosure may comprise a first consensus sequence and a second consensus sequence, wherein the second consensus sequence may be located 5′ relative to the first consensus sequence.

[0349] For example, 1) the first consensus sequence may be included in the second stem, the second carina, the third stem, the third carina and the fourth stem; while the second consensus sequence may be included in the fourth stem, the third carina, the third stem, the second carina and the second stem; 2) the first consensus sequence may be included in the first carina, the second stem, the second carina, the third stem, the third carina and the fourth stem; while the second consensus sequence may be included in the fourth stem, the third carina, the third stem, the second carina, the second stem and the first carina; or, 3) the first consensus sequence may be included in the nucleotide sequence forming the first stem, the second stem, the second carina, the third stem, the third carina and the fourth stem; while the second consensus sequence may be included in the fourth stem, the third carina, the third stem, the second carina and the second stem, the first carina and the first stem.

[0350] For example, 1) the second consensus sequence may be included in the second stem, the second carina, the third stem, the third carina and the fourth stem; while the first consensus sequence may be included in the fourth stem, the third carina, the third stem, the second carina and the second stem; 2) the second consensus sequence may be included in the first carina, the second stem, the second carina, the third stem, the third carina and the fourth stem; while the first consensus sequence may be included in the fourth stem, the third carina, the third stem, the second carina, the second stem and the first carina; or, 3) the second consensus sequence may be included in the nucleotide sequence forming the first stem, the second stem, the second carina, the third stem, the third carina and the fourth stem; while the first consensus sequence may be included in the fourth stem, the third carina, the third stem, the second carina and the second stem, the first carina and the first stem.

[0351] The first loop of an anti-Ang2 reference aptamer or an anti-Ang2 aptamer of the present disclosure may comprise 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).

[0352] The anti-Ang2 reference aptamer or the anti-Ang2 aptamer of the present disclosure may comprise a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12 or a variant or truncated form thereof. The variant may comprise a nucleotide sequence that is at least 50% identical to the nucleotide sequence as shown in any one of SEQ ID NOs: 1-12. The variant may comprise a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, 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 additions, deletions and / or substitutions.

[0353] 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.

[0354] Anti-Ang2 aptamers can be used to modulate the biological activity of Ang2 or Tie2.

[0355] Anti-Ang2 aptamers can be used to prevent, treat and / or improve Ang2-related diseases, disorders or conditions. For example, Ang2-related diseases, disorders or conditions can be neovascular diseases, disorders or conditions. For example, Ang2-related diseases, disorders or conditions can be ocular neovascular diseases, disorders or conditions. For example, Ang2-related diseases, disorders or conditions can be selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion and diabetic retinopathy. For example, Ang2-related diseases, disorders or conditions can be selected from wet age-related macular degeneration, myopic choroidal neovascularization and proliferative diabetic retinopathy.

[0356] In one aspect, the present disclosure provides an anti-Ang2 agent comprising an 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' end and / or the 3' end of the anti-Ang2 aptamer.

[0357] In another aspect, the present disclosure provides a composition comprising the anti-Ang2 aptamer and / or anti-Ang2 agent of the present application.

[0358] In some cases, the composition can be a pharmaceutical composition. For example, the composition can include a pharmaceutically acceptable excipient or carrier.

[0359] In some cases, the composition may be presented in discrete dosage forms, each containing a predetermined amount of the active ingredient in powder form, or in the form of granules, a solution or suspension in an aqueous or non-aqueous liquid. Such dosage forms may be prepared by any method known to the skilled artisan, for example, it may include the step of bringing the active ingredient into association with a carrier that constitutes one or more other ingredients. In general, the composition is prepared by uniformly and intimately mixing the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired appearance.

[0360] The composition may include a therapeutically effective amount of an active composition (e.g., an anti-Ang2 aptamer or anti-Ang2 agent of the present disclosure). A therapeutically effective amount may be an amount of the subject composition that is capable of preventing and / or curing (at least in part) the condition or disorder and / or any complications thereof in a subject suffering from a neovascular disease or disorder or at risk of developing the disease or disorder. The specific amount / concentration of the active agent included may vary depending on the method of administration and the needs of the patient, and may be determined based on, for example, the patient's volume, viscosity, and / or body weight. For example, a suitable dose may be from about 0.1 mg or 1 mg / kg per day to about 50 mg / kg per day; sometimes, the dose may be higher. It should be understood that these specific doses are preferably adjusted by a skilled person in the art (e.g., a physician or pharmacist) based on the condition, formulation, and / or disease of the particular patient.

[0361] The composition may also contain an effective amount of additional therapeutically active components, such as additional therapeutically active components for preventing, treating and / or ameliorating neovascular diseases or conditions. Each active component 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 additional active components.

[0362] In some cases, the additional therapeutically active component may be an anti-VEGF agent. The anti-VEGF agent may include aflibercept. The anti-VEGF agent may also include an anti-VEGF aptamer of the present disclosure.

[0363] Non-limiting exemplary compositions and methods for preparing such compositions are described below. The composition can be, for example, in a form suitable for oral administration, such as tablets, capsules, pills, powders, sustained release formulations, solutions, suspensions; suitable for parenteral injection, such as sterile solutions, suspensions or emulsions; suitable for topical administration, such as ointments or creams; or suitable for rectal administration, such as suppositories. The composition can be in a unit dosage form suitable for a single administration of a precise dose. In some embodiments, the composition can be a liquid pharmaceutical composition.

[0364] In the composition, the anti-Ang2 aptamer can be combined with a pharmaceutical carrier in an intimate mixture according to conventional pharmaceutical compounding techniques. The carrier can take a variety of forms, depending on the form of preparation desired for administration.

[0365] The composition may also include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, but are not limited to, anti-adherents, defoaming compositions, buffer compositions, polymers, antioxidants, preservatives, chelating compositions, viscosity modifiers, tonicity agents, flavoring agents, coloring agents, flavor enhancers, opacifiers, suspending compositions, adhesives, fillers, plasticizers, lubricants and / or mixtures thereof.

[0366] In one aspect, the present disclosure provides a method for modulating the biological activity of Ang2 or Tie2, comprising administering an effective amount of an anti-Ang2 aptamer or anti-Ang2 agent of the present disclosure to a subject in need thereof.

[0367] In one aspect, the present disclosure provides a method for preventing, treating and / or ameliorating an Ang2-related disease, disorder or condition, comprising administering an effective amount of an anti-Ang2 aptamer or anti-Ang2 agent of the present disclosure to a subject in need thereof.

[0368] For example, an Ang2-related disease, disorder or condition may be a neovascular disease, disorder or condition, and may be a neovascular disease, disorder or condition. An Ang2-related disease, disorder or condition may be an ocular neovascular disease, disorder or condition. An 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. An Ang2-related disease, disorder or condition may be selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0369] The method may further comprise administering an anti-VEGF agent to the subject. The anti-VEGF agent may comprise aflibercept and / or an anti-VEGF aptamer of the present disclosure.

[0370] In another aspect, the present disclosure provides use of the anti-Ang2 aptamer or anti-Ang2 agent of the present disclosure for the manufacture of a medicament for modulating the biological activity of Ang2 or Tie2.

[0371] In one aspect, the present disclosure provides use of the aptamer of the present disclosure or the anti-Ang2 agent of the present disclosure for the manufacture of a medicament for preventing, treating and / or ameliorating Ang2-related diseases, disorders or conditions.

[0372] For example, an Ang2-related disease, disorder or condition may be a neovascular disease, disorder or condition, and may be a neovascular disease, disorder or condition. An Ang2-related disease, disorder or condition may be an ocular neovascular disease, disorder or condition. An 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. An Ang2-related disease, disorder or condition may be selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0373] In one aspect, the present disclosure provides use of 1) an anti-Ang2 aptamer or 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) for the manufacture of a medicament for preventing, treating and / or ameliorating a neovascular disease, disorder or condition.

[0374] The anti-Ang2 aptamers or anti-Ang2 agents of the present disclosure can be administered simultaneously or sequentially with the anti-VEGF agent.

[0375] 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.

[0376] Bispecific Aptamers

[0377] In one aspect, the present disclosure provides a bispecific aptamer, which comprises an anti-Ang2 aptamer of the present disclosure and an anti-VEGF aptamer of the present disclosure. The bispecific aptamer may be included in the composition of the present disclosure.

[0378] The bispecific aptamer can have the formula A1-(L)n-A2, wherein: A1 is an anti-VEGF aptamer of the disclosure and A2 is an anti-Ang2 aptamer of the disclosure; or A1 is an anti-Ang2 aptamer of the disclosure and A2 is an anti-VEGF aptamer of the disclosure; L is a linker; and n is a number of at least 0. For example, n can be 0 to 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0379] Bispecific aptamers are able to specifically bind to both VEGF and Ang2.

[0380] For example, a bispecific aptamer may have a K of about 500 pM or less.D The bispecific aptamer may bind to VEGF-121 with a K 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. D values ​​for binding to VEGF-121) and the bispecific aptamer can bind to VEGF-121 with a K of about 500 pM or less. D The bispecific aptamer may bind to Ang2 with a K 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. D Value combined with Ang2).

[0381] L may be selected from naturally occurring nucleotide linkers, modified nucleotide linkers, hydrocarbon linkers, polyethylene glycol linkers, and combinations thereof.

[0382] In some cases, at least one L can be a polyethylene glycol linker. In some cases, at least one L can be a hexaethylene glycol (also indicated as H or PEG6) linker. For example, L can be a hexaethylene glycol linker, and n can be 0 to 10 (e.g., n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0383] For example, A1 can be an anti-VEGF aptamer comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, or a variant or truncated form thereof; and A2 can be an anti-Ang2 aptamer comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, or a variant or truncated form thereof.

[0384] As another example, A1 can be an anti-Ang2 aptamer comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, or a variant or truncated form thereof; and A2 can be an anti-VEGF aptamer comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, or a variant or truncated form thereof.

[0385] In some cases of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising the nucleotide sequence as shown in SEQ ID NO:83 or a truncated form thereof, A2 is an anti-Ang2 aptamer comprising the nucleotide sequence as shown in SEQ ID NO:2 or a truncated form thereof, L is a hexaethylene glycol linker and n is 0 to 10.

[0386] In some cases of the bispecific aptamer, A1 is an anti-VEGF aptamer comprising the nucleotide sequence as shown in SEQ ID NO:128 or a truncated form thereof, A2 is an anti-Ang2 aptamer comprising the nucleotide sequence as shown in SEQ ID NO:6 or a truncated form thereof, L is a hexaethylene glycol linker and n is 0 to 10.

[0387] In some cases of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising the nucleotide sequence as shown in SEQ ID NO:2 or a truncated form thereof, A2 is an anti-VEGF aptamer comprising the nucleotide sequence as shown in SEQ ID NO:83 or a truncated form thereof, L is a hexaethylene glycol linker and n is 0 to 10.

[0388] In some cases of the bispecific aptamer, A1 is an anti-Ang2 aptamer comprising the nucleotide sequence as shown in SEQ ID NO:6 or a truncated form thereof, A2 is an anti-VEGF aptamer comprising the nucleotide sequence as shown in SEQ ID NO:128 or a truncated form thereof, L is a hexaethylene glycol linker and n is 0 to 10.

[0389] In some cases, the bispecific aptamer may comprise a nucleotide sequence as shown in any one of SEQ ID NOs: 140-195, SEQ ID NOs: 202-204.

[0390] The bispecific aptamer can be selected from r-AMSB107.0, r-AMSB108.0, r-AMSB107.1, r-AMSB107.2, r-AMSB107, r-AMSB107.4, r-AMSB107.5, r-AMSB107.6, r-AMSB107.7, r-AMSB107.8, r-AMSB107.9, r-AMSB107.10, r-AMSB107.11, r-AMSB 107.12, r-AMSB107.13, r-AMSB107.14, r-AMSB107.15, r-AMSB107.16, r-AMSB107.17, r-AMSB107.18, r -AMSB107.19, r-AMSB107.22, r-AMSB107.23, r-AMSB107.24, r-AMSB107.25, r-AMSB107.26, r-AMSB107 .27, AMSB107.0, AMSB108.0, AMSB107.1, AMSB107.2, AMSB107, AMSB107.4, AMSB107.5, AMSB107.6, AMS B107.7, AMSB107.8, AMSB107.9, AMSB107.10, AMSB107.11, AMSB107.12, AMSB107.13, AMSB107.14, AMSB 107.15, AMSB107.16, AMSB107.17, AMSB107.18, AMSB107.19, AMSB107.20, AMSB107.21, AMSB107.22, AMSB107.23, AMSB107.24, AMSB107.25, AMSB107.26, AMSB107.P6, AMSB107.P40K, AMSB107P and AMSB107.27.

[0391] The bispecific aptamers r-AMSB107.22, r-AMSB107.23, r-AMSB107.24, r-AMSB107.25, AMSB107.22, AMSB107.23, AMSB107.24 and AMSB107.25 each comprise one or more hexaethylene glycol (also known as PEG6) as a linker (L) or modification.

[0392] Composition

[0393] In one aspect, the present disclosure provides a composition comprising an anti-Ang2 aptamer of the present disclosure, an anti-VEGF aptamer of the present disclosure and / or a bispecific aptamer of the present disclosure. In some cases, the composition comprises an effective amount of an anti-Ang2 aptamer of the present disclosure and an effective amount of an anti-VEGF aptamer of the present disclosure. In some cases, the composition comprises an effective amount of a bispecific aptamer of the present disclosure.

[0394] The composition may be a pharmaceutical composition. The composition may include a pharmaceutically acceptable excipient or carrier.

[0395] The composition can be used to prevent, treat and / or improve neovascular diseases, disorders or conditions. The neovascular diseases, disorders or conditions can be ocular neovascular diseases, disorders or conditions. In some embodiments, the neovascular diseases, disorders or conditions are selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion and diabetic retinopathy. In some embodiments, the neovascular diseases, disorders or conditions are selected from wet age-related macular degeneration, myopic choroidal neovascularization and proliferative diabetic retinopathy.

[0396] The composition can be, for example, in a form suitable for oral administration, such as tablets, capsules, pills, powders, sustained release formulations, solutions, suspensions; suitable for parenteral injection, such as sterile solutions, suspensions or emulsions; suitable for topical administration, such as ointments or creams; or suitable for rectal administration, such as suppositories. The composition can be in a unit dosage form suitable for single administration of a precise dose. In some cases, the composition can be a liquid pharmaceutical composition.

[0397] Pharmaceutically acceptable excipients or carriers may include, but are not limited to, anti-adherents, anti-foaming agents, buffers, polymers, antioxidants, preservatives, chelating agents, viscosity modifiers, tonicity agents, flavoring agents, colorants, flavor enhancers, opacifying agents, suspending agents, binders, fillers, plasticizers, lubricants and / or mixtures thereof.

[0398] In some cases, the composition may be presented in discrete dosage forms, each containing a predetermined amount of the active ingredient in powder form, or in the form of granules, a solution or suspension in an aqueous or non-aqueous liquid. Such dosage forms may be prepared by any method known to the skilled artisan, for example, it may include the step of bringing the active ingredient into association with a carrier that constitutes one or more other ingredients. In general, the composition is prepared by uniformly and intimately mixing the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired appearance.

[0399] The composition may include a therapeutically effective amount of an active agent (e.g., an agent of the present disclosure). A therapeutically effective amount may be an amount by which the subject composition can prevent and / or cure (at least in part) the condition or illness and / or any complications thereof of a subject suffering from a neovascular disease or disorder or having a risk of developing the disease or disorder. The specific amount / concentration of the active agent included may vary according to the method of administration and the needs of the patient, and may be determined based on, for example, the patient's volume, viscosity, and / or body weight. For example, a suitable dosage may be about 0.1 mg or 1 mg / kg to about 50 mg / kg per day; sometimes, the dosage may be higher. It should be understood that these specific dosages are preferably adjusted by a skilled person in the art (e.g., a doctor or pharmacist) based on the condition, formulation, and / or disease of a particular patient.

[0400] Medical uses and methods

[0401] 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 an anti-Ang2 aptamer of the present disclosure, an effective amount of an anti-VEGF aptamer of the present disclosure, an effective amount of a bispecific aptamer of the present disclosure and / or an effective amount of a composition of the present disclosure.

[0402] 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 for the manufacture of a medicament for preventing, treating and / or ameliorating a neovascular disease, disorder or condition.

[0403] The neovascular disease, disorder or condition can 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.

[0404] Additional embodiments

[0405] The present disclosure also provides the following embodiments:

[0406] AMSB107.25, AMSB107.26, AMSB107.P6, AMSB107.P40K, AMSB107P

[0407] The present disclosure also relates to the following sequence:

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426] 1. An anti-Ang2 aptamer that specifically binds to Ang2 with a KD value of about 500 picomolar (pM) or less.

[0427] 2. The anti-Ang2 aptamer according to embodiment 1, which specifically binds to Ang2 with a KD value of about 100 picomolar (pM) or less.

[0428] 3. The anti-Ang2 aptamer of any one of embodiments 1-2, wherein the Ang2 is human Ang2, mouse Ang2, monkey Ang2, rabbit Ang2 and / or rat Ang2.

[0429] 4. The anti-Ang2 aptamer according to any one of embodiments 1 to 3, which does not substantially bind to human Ang1.

[0430] 5. The anti-Ang2 aptamer according to any one of embodiments 1 to 4, which inhibits the interaction between Ang2 and Tie2.

[0431] 6. The anti-Ang2 aptamer according to any one of embodiments 1 to 5, which is capable of reducing neovascularization and / or lowering vascular permeability in a mouse oxygen-induced ischemic retinopathy (OIR) model.

[0432] 7. The anti-Ang2 aptamer of any one of embodiments 1-6, which is highly soluble.

[0433] 8. The anti-Ang2 aptamer according to any one of embodiments 1 to 7, which is an RNA aptamer, a DNA aptamer, or a combination thereof.

[0434] 9. The anti-Ang2 aptamer of any one of embodiments 1-8, comprising one or more modified nucleotides.

[0435] 10. The anti-Ang2 aptamer of any one of embodiments 1-9, which is resistant to nuclease.

[0436] 11. The anti-Ang2 aptamer of any one of embodiments 1-10, wherein all nucleotides of the anti-Ang2 aptamer are modified nucleotides and / or one or more substitutions (e.g., substituted 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-Ang2 aptamer can be substituted with a polyethylene glycol moiety or a linker comprising a plurality (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) of polyethylene glycol moieties. In some cases, the linker can be a hexaethylene glycol (H or PEG6) linker. Such substitutions can occur in any nucleotide of the anti-Ang2 aptamer, as long as it does not substantially affect the activity (e.g., binding activity or inhibitory 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 nucleotides included in the protuberance region or loop region of the secondary structure of the anti-Ang2 aptamer can be substituted. In some cases, the substitution does not occur in any nucleotide included in the stem region of the secondary structure of the anti-Ang2 aptamer.

[0437] 12. The anti-Ang2 aptamer of any one of embodiments 9 to 11, wherein 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.

[0438] 13. An anti-Ang2 aptamer as described in any one of embodiments 9-12, wherein the modified nucleotide comprises one or more modifications independently selected from the following: 2′-position sugar modification, 2′-amino (2′-NH2) modification, 2′-fluoro (2′-F) modification, 2′-O-methyl (2′-OMe) modification, 2′-O-(2-methoxyethyl) (2′-O-MOE) modification, 5-position modified pyrimidine, modification at the amine outside the cytosine ring, substitution with 5′-bromouracil, substitution with 5′-bromodeoxyuridine, substitution with 5′-bromodeoxycytidine, backbone modification, locked nucleic acid (LNA), methylation, 3′ cap and 5′ cap.

[0439] 14. The anti-Ang2 aptamer according to embodiment 13, wherein 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-benzylcarboxamide)-2'-deoxyuridine, 5-(N-isobutylcarboxamide)-2'-deoxyuridine, 5-(N-naphthylmethylcarboxamide)-2'-deoxyuridine and 5-(N-tryptaminecarboxamide)-2'-deoxyuridine.

[0440] 15. The anti-Ang2 aptamer of any one of embodiments 1-14, comprising one or more 2'-modified nucleotides.

[0441] 16. The anti-Ang2 aptamer of any one of embodiments 1-15, wherein all nucleotides of the anti-Ang2 aptamer are 2'-modified nucleotides.

[0442] 17. The anti-Ang2 aptamer of any one of embodiments 15-16, wherein the 2'-modified nucleotides are selected from 2'-amino-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides and 2'-O-(2-methoxyethyl)-modified nucleotides.

[0443] 18. The anti-Ang2 aptamer of any one of embodiments 1-17, comprising at least one 2'-fluoro-modified nucleotide, at least one 2'-O-methyl-modified nucleotide and / or at least one LNA.

[0444] 19. The anti-Ang2 aptamer of any one of embodiments 1-18, wherein all cytidines of the anti-Ang2 aptamer are 2′-modified cytidines.

[0445] 20. The anti-Ang2 aptamer of any one of embodiments 1-19, wherein all cytidines of the anti-Ang2 aptamer are 2'-fluoro-modified cytidine and / or 2'-O-methyl-modified cytidine.

[0446] 21. The anti-Ang2 aptamer of any one of embodiments 1-20, wherein all cytidines of the anti-Ang2 aptamer are 2'-deoxy-2'-fluorocytidine.

[0447] 22. The anti-Ang2 aptamer of any one of embodiments 1-21, wherein all uridines of the anti-Ang2 aptamer are 2'-modified uridines.

[0448] 23. The anti-Ang2 aptamer of any one of embodiments 1-22, wherein all uridines of the anti-Ang2 aptamer are 2'-fluoro-modified uridine and / or 2'-O-methyl-modified uridine.

[0449] 24. The anti-Ang2 aptamer of any one of embodiments 1-23, wherein all uridines of the anti-Ang2 aptamer are 2'-deoxy-2'-fluorouridine.

[0450] 25. The anti-Ang2 aptamer of any one of embodiments 1-24, wherein all adenosines of the anti-Ang2 aptamer are 2'-modified adenosines.

[0451] 26. The anti-Ang2 aptamer of any one of embodiments 1-25, wherein all adenosines of the anti-Ang2 aptamer are 2'-fluoro-modified adenosine and / or 2'-O-methyl-modified adenosine.

[0452] 27. The anti-Ang2 aptamer of any one of embodiments 1-26, wherein all adenosines of the anti-Ang2 aptamer are 2'-deoxy-2'-fluoroadenosine.

[0453] 28. The anti-Ang2 aptamer of any one of embodiments 1-27, wherein all guanosines of the anti-Ang2 aptamer are 2'-modified guanosines.

[0454] 29. The anti-Ang2 aptamer of any one of embodiments 1-28, wherein all guanosines of the anti-Ang2 aptamer are 2'-fluoro-modified guanosine and / or 2'-O-methyl-modified guanosine.

[0455] 30. The anti-Ang2 aptamer of any one of embodiments 1-29, wherein all guanosines of the anti-Ang2 aptamer are 2'-deoxy-2'-fluoroguanosine.

[0456] 31. The anti-Ang2 aptamer of any one of embodiments 1-30, wherein all nucleotides of the anti-Ang2 aptamer are 2'-modified nucleotides.

[0457] 32. The anti-Ang2 aptamer of any one of embodiments 1-31, wherein all nucleotides of the anti-Ang2 aptamer are 2'-fluoro-modified nucleotides and / or 2'-O-methyl-modified nucleotides.

[0458] 33. The anti-Ang2 aptamer of any one of embodiments 1-32, wherein all nucleotides of the anti-Ang2 aptamer are 2'-fluoro-modified nucleotides.

[0459] 34. The anti-Ang2 aptamer of any one of embodiments 1-33, which does not comprise any natural nucleotides.

[0460] 35. The anti-Ang2 aptamer of any one of embodiments 1-34, comprising about 10 to about 150 nucleotides.

[0461] 36. The anti-Ang2 aptamer of any one of embodiments 1-35, which 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.

[0462] 37. The anti-Ang2 aptamer of embodiment 36, wherein the anti-Ang2 reference aptamer comprises a secondary structure consisting of 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 from 5' to 3' direction.

[0463] 38. The anti-Ang2 aptamer of any one of embodiments 36-37, wherein the anti-Ang2 reference aptamer comprises a first consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 13-20.

[0464] 39. The anti-Ang2 aptamer of any one of embodiments 36-38, wherein the anti-Ang2 reference aptamer comprises a second consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 21-28.

[0465] 40. The anti-Ang2 aptamer of any one of embodiments 38-39, wherein the first consensus sequence is contained 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.

[0466] 41. The anti-Ang2 aptamer of any one of embodiments 38-40, wherein the first consensus sequence is not included in the nucleotide sequence forming the first loop of the anti-Ang2 reference aptamer.

[0467] 42. The anti-Ang2 aptamer of any one of embodiments 39-41, wherein the second consensus sequence is contained 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.

[0468] 43. The anti-Ang2 aptamer of any one of embodiments 39-42, wherein the second consensus sequence is not contained in the nucleotide sequence forming the first loop of the anti-Ang2 reference aptamer.

[0469] 44. The anti-Ang2 aptamer of any one of embodiments 36-43, wherein the first loop of the anti-Ang2 reference aptamer comprises 2-30 nucleotides.

[0470] 45. The anti-Ang2 aptamer of any one of embodiments 36-44, wherein the first loop of the anti-Ang2 reference aptamer comprises 3-10 nucleotides.

[0471] 46. ​​The anti-Ang2 aptamer of any one of embodiments 36-45, wherein the anti-Ang2 reference aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, or a variant or truncated form thereof.

[0472] 47. The anti-Ang2 aptamer of embodiment 46, wherein the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 1-12.

[0473] 48. The anti-Ang2 aptamer of any one of embodiments 46-47, wherein the variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, having one or more nucleotide additions, deletions and / or substitutions.

[0474] 49. The anti-Ang2 aptamer of any one of embodiments 36-48, wherein the anti-Ang2 reference aptamer comprises about 10 to about 150 nucleotides.

[0475] 50. The anti-Ang2 aptamer of any one of embodiments 38-49, wherein the anti-Ang2 reference aptamer comprises the first consensus sequence and the second consensus sequence, and wherein the first consensus sequence is located 5′ relative to the second consensus sequence.

[0476] 51. The anti-Ang2 aptamer of any one of embodiments 38-49, wherein the anti-Ang2 reference aptamer comprises the first consensus sequence and the second consensus sequence, and wherein the second consensus sequence is located 5′ relative to the first consensus sequence.

[0477] 52. The anti-Ang2 aptamer of any one of embodiments 1-51, wherein 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.

[0478] 53. The anti-Ang2 aptamer of any one of embodiments 1-52, wherein the anti-Ang2 aptamer comprises a secondary structure consisting of 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 from 5' to 3' direction.

[0479] 54. The anti-Ang2 aptamer of any one of embodiments 1-53, wherein the anti-Ang2 aptamer comprises a first consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 13-20.

[0480] 55. The anti-Ang2 aptamer of any one of embodiments 1-54, wherein the anti-Ang2 aptamer comprises a second consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 21-28.

[0481] 56. The anti-Ang2 aptamer of any one of embodiments 54-55, wherein the first consensus sequence is contained 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.

[0482] 57. The anti-Ang2 aptamer of any one of embodiments 54-56, wherein the first consensus sequence is not included in the nucleotide sequence forming the first loop of the anti-Ang2 aptamer.

[0483] 58. The anti-Ang2 aptamer of any one of embodiments 55-57, wherein the second consensus sequence is contained 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.

[0484] 59. The anti-Ang2 aptamer of any one of embodiments 55-58, wherein the second consensus sequence is not included in the nucleotide sequence forming the first loop of the anti-Ang2 aptamer.

[0485] 60. The anti-Ang2 aptamer of any one of embodiments 52-59, wherein the first loop of the anti-Ang2 aptamer comprises 2-30 nucleotides.

[0486] 61. The anti-Ang2 aptamer of any one of embodiments 52-60, wherein the first loop of the anti-Ang2 aptamer comprises 3-10 nucleotides.

[0487] 62. The anti-Ang2 aptamer of any one of embodiments 1-61, wherein the anti-Ang2 aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, or a variant or truncated form thereof.

[0488] 63. The anti-Ang2 aptamer of embodiment 62, wherein the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 1-12.

[0489] 64. The anti-Ang2 aptamer of any one of embodiments 62-63, wherein the variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, having one or more nucleotide additions, deletions and / or substitutions.

[0490] 65. The anti-Ang2 aptamer of any one of embodiments 54-64, wherein the anti-Ang2 aptamer comprises the first consensus sequence and the second consensus sequence, and wherein the first consensus sequence is located 5′ relative to the second consensus sequence.

[0491] 66. The anti-Ang2 aptamer of any one of embodiments 54-64, wherein the anti-Ang2 aptamer comprises the first consensus sequence and the second consensus sequence, and wherein the second consensus sequence is located 5′ relative to the first consensus sequence.

[0492] 67. An agent comprising the anti-Ang2 aptamer of any one of embodiments 1-66, wherein the anti-Ang2 aptamer is conjugated to a polyethylene glycol (PEG) moiety.

[0493] 68. The agent of embodiment 67, wherein at least one PEG moiety is conjugated to the 5′ end of the anti-Ang2 aptamer.

[0494] 69. The agent of any one of embodiments 67-68, wherein at least one PEG moiety is conjugated to the 3' end of the anti-Ang2 aptamer.

[0495] 70. An anti-VEGF aptamer that binds to VEGF-121 with a KD value of about 20 nanomolar (nM) or less and binds to VEGF-165 with a KD value of about 20 nanomolar (nM) or less.

[0496] 71. The anti-VEGF aptamer of embodiment 70, which binds to VEGF-121 with a KD value of about 2 nanomolar (nM) or less.

[0497] 72. The anti-VEGF aptamer of any one of embodiments 70-71, which binds to VEGF-165 with a KD value of about 2 nanomolar (nM) or less.

[0498] 73. The anti-VEGF aptamer of any one of embodiments 70-72, wherein the VEGF-121 is human VEGF-121, mouse VEGF-120, monkey VEGF-121, rabbit VEGF-121 and / or rat VEGF-120.

[0499] 74. The anti-VEGF aptamer of any one of embodiments 70-73, wherein the VEGF-165 is human VEGF-165, mouse VEGF-164, monkey VEGF-165, rabbit VEGF-165 and / or rat VEGF-164.

[0500] 75. The anti-VEGF aptamer of any one of embodiments 70-74, which specifically binds to both the VEGF receptor binding domain of VEGF-121 and the VEGF receptor binding domain of VEGF-165.

[0501] 76. The anti-VEGF aptamer of embodiment 75, wherein the VEGF receptor binding domain of VEGF-121 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 30-34, 40, 41 and 45.

[0502] 77. The anti-VEGF aptamer of any one of embodiments 75-76, wherein the VEGF receptor binding domain of VEGF-165 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 30-34, 40 and 41.

[0503] 78. The anti-VEGF aptamer of any one of embodiments 70-77, which inhibits the interaction between VEGF-121 and VEGF-R1.

[0504] 79. The anti-VEGF aptamer of any one of embodiments 70-78, which inhibits the interaction between VEGF-121 and VEGF-R2.

[0505] 80. The anti-VEGF aptamer of any one of embodiments 70-79, which inhibits the interaction between VEGF-165 and VEGF-R1.

[0506] 81. The anti-VEGF aptamer of any one of embodiments 70-80, which inhibits the interaction between VEGF-165 and VEGF-R2.

[0507] 82. The anti-VEGF aptamer of any one of embodiments 70-81, which is capable of reducing and / or ameliorating damage and / or leakage in a laser-induced choroidal neovascularization (CNV) rat model.

[0508] 83. The anti-VEGF aptamer of any one of embodiments 70-82, which is highly soluble.

[0509] 84. The anti-VEGF aptamer of any one of embodiments 70-83, which is an RNA aptamer, a DNA aptamer, or a combination thereof.

[0510] 85. The anti-VEGF aptamer of any one of embodiments 70-84, which is resistant to nucleases.

[0511] 86. The anti-VEGF aptamer of any one of embodiments 70-85, comprising one or more modified nucleotides, and / or one or more substitutions (e.g., substitutions 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 a plurality (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2) of polyethylene glycol moieties. In some cases, the linker may be a hexaethylene glycol (H or PEG6) linker. Such substitutions may occur at any nucleotide of the anti-VEGF aptamer, as long as they do not substantially affect the activity (e.g., binding activity or inhibitory activity) and / or function of the anti-VEGF aptamer. In some cases, the substitutions do not substantially affect the secondary structure of the anti-VEGF aptamer, for example, the substitutions may occur at nucleotides included in the knob region or loop region of the secondary structure of the anti-VEGF aptamer. In some cases, the substitutions do not occur at any nucleotide included in the stem region of the secondary structure of the anti-VEGF aptamer.

[0512] 87. The anti-VEGF aptamer of embodiment 86, wherein all nucleotides of the anti-VEGF aptamer are modified nucleotides.

[0513] 88. An anti-VEGF aptamer as described in any of embodiments 86-87, wherein 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.

[0514] 89. An anti-VEGF aptamer as described in any of embodiments 86-88, wherein the modified nucleotide comprises one or more modifications independently selected from the following: 2′-position sugar modification, 2′-amino (2′-NH2) modification, 2′-fluoro (2′-F) modification, 2′-O-methyl (2′-OMe) modification, 2′-O-(2-methoxyethyl) (2′-O-MOE) modification, 5-position modified pyrimidine, modification at the amine outside the cytosine ring, substitution with 5′-bromouracil, substitution with 5′-bromodeoxyuridine, substitution with 5′-bromodeoxycytidine, backbone modification, locked nucleic acid (LNA), methylation, 3′ cap and 5′ cap.

[0515] 90. An anti-VEGF aptamer as described in embodiment 89, wherein 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-benzylcarboxamide)-2'-deoxyuridine, 5-(N-isobutylcarboxamide)-2'-deoxyuridine, 5-(N-naphthylmethylcarboxamide)-2'-deoxyuridine and 5-(N-tryptaminecarboxamide)-2'-deoxyuridine.

[0516] 91. The anti-VEGF aptamer of any one of embodiments 70-90, comprising at least one 2'-modified nucleotide.

[0517] 92. The anti-VEGF aptamer of embodiment 91, wherein all nucleotides of the anti-VEGF aptamer are 2'-modified nucleotides.

[0518] 93. An anti-VEGF aptamer as described in any one of embodiments 91-92, wherein the 2'-modified nucleotides are selected from 2'-amino modified nucleotides, 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides and 2'-O-(2-methoxyethyl)-modified nucleotides.

[0519] 94. The anti-VEGF aptamer of any one of embodiments 70-93, comprising at least one 2'-fluoro-modified nucleotide, at least one 2'-O-methyl-modified nucleotide and / or at least one LNA.

[0520] 95. The anti-VEGF aptamer of any one of embodiments 70-94, wherein all cytidines of the anti-VEGF aptamer are 2'-modified cytidines.

[0521] 96. The anti-VEGF aptamer of any one of embodiments 70-95, wherein all cytidines of the anti-VEGF aptamer are 2'-fluoro-modified cytidine and / or 2'-O-methyl-modified cytidine.

[0522] 97. The anti-VEGF aptamer of any one of embodiments 70-96, wherein all cytidines of the anti-VEGF aptamer are 2'-deoxy-2'-fluorocytidine.

[0523] 98. The anti-VEGF aptamer of any one of embodiments 70-97, wherein all uridines of the anti-VEGF aptamer are 2'-modified uridines.

[0524] 99. The anti-VEGF aptamer of any one of embodiments 70-98, wherein all uridines of the anti-VEGF aptamer are 2'-fluoro-modified uridine and / or 2'-O-methyl-modified uridine.

[0525] 100. The anti-VEGF aptamer of any one of embodiments 70-99, wherein all uridines of the anti-VEGF aptamer are 2'-deoxy-2'-fluorouridine.

[0526] 101. The anti-VEGF aptamer of any one of embodiments 70-100, wherein all adenosines of the anti-VEGF aptamer are 2'-modified adenosines.

[0527] 102. The anti-VEGF aptamer of any one of embodiments 70-101, wherein all adenosines of the anti-VEGF aptamer are 2'-fluoro-modified adenosine and / or 2'-O-methyl-modified adenosine.

[0528] 103. The anti-VEGF aptamer of any one of embodiments 70-102, wherein all adenosines of the anti-VEGF aptamer are 2'-deoxy-2'-fluoroadenosine.

[0529] 104. The anti-VEGF aptamer of any one of embodiments 70-103, wherein all guanosines of the anti-VEGF aptamer are 2'-modified guanosines.

[0530] 105. The anti-VEGF aptamer of any one of embodiments 70-104, wherein all guanosines of the anti-VEGF aptamer are 2'-fluoro-modified guanosine and / or 2'-O-methyl-modified guanosine.

[0531] 106. The anti-VEGF aptamer of any one of embodiments 70-105, wherein all guanosines of the anti-VEGF aptamer are 2'-deoxy-2'-fluoroguanosine.

[0532] 107. The anti-VEGF aptamer of any one of embodiments 70-106, wherein all nucleotides of the anti-VEGF aptamer are 2'-modified nucleotides.

[0533] 108. The anti-VEGF aptamer of any one of embodiments 70-107, wherein all nucleotides of the anti-VEGF aptamer are 2'-fluoro-modified nucleotides and / or 2'-O-methyl-modified nucleotides.

[0534] 109. The anti-VEGF aptamer of any one of embodiments 70-108, wherein all nucleotides of the anti-VEGF aptamer are 2'-fluoro-modified nucleotides.

[0535] 110. The anti-VEGF aptamer of any one of embodiments 70-109, wherein the anti-VEGF aptamer does not comprise any natural nucleotides.

[0536] 111. The anti-VEGF aptamer of any one of embodiments 70-110, comprising about 10 to about 150 nucleotides.

[0537] 112. An anti-VEGF aptamer as described in any one of embodiments 70-111, wherein the anti-VEGF aptamer competes with an anti-VEGF reference aptamer for binding to the VEGF-165 and the VEGF-121, wherein the anti-VEGF reference aptamer comprises a secondary structure, and the secondary structure comprises a first stem, a first loop, a second stem, and a second loop from the 5' to the 3' direction.

[0538] 113. The anti-VEGF aptamer of embodiment 112, wherein the anti-VEGF reference aptamer comprises a secondary structure consisting of a first stem, a first loop, a second stem, and a second loop from 5' to 3' direction.

[0539] 114. The anti-VEGF aptamer of any one of embodiments 112-113, wherein the anti-VEGF reference aptamer comprises a first consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 136-137.

[0540] 115. The anti-VEGF aptamer of any one of embodiments 112-114, wherein the anti-VEGF reference aptamer comprises a second consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 138-139.

[0541] 116. The anti-VEGF aptamer of any one of embodiments 114-115, wherein the first consensus sequence comprises a nucleotide sequence that forms the first stem and / or the first loop of the anti-VEGF reference aptamer.

[0542] 117. The anti-VEGF aptamer of any one of embodiments 114-116, wherein the first consensus sequence is not contained in the nucleotide sequence forming the second stem or the second loop of the anti-VEGF reference aptamer.

[0543] 118. The anti-VEGF aptamer of any one of embodiments 115-117, wherein the second consensus sequence is comprised in the nucleotide sequence forming the second stem and the second loop of the anti-VEGF reference aptamer.

[0544] 119. The anti-VEGF aptamer of any one of embodiments 115-118, wherein the second consensus sequence is not contained in the nucleotide sequence forming the first stem or the first loop of the anti-VEGF reference aptamer.

[0545] 120. The anti-VEGF aptamer of any one of embodiments 112-119, wherein the anti-VEGF reference aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, or a variant or truncated form thereof.

[0546] 121. The anti-VEGF aptamer of embodiment 120, wherein the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201.

[0547] 122. The anti-VEGF aptamer of any one of embodiments 120-121, wherein the variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, having one or more nucleotide additions, deletions and / or substitutions.

[0548] 123. An anti-VEGF aptamer as described in any of embodiments 112-122, wherein the anti-VEGF reference aptamer comprises about 10 to about 150 nucleotides.

[0549] 124. The anti-VEGF aptamer of any one of embodiments 114-123, wherein the anti-VEGF reference aptamer comprises the first consensus sequence and the second consensus sequence, and wherein the first consensus sequence is located 5′ relative to the second consensus sequence.

[0550] 125. The anti-VEGF aptamer of any one of embodiments 114-123, wherein the anti-VEGF reference aptamer comprises the first consensus sequence and the second consensus sequence, and wherein the second consensus sequence is located 5′ relative to the first consensus sequence.

[0551] 126. The anti-VEGF aptamer of any one of embodiments 70-125, wherein the anti-VEGF aptamer comprises a secondary structure comprising, from 5' to 3' direction, a first stem, a first loop, a second stem, and a second loop.

[0552] 127. The anti-VEGF aptamer of any one of embodiments 70-126, wherein the anti-VEGF aptamer comprises a secondary structure consisting of a first stem, a first loop, a second and a second loop from the 5' to the 3' direction.

[0553] 128. The anti-VEGF aptamer of any one of embodiments 70-127, wherein the anti-VEGF aptamer comprises a first consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 136-137.

[0554] 129. The anti-VEGF aptamer of any one of embodiments 70-128, wherein the anti-VEGF aptamer comprises a second consensus sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 138-139.

[0555] 130. The anti-VEGF aptamer of any one of embodiments 128-129, wherein the first consensus sequence comprises nucleotide sequences that form the first stem and the first loop of the anti-VEGF aptamer.

[0556] 131. An anti-VEGF aptamer as described in any of embodiments 128-130, wherein the first consensus sequence is not contained in the nucleotide sequence forming the second stem or the second loop of the anti-VEGF aptamer.

[0557] 132. An anti-VEGF aptamer as described in any of embodiments 129-131, wherein the second consensus sequence comprises nucleotide sequences that form the second stem and the second loop of the anti-VEGF aptamer.

[0558] 133. The anti-VEGF aptamer of any one of embodiments 129-132, wherein the second consensus sequence is not contained in the nucleotide sequence forming the first stem or the first loop of the anti-VEGF aptamer.

[0559] 134. The anti-VEGF aptamer of any one of embodiments 70-133, wherein the anti-VEGF aptamer comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, or a variant or truncated form thereof.

[0560] 135. The anti-VEGF aptamer of embodiment 134, wherein the variant comprises a nucleotide sequence that is at least 50% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201.

[0561] 136. The anti-VEGF aptamer of any one of embodiments 134-135, wherein the variant comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, having one or more nucleotide additions, deletions and / or substitutions.

[0562] 137. The anti-VEGF aptamer of any one of embodiments 128-136, wherein the anti-VEGF aptamer comprises the first consensus sequence and the second consensus sequence, and wherein the first consensus sequence is located 5′ relative to the second consensus sequence.

[0563] 138. The anti-VEGF aptamer of any one of embodiments 128-136, wherein the anti-VEGF aptamer comprises the first consensus sequence and the second consensus sequence, and wherein the second consensus sequence is located 5′ relative to the first consensus sequence.

[0564] 139. An agent comprising the anti-VEGF aptamer of any one of embodiments 70-138, wherein the anti-VEGF aptamer is conjugated to a polyethylene glycol (PEG) moiety.

[0565] 140. The agent of embodiment 139, wherein at least one PEG moiety is conjugated to the 5' end of the anti-VEGF aptamer.

[0566] 141. The agent of any one of embodiments 139-140, wherein at least one PEG moiety is conjugated to the 3' end of the anti-VEGF aptamer.

[0567] 142. 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 70-141.

[0568] 143. The bispecific aptamer of embodiment 142, which has the formula A1-(L)n-A2, wherein:

[0569] 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;

[0570] L is a connector; and

[0571] n is a number at least 0.

[0572] 144. The bispecific aptamer of any one of embodiments 142-143, which is capable of specifically binding to both VEGF and Ang2.

[0573] 145. The bispecific aptamer of any one of embodiments 142-144, which specifically binds to VEGF-121 with a KD value of about 500 pM or less, and binds to Ang2 with a KD value of about 500 pM or less.

[0574] 146. The bispecific aptamer of any one of embodiments 142-145, which binds to VEGF-121 with a KD value of about 200 pM or less, and binds to Ang2 with a KD value of about 200 pM or less.

[0575] 147. The bispecific aptamer of any one of embodiments 142-146, which binds to VEGF-121 with a KD value of about 100 pM or less, and binds to Ang2 with a KD value of about 100 pM or less.

[0576] 148. The bispecific aptamer of any one of embodiments 143-147, wherein n is 0 to 20.

[0577] 149. The bispecific aptamer of any one of embodiments 143-148, wherein n is 0 to 10.

[0578] 150. The bispecific aptamer of any one of embodiments 143-149, wherein L is selected from the group consisting of a naturally occurring nucleotide linker, a modified nucleotide linker, a hydrocarbon linker, a polyethylene glycol linker, and combinations thereof.

[0579] 151. The bispecific aptamer of any one of embodiments 143-150, wherein at least one L is a polyethylene glycol linker.

[0580] 152. The bispecific aptamer of any one of embodiments 143-151, wherein at least one L is a hexaethylene glycol linker.

[0581] 153. The bispecific aptamer of any one of embodiments 143-152, wherein L is a hexaethylene glycol linker and n is 0 to 10.

[0582] 154. The bispecific aptamer of any one of embodiments 143-153, wherein A1 is an anti-VEGF aptamer comprising a nucleotide sequence as shown in any one of SEQ ID NOs:46-135, SEQ ID NOs:200-201, or a variant or truncated form thereof; and A2 is an anti-Ang2 aptamer comprising a nucleotide sequence as shown in any one of SEQ ID NOs:1-12, or a variant or truncated form thereof.

[0583] 155. The bispecific aptamer of any one of embodiments 143-154, wherein A1 is an anti-Ang2 aptamer comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 1-12, or a variant or truncated form thereof; and A2 is an anti-VEGF aptamer comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 46-135, SEQ ID NOs: 200-201, or a variant or truncated form thereof.

[0584] 156. The bispecific aptamer of any one of embodiments 143-155, wherein:

[0585] 1) A1 is an anti-VEGF aptamer comprising the nucleotide sequence as shown in SEQ ID NO: 83 or a truncated form thereof, A2 is an anti-Ang2 aptamer comprising the nucleotide sequence as shown in SEQ ID NO: 2 or a truncated form thereof, L is a hexaethylene glycol linker, and n is 0 to 10;

[0586] 2) A1 is an anti-VEGF aptamer comprising the nucleotide sequence as shown in SEQ ID NO: 128 or a truncated form thereof, A2 is an anti-Ang2 aptamer comprising the nucleotide sequence as shown in SEQ ID NO: 6 or a truncated form thereof, L is a hexaethylene glycol linker, and n is 0 to 10;

[0587] 3) A1 is an anti-Ang2 aptamer comprising the nucleotide sequence as shown in SEQ ID NO: 2 or a truncated form thereof, A2 is an anti-VEGF aptamer comprising the nucleotide sequence as shown in SEQ ID NO: 83 or a truncated form thereof, L is a hexaethylene glycol linker, and n is 0 to 10; or

[0588] 4) A1 is an anti-Ang2 aptamer comprising the nucleotide sequence as shown in SEQ ID NO:6 or a truncated form thereof, A2 is an anti-VEGF aptamer comprising the nucleotide sequence as shown in SEQ ID NO:128 or a truncated form thereof, L is a hexaethylene glycol linker, and n is 0 to 10.

[0589] 157. The bispecific aptamer of any one of embodiments 142-156, which is selected from r-AMSB107.0,

[0590] r-AMSB108.0, r-AMSB107.1, r-AMSB107.2, r-AMSB107, r-AMSB107.4, r-AMSB107.5,

[0591] r-AMSB107.6, r-AMSB107.7, r-AMSB107.8, r-AMSB107.9, r-AMSB107.10, r-AMSB107.11,

[0592] r-AMSB107.12, r-AMSB107.13, r-AMSB107.14, r-AMSB107.15, r-AMSB107.16,

[0593] r-AMSB107.17, r-AMSB107.18, r-AMSB107.19, r-AMSB107.22, r-AMSB107.23,

[0594] r-AMSB107.24, r-AMSB107.25, r-AMSB107.26, r-AMSB107.27, AMSB107.0, AMSB108.0, AMSB107.1, AMSB107.2, AMSB107, AMSB107.4, AMSB107.5, AMSB107.6, AMSB107.7,

[0595] AMSB107.8, AMSB107.9, AMSB107.10, AMSB107.11, AMSB107.12, AMSB107.13, AMSB107.14, AMSB107.15, AMSB107.16, AMSB107.17, AMSB107.18, AMSB107.19,

[0596] AMSB107.20, AMSB107.21, AMSB107.22, AMSB107.23, AMSB107.24, AMSB107.25,

[0597] AMSB107.26, AMSB107.P6, AMSB107.P40K, AMSB107P and AMSB107.27.

[0598] 158. The bispecific aptamer of any one of embodiments 142-157, comprising SEQ ID NO:

[0599] 140-195, the nucleotide sequence shown in any one of SEQ ID NOs: 202-204.

[0600] 159. A composition comprising the anti-Ang2 aptamer of any one of embodiments 1-69, the anti-VEGF aptamer of any one of embodiments 70-141, and / or the bispecific aptamer of any one of embodiments 142-158.

[0601] 160. The composition of embodiment 159, which is a pharmaceutical composition.

[0602] 161. The composition of any one of embodiments 159-160, comprising a pharmaceutically acceptable excipient or carrier.

[0603] 162. The composition of any one of embodiments 159-161, for use in treating a neovascular disease, disorder or condition.

[0604] 163. The composition of embodiment 162, wherein the neovascular disease, disorder or condition is an ocular neovascular disease, disorder or condition.

[0605] 164. A composition as described in any of embodiments 162-163, wherein the neovascular disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion and diabetic retinopathy.

[0606] 165. A composition as described in any of embodiments 162-164, wherein the neovascular disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization and proliferative diabetic retinopathy.

[0607] 166. A method for treating a neovascular disease, disorder or condition, comprising administering to a subject in need thereof an effective amount of an anti-Ang2 aptamer as described in any one of embodiments 1-69, an effective amount of an anti-VEGF aptamer as described in any one of embodiments 70-141, an effective amount of a bispecific aptamer as described in any one of embodiments 142-158, and / or an effective amount of a composition as described in any one of embodiments 159-165.

[0608] 167. The method of embodiment 166, wherein the neovascular disease, disorder or condition is an ocular neovascular disease, disorder or condition.

[0609] 168. A method as described in any of embodiments 166-167, wherein the neovascular disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion and diabetic retinopathy.

[0610] 169. A method as described in any of embodiments 166-168, wherein the neovascular disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0611] 170. Use of the anti-Ang2 aptamer of any one of embodiments 1-69, the anti-VEGF aptamer of any one of embodiments 70-141, the bispecific aptamer of any one of embodiments 142-158 and / or the composition of any one of embodiments 159-165 for the manufacture of a medicament for treating a neovascular disease, disorder or condition.

[0612] 171. The use of embodiment 170, wherein the neovascular disease, disorder or condition is an ocular neovascular disease, disorder or condition.

[0613] 172. The use of any one of embodiments 170-171, wherein the neovascular disease, disorder or condition is selected from age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion and diabetic retinopathy.

[0614] 173. The use of any one of embodiments 170-172, wherein the neovascular disease, disorder or condition is selected from wet age-related macular degeneration, myopic choroidal neovascularization, and proliferative diabetic retinopathy.

[0615] Example

[0616] The following examples are set forth in order to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are neither intended to limit the scope of what the inventors regard as their invention nor to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pairs; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; nt, nucleotides; im, intramuscular; ip, intraperitoneal; sc, subcutaneous, etc.

[0617] Example 1 Selection of anti-VEGF aptamers

[0618] The anti-VEGF aptamers of the present disclosure were identified using chemically enhanced particle display (CAPD), which is a platform described below (see Wang et al. WO 2018064086A1; Wang et al. WO2018102115A1; Wang et al. PCT / US2013 / 071318). Detailed methods and steps are described in, for example, WO2018064086A1 (incorporated herein in its entirety by reference).

[0619] Briefly, recombinant human VEGF-121 and VEGF-165 proteins from multiple suppliers (listed in Table 1) were used as target proteins for aptamer discovery. Typical initial selection libraries contained a random region of 40-50 nucleotides, in which all nucleotides contained 2'-fluorine (2'-F) modifications. For example, for selection against human VEGF-121 (R&D systems, 4644-vs-CF), the forward primer used was 5'-CGCCCTCGTCCCATCTC-3' (SEQ ID NO: 196) and the reverse primer used was 5'-CGTTCTCGGTTGGTGTTC-3' (SEQ ID NO: 197).

[0620] Table 1 Recombinant VEGFA protein used for aptamer selection

[0621]

[0622]

[0623] The selection begins with a library of synthetic monoclonal modified aptamer particles (MAPs), each of which displays multiple copies of the complete 2'-fluoro-modified aptamer candidate sequence on its surface. The emulsion is then broken, demixing the reverse strands, leaving the single-stranded 2'-fluoro-modified aptamer (about 90%) and the native DNA template sequence (about 10%) on the particle, completing the formation of the MAP, ready for screening. Like the template particles, MAPs are monoclonal, with each particle displaying about 10 5 copies of a single modified aptamer sequence and about 10 4 Copies of amplifiable natural DNA sequences.

[0624] The MAPs are then incubated at 37°C with one or more selected VEGF targets from Table 1. Subsequently, the MAPs are labeled using fluorescently labeled (Alexa Fluor 488 or 647) anti-VEGF antibodies (for untagged VEGF), anti-His antibodies (for His-tagged VEGF), or streptavidin (for biotin-labeled VEGF). The fluorescence intensity of each MAP reflects the relative affinity of the 2'-fluoro-modified aptamer to the VEGF target, which allows for quantitative separation of aptamers with high affinity to the target via a Fluorescence Activated Cell Sorter (FACS). In order to repeat several rounds of CAPD to focus the pool on the best binders, PCR is used to amplify the native DNA coding strand on the enriched MAPS via a high-fidelity polymerase, followed by several rounds of screening or sequencing. Anti-VEGF aptamers with higher affinity are preferentially selected by shortening the incubation time, reducing the VEGF concentration, and increasing the washing time in consecutive rounds. To avoid the generation of anti-VEGF aptamers that bind to VEGF primarily via less specific polar interactions, increasing concentrations of polyanionic competitors such as salmon sperm DNA (Thermo Fisher Scientific, 15632011), herring sperm DNA (Sigma-Aldrich, D3159) and dextran sulfate (Sigma-Aldrich, 31404) were introduced in successive rounds.

[0625] To ensure binding specificity to the selected target, increasing concentrations of non-target proteins, such as bovine serum albumin (BSA) or casein, or non-target protein mixtures, such as dilute human serum (0%, 0.1%, 1%, and 10%), can be introduced into the selection to ensure that the resulting anti-VEGF aptamer can specifically recognize VEGF with great affinity even in the presence of excess (up to 10% compared to VEGF). 9 times the concentration) of interfering proteins.

[0626] A method for enriching anti-VEGF aptamers with slow off-rates was also incorporated in some selections. To do this, the enriched high-affinity MAPs were incubated with saturating concentrations of biotin-labeled VEGF (ACROBiosystems, VE1-H82E7 or VE5-H82Q0) targets, followed by washing with excess unlabeled VEGF (ACROBiosystems, VE1-H5246 or VE5-H5248) to increase the duration (from 1 minute to 24 hours) in consecutive rounds. Unlabeled VEGF can replace biotin-labeled VEGF dissociated from MAPs and will not show any fluorescent signal after MAPs are labeled with fluorescent streptavidin. After the washing procedure, MAPs displaying anti-VEGF aptamers with slower off-rates can retain more biotin-labeled VEGF targets and show higher fluorescence intensity, which allows for quantitative separation of anti-VEGF aptamers with slow off-rates to VEGF via fluorescence activated cell sorter (FACS).

[0627] For some selections against VEGF-165, excess heparin (Sigma-Aldrich H3393) was introduced as a competitor in the selection to ensure that anti-VEGF aptamers with higher affinity for the receptor binding domain of VEGF-165 were preferentially selected and anti-VEGF aptamers targeting the heparin binding domain of VEGF-165 were avoided. By this approach, the resulting anti-VEGF aptamers are likely to recognize both VEGF-165 and VEGF-121.

[0628] To generate anti-VEGF aptamers that recognize both VEGF-121 and VEGF-165 with similarly high affinity, some selections were performed by switching the protein target between human VEGF-121 and human VEGF-165 during alternating rounds of selection. The "switching" selection process generated a family of anti-VEGF aptamers that bind VEGF-121 and VEGF-165 with high affinity. Species cross-reactivity aids in preclinical evaluation of potential therapeutic anti-VEGF aptamers in animal models. To generate aptamers that bind both human VEGF and rat / mouse VEGF, some selections were also performed by switching 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.

[0629] To ensure that the selected anti-VEGF aptamer can inhibit the interaction between VEGF and its receptor, the VEGF binding agents that recognize the receptor binding domain of VEGF antigen, such as recombinant human VEGF receptor 2 (VEGFR2, R&D Systems, 357-KD-050), recombinant human VEGF receptor 1 (VEGFR1, R&D Systems, 321-FL-050), ranibizumab ( ) or aflibercept Several selections were performed. Existing VEGF binders were used as competitors during the particle display screen.

[0630] Next generation sequencing using Illumina MiSeq TM The selected aptamer pools are sequenced by the system and analyzed bioinformatically. Because these aptamers are generated using advanced selection methods as described above, such as including polyanions and / or protein competitors, improved dissociation rates, switching between VEGF-121 and VEGF-165, the resulting aptamers can recognize both VEGF-121 and VEGF-165 with high affinity.

[0631] Example 2 Identification and truncation of anti-VEGF aptamers

[0632] Truncated and modified variants of the anti-VEGF aptamers disclosed herein were prepared by LGC Biosearch Technologies (Novato, CA) and their binding affinities to the corresponding targets were examined. Briefly, the equilibrium binding constants of the enriched libraries were measured using magnetic bead separation. Biotinylated anti-VEGF aptamers were conjugated to 1 μm MyOne Streptavidin C1 magnetic beads coated with streptavidin (Thermo Fisher Scientific, 65002). A fixed concentration of anti-VEGF aptamers (0.01 nM) was added to PBSMCT buffer (DPBS, pH 7.2, with 2.5 mM MgCl2, 1 mM CaCl2, and 0.01% TWEEN-20) at a concentration of 10 -12 Up to 10 -8Binding assays were performed by incubating target proteins in the range of 1 μM at 37°C for 90 minutes. Bound target proteins were separated by magnetic separation and subsequently labeled via fluorescent anti-VEGF or anti-His-tag antibodies. The fraction of bound anti-VEGF aptamers was quantified by flow cytometry (BD Accuri C6 Plus). The raw binding data were corrected for nonspecific background binding of target protein to empty magnetic beads. The fraction of bound anti-VEGF aptamers was plotted as a function of protein concentration, and the equilibrium binding constant (K) was extracted from the data using a nonlinear curve fitting algorithm (Langmuir one-site binding model). D value)( Figure 1A-1B ).

[0633] Table 2 shows truncated variants of representative anti-VEGF aptamer sequences of the present disclosure and indicates their K values ​​for VEGF-121 and VEGF-165. D value.

[0634] Table 2 Mutations and truncations of anti-VEGF aptamer AMS0433.0

[0635]

[0636]

[0637] The only FDA-approved aptamer-based anti-VEGF therapeutic that binds only to VEGF-165 In comparison, the anti-VEGF aptamers of the present disclosure bind to human VEGF-121 and human VEGF-165 ( Figure 1A-1B ). FDA-approved protein-based anti-VEGF therapeutics that bind to VEGF-121 and VEGF-165 with similar affinity In comparison, the anti-VEGF aptamers of the present disclosure showed a binding affinity improvement of more than 10-fold.

[0638] Example 3 In vitro VEGF-R competition assay and secondary structure prediction of anti-VEGF aptamers

[0639] To demonstrate that the anti-VEGF aptamers of the present disclosure are effective inhibitors of vascular endothelial growth factor-A (VEGF) signaling, an in vitro VEGF receptor competition ELISA assay was performed to confirm that the anti-VEGF aptamers can effectively block the interaction between VEGF-A and VEGF receptors, and their performance was compared with that of the FDA-approved protein-based drug ranibizumab. Briefly, PBS-MCT buffer (DPBS, pH 7.2, with 2.5 mM MgCl2, 1 mM CaCl2, and 0.01% TWEEN-20) was added at a concentration of 10 -12 Up to 10 -8 AMS0433 (as an example of an anti-VEGF aptamer disclosed herein) or Lucentis in the M range was mixed with 50 pM human VEGF-165 (R&D Systems, 293-VE-050) and transferred to a plate immobilizing human VEGFR2 receptor (R&D Systems, 357-KD-050) and incubated at 37°C for 1 hour. After washing, VEGF-165 that captured VEGFR2 was detected by 1 μg / ml anti-VEGF antibody (R&D Systems, AF-293-NA) and horseradish peroxidase (HRP)-conjugated anti-goat IgG, followed by TMB substrate. VEGF bound to VEGFR2 was quantified by measuring the optical density (OD) at 405 nm using a Biotek Synergy H1 hybrid multimode reader. The raw data was normalized to the absorbance signal from a sample with 50 pM VEGF-165 but without aptamer competition (0 nM aptamer). VEGFR2 binding to VEGF was plotted as a function of aptamer concentration and IC was extracted from the data using a nonlinear curve fitting algorithm (dose-response inhibition). 50 value( Figure 2 ).

[0640] It can be seen that the anti-VEGF aptamer disclosed in the present invention can be used with an IC of about 50 pM. 50 The value effectively inhibited the interaction between VEGF-A and VEGFR2 ( Figure 2 ), which demonstrated that the anti-VEGF aptamer binds to and blocks the receptor binding domain on VEGF and shows similarity to FDA-approved protein-based anti-VEGF therapeutics The potency was increased by about 10-fold compared to Table 3. Exemplary potencies of anti-VEGF aptamers of the present disclosure are listed in Table 3, as measured by a VEGFR2 receptor blocking assay.

[0641] Table 3 In vitro efficacy of anti-VEGF aptamers

[0642] Aptamer ID <![CDATA[VEGFR2 IC 50 (nM)]]> AMS0433.0 27.46 AMS0433.1 1.729 AMS0433.2 9.262 AMS0433.3 2.099 AMS0433.4 1.959 AMS0433.5 2.499 AMS0433.6 3.494 AMS0433.7 8.231 AMS0433.8 42.94 AMS0433.9 >100 AMS0433.10 27.17 AMS0433.11 29.53 AMS0433.12 >100 AMS0433.13 12.28 AMS0433 0.037 AMS0433.15 2.165 AMS0433.16 1.195 AMS0433.17 0.983 AMS0433.18 8.34 AMS0433.19 0.056 AMS0433.20 0.044 AMS0433.21 0.064 AMS0433.m4 0.040 AMS0433.m6 0.041

[0643] In addition, 2-dimensional structural analysis of the selected anti-VEGF aptamers was performed using the NUPACK software suite. Figure 5A The results for AMS0433 are shown in .

[0644] In addition, it was demonstrated that several 2'-F nucleotides were replaced with 2'-Ome nucleotides without compromising performance. A key advantage of the aptamers of the present application is that they are flexible for a variety of chemical substitutions or modifications. Such modifications are easily achieved during the chemical synthesis process. For example, VEGF aptamer AMS0433 (having a nucleotide sequence as shown in SEQ ID No. 128) and 2 additional variants (AMS0433.m4 and AMS0433.m6, having a nucleotide sequence as shown in SEQ ID No. 200 or SEQ ID No. 201, respectively), each containing multiple 2'-Ome-modified nucleotides in place of the previous 2'-Fluoro-modified nucleotides. Both variants showed similar potency to AMS0433 in an in vitro VEGF-R competition assay (Table 3), and had additional benefits compared to AMS0433, such as better manufacturability.

[0645] Example 4 In vivo efficacy testing of anti-VEGF aptamers in non-human primate (NHP) laser-induced CNV model

[0646] This study was conducted essentially according to previously established procedures. Adult cynomolgus macaques (2-4 kg) were used in the study. The monkeys were anesthetized with ketamine hydrochloride (10 mg / kg, intramuscular injection) and sodium pentobarbital (12 mg / kg, intravenous injection), the pupils were dilated, and CNV was induced in the monkeys by rupturing the Bruch's membrane around the macula with a laser (6-8 burns per eye. Laser settings: 532 nm wavelength, 400-500 mW, 50 μm diameter, 100 ms duration). Only laser spots that formed bubbles but did not bleed were considered effective CNV lesions and were included in the study. Two weeks after laser treatment, fluorescein angiography (FA) was performed to visually observe CNV lesions: 100 mg / ml sodium fluorescein was injected at 10 mg / kg body weight; after injection, a series of rapid images were taken in the first minute (early images) and 5 minutes later (late images). Leakage was visually assessed for each lesion by comparing late images to early images, and the severity of leakage was scored according to a scale of grades I to IV: Grade I - no hyperfluorescence; Grade II - hyperfluorescence, no leakage; Grade III - hyperfluorescence and late leakage in early or mid-course images; Grade IV - bright hyperfluorescence mid-course, late leakage beyond the treated area. Grade IV lesions were considered clinically significant. Optical coherence tomography (OCT) was also performed to measure the height of subretinal hyperreflective material (SHRM) at the laser burn, which resembles human pathological CNV. The following agents were then administered via intravitreal injection on the same day: Group 1: 50% AMS0433; Group 2: 50% S0 negative control aptamer. One and two weeks after the test article injection, the CNV lesions were measured again according to the above procedure, and the aptamer effect was measured by comparing the leakage area of ​​Grade IV lesions before and after injection. In addition, OCT was also performed, and the height of SHRM was compared before and after injection.

[0647] The results are shown in Figure 7. As expected, the negative control aptamer showed little effect on the thickness and leakage area of ​​SHRM. In contrast, injection of the anti-VEGF aptamer (e.g., AMS0433) of the present disclosure caused a sharp reduction in the thickness and leakage area of ​​SHRM and caused complete regression of grade IV lesions, confirming its efficacy in effectively inhibiting CNV of NHPs.

[0648] Interestingly, this model was also tested But it has been reported that it has no effect on inhibiting CNV, probably because it does not inhibit some VEGF isoforms, such as VEGF-121 (see RTTzekov et al., Evaluation of intravitreal application of Pegaptanib Sodium on Laser-induced CNV in Cynomolgus Monkeys, ARVO Annual Meeting Abstract, May 2006). However, the aptamers of the present disclosure showed effective efficacy in this model.

[0649] Example 5 Anti-Ang2 Aptamer Selection

[0650] The selection method is similar to that of Example 1.

[0651] Briefly, recombinant human angiopoietin-2 protein (R&D Systems, 623-AN) was used as a target protein for the discovery of anti-Ang2 aptamers. A typical initial selection library contained a 40-nucleotide random region, in which all nucleotides contained 2'-fluorine (2'-F) modifications. The forward primer was 5'-ATCCAGAGTGACG-CTCTTCAGCA-3' (SEQ ID NO: 198) and the reverse primer was 5'-TGAAGAGCGTCACTCTGGAT-3' (SEQ ID NO: 199). The following example describes the selection and generation of aptamers that specifically bind to recombinant human Ang2.

[0652] Each selection starts with a library of synthetic monoclonal modified aptamer particles (MAPs), each of which displays multiple copies of a fully 2′-fluoro-modified aptamer candidate sequence on its surface. The MAPs are then incubated with the Ang2 target at 37°C. Subsequently, the MAPs are labeled using fluorescently labeled (Alexa Fluor 488 or 647) anti-Ang2 antibodies or anti-His antibodies (for His-tagged Ang2) or streptavidin (for biotin-labeled Ang2). The fluorescence intensity of each MAP reflects the relative affinity of the 2′-fluoro-modified aptamer to the Ang2 target, which allows for quantitative separation of aptamers with high affinity to the target via a fluorescence activated cell sorter (FACS). In order to repeat several rounds of CAPD to focus the pool on the best binder, PCR is performed, and the native DNA coding strand is amplified via a high-fidelity polymerase on the enriched MAPS, followed by several rounds of screening or sequencing. Anti-Ang2 aptamers with higher affinity are preferentially selected by shortening the incubation time, reducing the Ang2 concentration, and increasing the washing time in consecutive rounds. To avoid the generation of anti-Ang2 aptamers that bind to Ang2 primarily via less specific polar interactions, increasing concentrations of polyanionic competitors such as salmon sperm DNA (ThermoFisher Scientific, 15632011), herring sperm DNA (Sigma-Aldrich, D3159) and dextran sulfate (Sigma-Aldrich, 31404) were introduced in successive rounds.

[0653] During some selections, advanced techniques were employed to ensure the extreme binding specificity requirement of the anti-Ang2 aptamers - an ideal anti-Ang2 aptamer should not bind to its homologue Ang1, which plays an opposite role to Ang2. For example, increasing concentrations of non-target proteins, such as bovine serum albumin (BSA) or casein or Ang1 (R&D Systems, 923-AN), or mixtures of non-target proteins, such as dilute human serum (0%, 0.1%, 1% and 10%), can be introduced into the selection to ensure that the resulting anti-Ang2 aptamers can specifically recognize Ang2 with great affinity even in the presence of excess (up to 10% compared to Ang2). 9 times the concentration) of interfering proteins.

[0654] Methods for enriching anti-Ang2 aptamers with slow off-rates were also incorporated in some selections. To do this, enriched high-affinity MAPs were incubated with saturating concentrations of biotin-labeled Ang2 targets, followed by washing with excess unlabeled Ang2 to increase the duration in consecutive rounds (from 1 minute to 24 hours). Unlabeled Ang2 can replace biotin-labeled Ang2 that dissociates from MAPs and will not exhibit any fluorescent signal after MAPs are labeled with fluorescent streptavidin. After the washing procedure, MAPs that display aptamers with slower off-rates can retain more biotin-labeled Ang2 targets and exhibit higher fluorescence intensity, which allows for quantitative separation of anti-VEGF aptamers with slow off-rates to Ang2 via a fluorescence activated cell sorter (FACS).

[0655] Species cross-reactivity facilitates preclinical evaluation of potential therapeutic anti-Ang2 aptamers in animal models. To generate anti-Ang2 aptamers that bind both human Ang2 and mouse Ang2, some selections were also performed by switching the protein target between human Ang2 and mouse Ang2 (R&D Systems, 7186-AN) during alternating rounds of selection. The switch selection process generated a family of anti-Ang2 aptamers that bind both human and mouse Ang2 with high affinity.

[0656] Example 6 Identification and truncation of anti-Ang2 aptamers and binding affinity determination

[0657] A variety of mutations and truncated variants of a representative anti-Ang2 aptamer (AMS0525) were obtained and their binding affinity to Ang2 was measured separately using magnetic beads. The biotinylated anti-Ang2 aptamer was conjugated to MyOne streptavidin C1 magnetic beads (Thermo Fisher Scientific, 65002) coated with streptavidin. The fixed concentration of aptamer (0.01 nM) was mixed with PBS MCT buffer (DPBS, pH 7.2, with 2.5 mM MgCl2, 1 mM CaCl2 and 0.01% TWEEN-20) at a concentration of 10 -12 Up to 10 -8 Binding assays were performed by incubating Ang2 proteins in the range of 1 μM at 37°C for 90 min. The bound target protein was separated by magnetic separation and subsequently labeled by fluorescent anti-His-tag antibody. The fraction of bound anti-Ang2 aptamers was quantified by flow cytometry (BD Accuri C6 Plus). The raw binding data were corrected for nonspecific background binding of target protein to empty magnetic beads. The fraction of bound anti-Ang2 aptamers was plotted as a function of protein concentration, and the equilibrium binding constant (K) was extracted from the data using a nonlinear curve fitting algorithm (Langmuir single-site binding model). D value)( Figure 3A ).

[0658] The obtained K for Ang2 is shown in Table 4. D In a series of mutation and deletion experiments, AMS0525 has been truncated to a 48-mer (AMS0526). This is similar to the previously reported partially modified AMS0525 with a K of 3.1 nM. D Compared with the anti-Ang2 aptamer that binds to human Ang2, AMS0526 is a fully modified aptamer that binds to human Ang2 with excellent affinity (K D <100 pM) binds to both human and mouse Ang2, and does not recognize human Ang1 ( Figure 3A ).

[0659] Table 4 Variants of anti-Ang2 aptamer AMS0525

[0660]

[0661]

[0662] Interestingly, it was noted that replacing the 2'F modified nucleotides with the corresponding nucleotides modified with 2'OMe or LNA did not significantly affect the binding of the resulting anti-Ang2 aptamers to target Ang2 (e.g., K of AMS0525.5 and AMS0525.6 D value).

[0663] In addition, the anti-Ang2 aptamer AMS0526 was analyzed in 2D using the NUPACK software suite and Figure 5B The predicted structure is shown in .

[0664] Example 7 Anti-Ang2 Aptamer In Vitro Tie2 Receptor Competition Assay

[0665] In order to demonstrate that the anti-Ang2 aptamers disclosed herein are effective inhibitors of Ang2 signaling, in vitro competition assays were performed to confirm that the selected anti-Ang2 aptamers can block the interaction between Ang2 and Tie-2. 50 0.01 nM His-tagged Ang2 was conjugated to Dynabeads His-tag separation and pull-down beads (Thermo Fisher Scientific, 10104D) and then mixed with 1 nM Tie2 (R&D Systems, 313-TI) and PBS MCT buffer (DPBS, pH 7.2, with 2.5 mM MgCl2, 1 mM CaCl2 and 0.01% TWEEN-20) at a concentration of 10 -12 Up to 10 -8Anti-Ang2 aptamers in the range of 1 μM were incubated at 37 ° C for 90 minutes. Ang2-bound Tie2 and anti-Ang2 aptamers were separated by magnetic separation, and then Ang2-bound Tie2 was labeled via fluorescent anti-human antibodies. The fraction of Ang2-bound receptors was quantified by flow cytometry (BD Accuri C6 Plus). The raw binding data were corrected for nonspecific background binding and normalized to the fluorescence intensity from samples with 1 nM Tie2 but no anti-Ang2 aptamer competition. The fraction of bound receptors was plotted as a function of the anti-Ang2 aptamer concentration, and the IC was extracted from the data using a nonlinear curve fitting algorithm (dose-response inhibition) 50 value( Figure 3B ). It can be seen that AMS0526 can effectively inhibit the interaction between Ang2 and Tie2, IC 50 The aptamer activity was 83.9 pM, which was 30 times better than the previously reported anti-Ang2 aptamer ( Figure 3B ).

[0666] Example 8 In vivo efficacy testing of anti-Ang2 aptamers in oxygen-induced retinopathy (OIR) model

[0667] Studies were performed essentially according to previously established procedures to test effects on angiogenesis and vascular permeability. OIR was induced by placing C57BL / 6 mouse pups in 75% O2 on postnatal day 7 (P7). On P12, mice were returned to room air. To test effects on angiogenesis, mice received intravitreal injections of the following agents on the same day: Group 1 (G1): PBS (negative control); G2: 20 μg aflibercept; G3: 20 μg AMS0525; G4: 20 μg aflibercept + 20 μg AMS0525. On P17, mice were sacrificed, fixed in 10% phosphate-buffered formalin for 4 h at 22°C, mouse eyes were dissected and retinal flat mounts were prepared. Retinas were then stained with FITC-labeled Griffonia simplicifolia (GSA) lectin (Vector Laboratories) for 45 min to visualize NV. Digital photographs were obtained by Zeiss fluorescence microscope at 5X magnification, and the images were merged into a single image using the photomerge option of Photoshop CS5.4 to show the entire retina. An independent researcher who was blinded to the identity of the samples measured the area of ​​retinal NV by Image-Pro Plus (MediaCybernetics). To test the effect on vascular permeability, OIR was induced via the same procedure as above, but mice received injections at P16, when new blood vessel formation (NV) had already developed. In this way, only the effect of the drug on vascular permeability was tested, not NV growth. At P17, vitreous samples were obtained from mice, and the amount of albumin leaked from the blood vessels was measured by ELISA.

[0668] The results are shown in Figure 6. While inhibition of Ang2 alone had very little effect (consistent with previous studies), the combination of the anti-Ang2 aptamer of the present disclosure and aflibercept almost completely eliminated NV, with significantly greater efficacy than aflibercept alone (Figure 6). As shown in Figure 6, despite the establishment of NV, the combination of the anti-Ang2 aptamer of the present disclosure and aflibercept reduced leakage to a greater extent than aflibercept alone.

[0669] Example 9 Obtaining a bispecific aptamer

[0670] To develop bispecific aptamers that can recognize both VEGF and Ang2 simultaneously, various aptamer constructs were prepared and tested, comprising an anti-VEGF aptamer (AMS0433 or its derivatives from Table 2) linked to an anti-Ang2 aptamer AMS0526 (shown in Table 4). The bispecific aptamer constructs were synthesized head-to-tail with either the anti-VEGF aptamer at the 5' end (AMSB107.0) or the anti-Ang2 aptamer at the 5' end (AMSB108.0) in both orientations, directly linked without any linker. The results are summarized in Table 5.

[0671] Table 5 Binding affinity of bispecific aptamers

[0672] Aptamer ID KD for VEGF-165 (nM) KD for Ang2 (nM) AMSB107.0 1.286 0.222 AMSB108.0 15.67 0.520

[0673] Based on the binding affinity to VEGF and Ang2, the bispecific aptamer AMSB107.0 appeared to give the best results and was selected for further truncation and mutation experiments. The VEGF / Ang2 bispecific aptamer AMSB107 showed K values ​​of 1.3 for VEGF and Ang2. D and IC 50 The binding affinity of AMSB107 was equal to or better than that of its precursor aptamer (Table 6). Based on the results of functional receptor inhibition assays, AMSB107 inhibited VEGF / VEGFR2 and Ang2 / Tie2 interactions, IC 50 The value was about 100 pM (Figure 4).

[0674] Table 6 Binding affinity and in vitro function of bispecific aptamers

[0675]

[0676]

[0677] The predicted secondary structures of the anti-VEGF aptamer AMS0433, the anti-Ang2 aptamer AMS0526, and the anti-VEGF / anti-Ang2 bispecific aptamer AMSB107 are shown in FIG5 . When AMSB107 was designed, the 3' bottom stem region of the anti-VEGF aptamer AMS0433 and the anti-Ang2 aptamer AMS0526 was carefully selected and shared to minimize the possibility of misfolding and interference between the two individual aptamers ( FIG5 ). Therefore, the structures of both the anti-VEGF aptamer AMS0433 and the anti-Ang2 aptamer AMS0526 were well preserved in the bispecific construct AMSB107.

[0678] Table 7 Binding affinity and in vitro function of bispecific aptamers

[0679] Aptamer ID <![CDATA[VEGF IC 50 (nM)]]> <![CDATA[Tier2 IC 50 (nM)]]> AMSB107.P6 0.039 0.035 AMSB107.P40K 0.036 0.045

[0680] In addition, it was confirmed that the addition of linkers or PEG polymers does not impair performance. Another key advantage of the aptamers of the present application is their modularity. Linkers such as hexaethylene glycol linkers (H or PEG6) can be added between different binding motifs during chemical synthesis, and large polymers such as high molecular weight polyethylene glycol (PEG) can be conjugated to the ends of the aptamers without affecting the performance of the aptamers. For example, the VEGF aptamer AMSB107 (having a nucleotide sequence as shown in SEQ ID No.171) and two other variants (AMSB107.P6 and AMSB107.P40K, having a nucleotide sequence as shown in SEQ ID No.202 or SEQ ID No.203, respectively) show similar potency in in vitro receptor competition assays for VEGF and Ang2. Compared with AMSB107, such variants may have additional benefits, such as adjustable spacing, lower manufacturing costs, and better in vivo pharmacokinetics. AMSB107.P6 uses an H linker to replace several nucleotides in AMSB107, and the H linker can be incorporated using well-known procedures during the chemical synthesis of the aptamer. Using well-known thiol-based conjugation chemistry, AMSB107.P40K was conjugated to the 5' end of AMSB107 using 40 kDa PEG.

[0681] PEGylation and purification procedures:

[0682] The process of adding trityl was used to synthesize an aptamer with a 5'-amino-modified C6 linker (Glen-research, 10-1906-02M) and purified via a Glen-pak purification cartridge (Glen-research, 60-5100-96) to remove the trityl and remove short failed synthetic products. The desalted aptamer was then concentrated using a rotary concentrator (Millipore, UFC901024). 1 mM 5'-amino-modified aptamer was mixed with 1.5 mM to 2 mM 40 kDa Y-type NHS ester modified PEG (JenKemTechnology USA Inc, Y-NHS-40K) in 50% DMSO and 50 mM Teorell and Stenhagen buffer at pH 8.2. The reaction mixture was thoroughly mixed and incubated at 37°C for 1 hour. The reaction mixture was analyzed on an Agilent 1260HPLC system using a DNAPac PA100 analytical column (Thermofisher, 043010). The formation of the PEGylated aptamer was confirmed by the appearance of a corresponding elution peak different from that of the non-PEGylated aptamer. The PEGylated aptamer was then purified using TSKgel SuperQ-5PW anion exchange resin (Tosoh Bioscience, 0043383) on an AKTA Explorer FPLC system. The PEGylated aptamer was then desalted using ultrafiltration, lyophilized and stored at -20°C.

[0683] The binding affinity of the aptamer of the present application to Tier 2 was measured using a method similar to the method for measuring the binding affinity of the aptamer of the present application to VEGF.

[0684] Example 10: PEGylated bispecific aptamers are more potent and longer lasting than aflibercept in the rabbit DL-a-aminoadipate (DL-AAA)-induced retinal NV model

[0685] AMSB107P (having the nucleotide sequence as shown in SEQ ID No. 204), a fully modified bispecific aptamer containing 2'-Fluoro-modified nucleotides and 2'-Ome-modified nucleotides, was constructed and conjugated to 40 kDa PEG at the 5' end using the amino-NHS conjugation chemistry as described for AMSB107.P40K. It was hypothesized that the PEGylated aptamer would have a longer half-life than the biologic; in addition, the VEGF / Ang2 dual targeting mechanism might also be more effective and last longer in vivo. This hypothesis was tested in a rabbit DL-a-aminoadipate (DL-AAA)-induced retinal NV model.

[0686] The study was performed by modifying previously established procedures (see J. Cao, TC MacPherson, BB V Iglesias, Y. Liu, N. Tirko, GD Yancopoulos, SJ Wiegand, C. Romano, Aflibercept Action in a Rabbit Model of Chronic Retinal Neovascularization: Reversible Inhibition of Pathologic Leakage With Dose-Dependent Duration, Investig. Opthalmology Vis. Sci. 59, 1033 (2018)). New Zealand white rabbits (NZW) aged 2-4 months (approximately 6 pounds) were used in this study. The rabbits were acclimated to the new environment for 1 week after arrival at the institution. To induce retinal NV and leakage, 8 mg / kg Zoletil was injected intramuscularly TM Rabbits were anesthetized with 50 and 1.6 mg / kg xylazine and injected intravitreally with 80 μl of freshly prepared 80 mM DL-a-aminoadipic acid (DL-AAA) solution in both eyes to induce retinal vascular leakage. After injection, the injection site was treated with antibiotic ointment using a clean cotton swab. The rabbits were then raised for at least 10 weeks to allow NV and leakage to stabilize.

[0687] Rabbits with stable leakage then received intravitreal injections of the following test articles: Group 1: 50 μl 10 mg / ml Aflibercept; Group 2: 50 μl 10 mg / ml AMSB107P. The efficacy of inhibition of leakage and resolution of NV was measured using the Spectralis HRA+OCT system (Heidelberg). To measure the effect of leakage inhibition, rabbits were injected with 10% sodium fluorescein (10 mg / kg) in the marginal ear vein for fluorescein angiography (FA) imaging; retinal NV was visualized using monochromatic imaging. FA and monochromatic imaging were performed one day before injection (as baseline) and weekly thereafter.

[0688] The safety of the test articles was assessed by weekly slit-lamp biomicroscopy.

[0689] AMSB107P was found to be well tolerated, and no test article-related toxicity was reported throughout the study.

[0690] Through monochrome imaging ( Figure 8) measured the effect on pathological NV. Before injection of the test article, NV was evident in all eyes (indicated by arrows). At 1 week post-injection, resolution of retinal NV was observed in the aflibercept arm, but residual NV was still evident (indicated by arrows). In contrast, AMSB107P caused almost complete resolution of retinal NV (arrows are located at the same location where NV had previously appeared, for orientation).

[0691] At 8 weeks post-injection, NV had grown back to baseline levels in aflibercept-treated eyes. The regrowth followed the same vascular pattern observed before injection. In contrast, AMSB107P still maintained its inhibitory effect on retinal NV (indicated by arrows).

[0692] NV regrowth was observed in eyes treated with AMSB107P 15 weeks after injection (indicated by arrows). The new NVs followed a different pattern compared to the NVs observed before injection, suggesting that the aptamer caused vascular remodeling. Similar effects of vascular remodeling were also reported in previous reports (see Bibiana, T. MacPherson, J. Cao, C. Romano, Aflibercept in combination with nesvacumab (anti-Ang2) induces vascular remodeling in a rabbit model of pathological neovascularization., Invest. Ophthalmol. Vis. Sci. 59, 1447-1447 (2018)), where the combination of aflibercept and Ang2 antibody (Nesvacumab) caused vascular remodeling, while aflibercept alone did not show such effects.

[0693] Effects on vascular leakage were measured by FA ( Fig. 9 ). Before injection of the test article, leakage was evident in all eyes (indicated by white arrows). At 1 week after injection, both aflibercept and AMSB107P completely inhibited leakage. At 7 weeks after injection, the aflibercept-treated eye began to leak again; leakage worsened at 8 weeks after injection, returning to baseline levels. In contrast, no leakage was observed in the aptamer-treated eye at 7 and 8 weeks after injection. A small amount of leakage began to be observed at 14 weeks after injection; the extent of leakage at both weeks 14 and 15 was significantly below baseline levels.

[0694] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only by way of example. It is not intended that the specific embodiments provided in this specification limit the present invention. Although the present invention has been described with reference to the above description, the description and illustration of the embodiments herein are not meant to be limiting. Without departing from the present invention, those skilled in the art will now make many changes, modifications and substitutions. In addition, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions set forth herein, depending on a variety of conditions and variables. It should be understood that a variety of alternatives to the embodiments of the present invention described herein can be used to practice the present invention. Therefore, it is contemplated that the present invention should also encompass any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the present invention, and therefore encompass methods and structures within the scope of these claims and their equivalents.

Claims

1. An anti-VEGF aptamer having a nucleotide sequence as shown in any one of SEQ ID NO: 128 and SEQ ID NO: 200-201.

2. An agent comprising the anti-VEGF aptamer of claim 1, wherein the anti-VEGF aptamer is conjugated to a polyethylene glycol (PEG) moiety.

3. The agent of claim 2, wherein at least one PEG moiety is conjugated to the 5' end of the anti-VEGF aptamer.

4. The agent as claimed in claim 3, wherein at least one PEG moiety is conjugated to the 3' end of the anti-VEGF aptamer.

5. A bispecific aptamer, which has a nucleotide sequence as shown in any one of SEQ ID NOs: 171, 202-204. 6 . A composition comprising the anti-VEGF aptamer according to claim 1 and / or the bispecific aptamer according to claim 5 .

7. The composition of claim 6, which is a pharmaceutical composition.

8. The composition of claim 7, comprising a pharmaceutically acceptable excipient or carrier.

9. Use of the bispecific aptamer according to claim 5 and / or the composition according to any one of claims 6 to 8 for the manufacture of a medicament for treating age-related macular degeneration, choroidal neovascularization, diabetic macular edema, retinal vein occlusion and diabetic retinopathy.

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