Bispecific Aptamer Compositions for the Treatment of Retinal Disorders

FIELD OF THE INVENTION

Disclosed herein are bispecific aptamers, pharmaceutical compositions comprising the same as well as methods of treating retinal disorders with bispecific aptamers and pharmaceutical compositions. Methods of manufacturing such bispecific aptamers and pharmaceutical compositions are also disclosed.

BACKGROUND

Wet Age-Related Macular Degeneration (wAMD) affects more than 1.7 million Americans with about 200,000 new cases of wet AMD diagnosed each year (National Eye Institute). Anti-VEGF therapy (Lucentis®, Eylea®, Avastin®) is the standard of care and generally results in significant visual gains. Unfortunately, not all patients respond fully with as many as 25-75% of treated patients maintaining persistent retinal fluid (Wells et al. Ophthalmology 123, 1351-1359 (2016); Group, C. R., New England Journal of Medicine 364, 1897-1908 (2011); Heier et al. Ophthalmology 119, 2537-2548 (2012)). Persistent retinal fluid is associated with worse long-term visual outcomes compared to patients with dry/normal retinas (Sharma, S. et al. Ophthalmology 123, 865-875 (2016); Brown et al., Retina 33, 23-34 (2013)). For patients that respond well, treatment can be conducted at the prescribed dosing intervals (q4w, q8w or q 12w depending on the drug) or “as needed” to improve or maintain visual gains. For patients that do not respond well, monthly dosing is required. For example, nearly one third of the patients in the HARBOR trial required near monthly dosing, a consequence of ≥5 letter decrease in vision, intraretinal fluid, subretinal fluid, or subretinal pigment epithelial fluid (Ho et al. Ophthalmology 121, 2181-2192 (2014)). For patients that don't fully respond (e.g., maintain fluid), the standard practice is currently to switch from one anti-VEGF therapy (usually Avastin® to start) to one of the alternatives (Lucentis® or Eylea®). In some instances, treatment dosing is increased to levels beyond what is prescribed. However, improvements gained with switching are usually minimal and are mostly anecdotal (Shah, C. P. Review of Ophthalmology (2018); You et al. Retina (Philadelphia, Pa.) 38, 1156 (2018)).

Diabetic Macular Edema (DME), which is a type of diabetic retinopathy (DR), affects more than 750,000 Americans and is a leading cause of vision loss for people with diabetes (Varma, R. et al. JAMA Ophthalmology 132, 1334-1340 (2014). Anti-VEGF therapies are only effective for ˜30-40% of patients. For example, an analysis of data from the DRCR Network's Protocol I revealed only 40% of eyes showed improvement in best corrected visual acuity [BCVA] (≥10 letters) by week 12 following 3 doses of Lucentis®. No further vision improvement was observed for most patients beyond what was observed in the initial 12 weeks even after a year of monthly dosing (Gonzalez, V. H. et al. American Journal of Ophthalmology 172, 72-79 (2016). Vascular and tissue inflammation contribute to DME, which is supported by studies correlating high levels of cytokines in the vitreous and aqueous humors of DME patients (Roh et al., Ophthalmology 116, 80-86 (2009); Funk, M. et al. Retina 30, 1412-1419 (2010); Feng, S. et al. Journal of Diabetes Research 2018 (2018); Jonas et al. Retina 32, 2150-2157 (2012)). Steroids (Ozurdex® and Iluvien®) are approved as second-line treatment alone or in combination with anti-VEGF therapy. However, the broad mechanism of action of these drugs leads to partial downregulation of a host of different cytokines, chemokines and growth factors. This contributes to side effects such as increased ocular pressure and cataracts, which limits their use (Schwartz et al., Clinical Ophthalmology (Auckland, NZ) 10, 1723 (2016); Regillo, C. D. et al. Ophthalmic Surgery, Lasers and Imaging Retina 48, 291-301 (2017)).

The initial pivotal randomized controlled trials supported monthly dosing for Lucentis® and Avastin® and bimonthly dosing after 3-monthly doses for Eylea®. In order to mitigate the treatment burden of wAMD and DME, attention has been placed on researching the optimal dosing regimen for these medications. Anti-VEGF therapy has been administered at regularly spaced fixed intervals in ‘continuous’ regimens or at varying intervals in ‘discontinuous’ regimens in an attempt to reduce the burden, risks and costs of repeated intravitreal injections. These discontinuous regimens include a ‘pro re nata’ (PRN) approach based on findings of exudation, or a ‘treat and extend’ (T&E) approach that gradually increases assessment and treatment intervals after exudation is controlled. However, recent real-world data have shown that patients who receive a low number of annual injections achieve meaningfully worse visual acuity outcomes than those in pivotal trials.

Although anti-VEGF therapies have been effective and revolutionized the way retinal diseases are treated, a significant portion of patients do not respond to treatment or are undertreated due to the injection burden of current therapies and are left with inflammation, retinal fluid and edema. New approaches are needed to enhance efficacy, reduce treatment burden and improve patient care.

SUMMARY OF THE INVENTION

Disclosed herein are bispecific aptamers (e.g., RNA aptamers) that specifically bind to two or more target molecules (e.g., VEGF, IL8, Ang2 and combinations thereof), as well as pharmaceutical compositions comprising such bispecific aptamers. Also disclosed are methods of using such bispecific aptamers and pharmaceutical compositions for the treatment of ocular disease and disorders (e.g., retinal diseases and disorders), as well as methods of making such bispecific aptamers and pharmaceutical compositions.

In one aspect, a bispecific RNA aptamer is disclosed comprising Formula I:

X
₁-(aptamer1)-X₂-(linker)-Y₁-(aptamer2)-Y₂-invdT Formula I

wherein the bispecific aptamer comprises at least one nucleotide sequence shown in in Table A or at least one nucleotide sequences sharing at least about 70% identify with the nucleotide sequences shown in Table A.

In one embodiment, the aptamer and aptamer 2 each comprise a nucleotide sequence selected from SEQ ID Nos identified in Table 1 and sequences sharing at least about 70% identity with such SEQ ID Nos.

In a particular embodiment, the bispecific RNA aptamer has a hydrodynamic radius of about between about 9 and about 15 nm and more particularly, about 13.5 nm.

In a particular embodiment, aptamer 1 comprises a nucleotide sequence selected from SEQ ID. Nos.: 1-54. In a particular embodiment, aptamer 2 comprises a nucleotide sequence selected from SEQ ID. Nos.: 1-54.

In one embodiment, aptamer 1 and aptamer 2 are between about 30 and about 40 nucleotides in length.

In one embodiment, an inverted deoxythymidine (invdT) is incorporated at the 3′-end of the bispecific aptamer of Formula I, leading to the formation of a 3′-3′ linkage which inhibits both degradation by 3′ exonucleases and extension by DNA polymerases.

In another embodiment, the bispecific RNA aptamer specifically binds to VEGF or an isoform thereof (e.g., VEGF-A) and IL8 and inhibits the function thereof by between about 90% and about 100%, more particularly, about 90%, about 95%, about 98% or about 100%.

In a particular embodiment, the bispecific RNA aptamer binds to VEGF or an isoform thereof (e.g., VEGF-A) and IL8 with a binding affinity of between about 250 pM and about 20 pM, between about 500 nM and about 10 pM, or between about 750 nM and about 1 pM. In certain embodiments, the bispecific RNA aptamer has a binding affinity of about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM or about 800 nM, about 850 nM, about 900 nM, about 950 nM or about 1 pM. In one embodiment, the bispecific RNA aptamer has a binding affinity less than about 20 pM, less than about 15 pM, less than about 10 pM, less than about 5 pM or about 1 pM or less.

In another embodiment the bispecific RNA aptamer specifically binds to VEGF or an isoform thereof (e.g., VEGF-A) and Ang2 and inhibits the function thereof by between about 90% and about 100%, more particularly, about 90%, about 95%, about 98% or about 100%.

In a particular embodiment, the bispecific RNA aptamer binds to VEGF or an isoform thereof (e.g., VEGF-a) and Ang2 with a binding affinity of about 250 pM and about 10 pM. In certain embodiments, the bispecific RNA aptamer has a binding affinity between about 500 nM and about 5 pM, or between about 750 nM and about 1 pM. In certain embodiments, the bispecific RNA aptamer has a binding affinity of about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM or about 800 nM, about 850 nM, about 900 nM, about 950 nM or about 1 pM. In one embodiment, the bispecific RNA aptamer has a binding affinity less than about 10 pM, less than about 5 pM, or less than about 10 pM.

In a further embodiment, the bispecific RNA aptamer specifically binds to IL8 and Ang2 and inhibits the function thereof by between about 90% and about 100%, more particularly, about 90%, about 95%, about 98% or about 100%.

In a particular embodiment, the bispecific RNA aptamer binds to IL8 and Ang2 with a binding affinity of between about 20 pM and about 10 pM. In one embodiment, the bispecific aptamer has a binding affinity of about 20 pM, about 18 pM, about 15 pM, about 13 pM, about 10 pM, about 8 pM, about 5 pM, about 3 pM, or about Rogallo 1 pM.

In certain embodiments, X₁comprises between 0-5 nucleotides, wherein the nucleotides are complementary to the nucleotides of X₂.

In certain embodiments, Y₁comprises between 0-5 nucleotides that are complementary to the nucleotides of Y₂.

In one embodiment, the linker is a nucleotide linker comprising between 0 and 20 nucleotides.

In a particular embodiment, the linker is a nucleotide linker comprising one or more 2′ OMe uridine residues.

In certain embodiments, the nucleotide linker comprises UUUUU, where U is 2′ OMe. In certain embodiments, the nucleotide linker comprises GCCGUGUUUUCACGGC (SEQ ID NO:443); where U, G, C and A are 2′ OMe.

In a particular embodiment, the linker is a nucleotide linker comprising one or more 5 mU residues.

In certain embodiment, the linker is a non-nucleotide linker as shown in Table B.

In a particular embodiment, the linker is a heterobifunctional linker comprising a thiol reactive moiety (e.g., maleimide) and an amine reactive moiety.

In a particular embodiment, the linker is a non-nucleotide linker selected from the group consisting of 1,3-propanediol, 1,6 hexanediol, 1,12 dodecyldiol, triethylene glycol or hexaethylene glycol.

In one embodiment, aptamer A and aptamer B joined by hybridization.

In one embodiment, the bispecific RNA aptamer is modified with polyethylene glycol.

In certain embodiment, the polyethylene glycol is coupled to the bispecific aptamer.

In certain embodiments, the polyethylene glycol is coupled to a second linker, wherein the second linker is coupled to the bispecific aptamer.

In one embodiment, the bispecific RNA aptamer is modified with one or more additional therapeutic agents.

In certain embodiments, the bispecific RNA aptamer comprises one or more nucleotides that are chemically modified.

In a particular embodiment, the one or more chemically modified nucleotides are selected from the group consisting of 2′F Guanosine, 2′ OMe Guanosine, 2′OMe Adenosine, 2′OMe Cytosine, 2′OMe Uridine and combinations thereof.

In certain embodiments, the one or more chemical modification(s) result in one or more improved characteristics selected from the group consisting of in vivo stability, stability against degradation, binding affinity for its target, and/or improved delivery characteristics in comparison to the same bispecific RNA aptamer having unmodified nucleotides.

In one embodiment, the one or more chemical modification results in an improvement in in vivo stability and more particularly, the half-life of the non-pegylated bispecific RNA aptamer is greater than about 10 hours or more particularly, greater than about 20 hours.

In certain embodiments, the half-life of the non-pegylated bispecific RNA aptamer is between about 10 and about 100 hours, more particularly, between about 300 and about 700 hours.

In certain embodiments, the half-life of the non-pegylated bispecific aptamer is between about 400 and about 700 hours, more particularly, between about 500 and about 600 hours and even more particularly, about 500, about 525, about 550, about 575, or about 600 hours.

In a particular embodiment, the one or more modifications enhance the affinity and specificity of the binding moiety for the target molecule compared to the bispecific RNA aptamer having a binding moiety with unmodified nucleotides.

In a particular embodiment, the one or chemical modifications provide additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and functionality to the bispecific aptamer.

In certain embodiments, the bispecific RNA aptamer comprises a VEGF Aptamer selected from the group consisting of Aptamer 285, Aptamer 481 and Aptamer 628 and an IL8 Aptamer selected from the group consisting of Aptamer 269 and Aptamer 248 and combinations thereof.

In certain embodiments, the bispecific RNA aptamer comprises VEGF Aptamer 285 and IL8 Aptamer 269.

In certain embodiments, the bispecific RNA aptamer comprises VEGF Aptamer 285 and aptamer 2 comprises IL8 Aptamer 248.

In certain embodiments, the bispecific RNA aptamer comprises VEGF Aptamer 481 and aptamer 2 comprises IL8 Aptamer 269.

In certain embodiments, the bispecific RNA aptamer comprises VEGF Aptamer 481 and aptamer 2 comprises IL8 Aptamer 248.

In certain embodiments, the bispecific RNA aptamer comprises VEGF Aptamer 628 and aptamer 2 comprises IL8 Aptamer 269.

In certain embodiments, the bispecific RNA aptamer comprises VEGF Aptamer 628 and aptamer 2 comprises IL8 Aptamer 248.

In certain embodiments, the bispecific aptamer comprises Aptamer 285 and Aptamer 269 linked by a non-nucleotide linker.

In a particular embodiment, the bispecific aptamer comprises Aptamer 285 and Aptamer 269 linked by hybridization.

In one embodiment, the bispecific RNA aptamer is associated with one or more additional molecules, which association may be covalent or non-covalent. In certain embodiments, the association comprises a linker.

In a particular embodiment, the one or more additional molecules is selected from the group consisting of antibodies, peptides, proteins, carbohydrates, enzymes, polymers, drugs, small molecules, gold nanoparticles, radiolabels, fluorescent labels, dyes, haptens, other aptamers, or nucleic acids.

In a particular embodiment, the one or more additional molecules is polyethylene glycol.

In a third aspect, a pharmaceutical composition is disclosed comprising the bispecific RNA aptamer disclosed herein and a pharmaceutically acceptable carrier.

In a particular embodiment, the pharmaceutical composition is formulated for intravitreal administration.

In a fourth aspect, a syringe is disclosed, wherein the syringe is pre-filed with the pharmaceutical composition disclosed herein.

In a fifth aspect, a method of modulating (e.g., inhibiting) the function of at least one target molecule is disclosed, comprising contacting the target molecule with the bispecific aptamer disclosed herein.

In a particular embodiment, the target molecule is selected from VEGF, IL8, Ang2 or a combination thereof.

In a sixth aspect, a method of treating a retinal disease or disorder is disclosed comprising administering an effective amount of the bispecific aptamer disclosed herein to a subject in need thereof, thereby treating the retinal disease or disorder.

In a particular embodiment, the retinal disease or disorder is the wet form of age-related macular degeneration (wAMD).

In a particular embodiment, the retinal disease or disorder is diabetic retinopathy.

In a particular embodiment, the diabetic retinopathy is diabetic macular edema.

In a particular embodiment, the retinal disease is retinal vein occlusion.

In a particular embodiment, the retinal vein occlusion is branched retinal vein occlusion.

In a particular embodiment, the retinal vein occlusion is central retinal vein occlusion.

In a particular embodiment, the retinal disease is retinopathy of prematurity.

In a particular embodiment, the retinal disease is radiation retinopathy.

In one embodiment, the subject in need thereof has been diagnosed with the retinal disease or disorder.

In a particular embodiment, the subject in need thereof has been previously treated with other anti-VEGF agent(s), but where the subject has shown a suboptimal response to such treatment.

In another embodiment, the subject in need thereof is at risk for the retinal disease or disorder.

In one embodiment, the administering is intraocular administration.

In a particular embodiment, the administering is by intravitreal injection.

In a particular embodiment, the intravitreal injection is part of kit containing a syringe that is prefilled with the bispecific composition.

In a particular embodiment, treatment results in an increase in overall best corrected visual acuity (BCVA) as measured on the Early Treatment Diabetic Retinopathy Study (ETDRS) chart by at least 3 letters, at least 4 letters, at least 5 letters, at least 6 letters, at least 7 letters, at least 8 letters, at least 9 letters, at least 10 letters, at least 11 letters, at least 12 letters, at least 13 letters, at least 14 letters, at least 15 letters, at least 16 letters, at least 17 letters, at least 18 letters, at least 19 letters, at least 20 letters, or more than 20 letters as compared to an untreated control subject over a defined period of time, selected from at least one of 2 weeks, one month, 2 months, 3 months, 6 months, one year, 2 years, or 5 years.

In one embodiment, treatment results in a percentage of patients gaining ≥15 letters in BCVA from baseline of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more as compared to an untreated control subject over a defined period of time, selected from at least one of 2 weeks, one month, 2 months, 3 months, 6 months, one year, 2 years, or 5 years.

In one embodiment, treatment results in a percentage of patients gaining ≥10 letters in BCVA from baseline of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more as compared to an untreated control subject over a defined period of time, selected from at least one of 2 weeks, one month, 2 months, 3 months, 6 months, one year, 2 years, or 5 years.

In one embodiment, treatment results in a percentage of patients gaining ≥5 letters in BCVA from baseline of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more as compared to an untreated control subject over a defined period of time, selected from at least one of 2 weeks, one month, 2 months, 3 months, 6 months, one year, 2 years, or 5 years.

In a particular embodiment, treatment results in a reduction of retinal fluid as measured by fluorescein angiography (FA) and optical coherence tomography (OCT) of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more as compared to an untreated control subject over a defined period of time, selected from at least one of 2 weeks, one month, 2 months, 3 months, 6 months, one year, 2 years, or 5 years.

In a particular embodiment, treatment results in a reduction of retinal thickness as measured by fluorescein angiography (FA) and optical coherence tomography (OCT) of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more as compared to an untreated control subject over a defined period of time, selected from at least one of 2 weeks, one month, 2 months, 3 months, 6 months, one year, 2 years, or 5 years.

In a particular embodiment, treatment results in a reduction of the total area of choroidal neovascular (CNV) lesions as measured by fluorescein angiography (FA) and optical coherence tomography (OCT) of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more as compared to an untreated control subject over a defined period of time, selected from at least one of 2 weeks, one month, 2 months, 3 months, 6 months, one year, 2 years, or 5 years. In one embodiment, the method further comprises co-administering to the subject in need thereof at least one additional therapeutic modality, e.g., at least one additional therapeutic agent.

In a particular embodiment, the at least additional therapeutic agent is selected from Illuvien® and Ozurdex.®

In a seventh aspect, a method of treating a population of subjects in need thereof is provided, comprising administering an effective amount of the bispecific aptamer disclosed herein to such subjects.

In one embodiment, the method results in effective treatment for more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90% or more than 95% of subjects treated. In a particular embodiment, effective treatment is measured by overall best corrected visual acuity (BCVA) as measured on the Early Treatment Diabetic Retinopathy Study (ETDRS).

In one embodiment, the method results in fewer than 30%, fewer than 25%, fewer than 20%, fewer than 15% or fewer than 10% of such subjects maintaining persistent retinal fluid.

In an eighth aspect, a method of making the bispecific RNA aptamer disclosed herein, comprising direct chemical synthesis, enzymatic synthesis, chemical synthesis followed by domain chemical conjugation, and/or domain hybridization.

In one embodiment, the bispecific aptamer is synthesized by direct chemical synthesis.

In one embodiment, the bispecific aptamer is synthesized by enzymatic synthesis.

In one embodiment, the bispecific aptamer is synthesized by chemical synthesis followed by domain chemical conjugation.

In one embodiment, the bispecific aptamer is synthesized by domain hybridization.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A Depicts structure of aptamer 285 as folded in mfold which is consistent with the experimentally derived structure.

FIG. 1B Depicts structure of aptamer 269 as folded in mfold which is consistent with the experimentally derived structure.

FIG. 1C Depicts structure of a bispecific aptamer comprised of aptamer 285 and aptamer 269 as folded in mfold. The structures of the aptamer domains are not consistent with the experimentally derived structure.

FIG. 1D Depicts structure of a bispecific aptamer comprised of a variant of aptamer 285 which has been extended by 2 base pairs (aptamer 285ex) and aptamer 269 as folded in mfold. The structures of the aptamer domains are consistent with the experimentally derived structure. The boxed region highlights the additional base pairs.

FIG. 1E Depicts the structure of a bispecific aptamer comprised of aptamer 285 and a variant of aptamer 269 which has been extended by 2 base pairs (aptamer 269ex) as folded in mfold. The structures of the aptamer domains are consistent with the experimentally derived structure. The boxed region highlights the additional base pairs.

FIG. 2: Depicts a plot of the experimentally derived relationship between molecular hydrodynamic radius and the intravitreal half-life. The target range for a bispecific aptamer is indicated.

FIG. 3: Depicts a flow diagram illustrating the steps involved in the synthesis, deprotection, PEGylation and purification of a bispecific aptamer by direct chemical synthesis.

FIG. 4: Depicts examples, approaches and parameters to link two aptamers by a nucleotide linker N(n). Two different aptamer domains can be linked by a linker comprised of nucleotides. The length of the linker can vary from 0 to 50 nucleotides in length. The linker can be unstructured or structured (e.g., designed to form a stem loop). When designed to form a stem loop the length of the stem can be varied from 2 to 10 nucleotides and then loop length varied from 3 to 10 nucleotides. A structured stem linker can be flanked by nucleotide linkers (X(n) and Y(n)) that are between 0 and 15 nucleotides in length.

FIG. 5A: Depicts examples, approaches and parameters to link two aptamers by a non-nucleotide linker. Aptamer domains can be linked with the 3′ end of the first aptamer attached to the 5′ end of the second aptamer.

FIG. 5B Depicts examples, approaches and parameters to link two aptamers by a non-nucleotide linker. Aptamer domains can be linked with the 3′ end of the first aptamer is attached to the 3′ end of the second aptamer.

FIG. 5C Depicts examples, approaches and parameters to link two aptamers by a non-nucleotide linker. Aptamer domains can be linked with the 5′ end of the first aptamer is attached to the 3′ end of the second aptamer.

FIG. 5D Depicts examples, approaches and parameters to link two aptamers by a non-nucleotide linker. Aptamer domains can be linked with the 5′ end of the first aptamer is attached to the 5′ end of the second aptamer.

FIG. 6: Depicts an exemplary bispecific aptamer composed of Aptamer 285 and Aptamer 269 linked by a nucleotide linkage composed of five mU residues produced by direct chemical synthesis. mA, mC, mU and mG are 2′OMe RNA, fG is 2′F RNA and sp3 is a 1,3 propanediol linker.

FIG. 7: Depicts an exemplary bispecific aptamer composed of aptamer 285 and aptamer 269 generated by post synthesis chemical conjugation. Depicted here, aptamer 285 and 269 are synthesized separately. Following synthesis aptamer 269 is PEGylated. Following PEGylation, the aptamers are chemically conjugated using a PEG linker. mA, mC, mU and mG are 2′OMe RNA, fG is 2′F RNA and sp3 is a 1,3 propanediol linker.

FIG. 8A: Depicts examples, approaches and parameters to link two aptamers by hybridization. Aptamer domains can be linked by hybridization in which the 3′ end the first aptamer is extended and designed to hybridize and form a duplex with a 3′ extension on the second aptamer. Or, aptamer domains can be linked by hybridization in which the 5′ end the first aptamer is extended and designed to hybridize and form a duplex with a 5′ extension on the second aptamer. The duplex length (DL) can vary between 3 and 35 nucleotides. The duplex may be separated from the aptamer by a nucleotidyl linker 0 to 25 nucleotides in length or a non-nucleotidyl linker.

FIG. 8B Depicts examples, approaches and parameters to link two aptamers by hybridization. Aptamer domains can be linked by hybridization in which the 3′ end the first aptamer is extended and designed to hybridize and form a duplex with a 5′ extension on the second aptamer. The duplex length (DL) can vary between 3 and 35 nucleotides. The duplex may be separated from the aptamer by a nucleotidyl linker 0 to 25 nucleotides in length or a non-nucleotidyl linker.

FIG. 8C Depicts examples, approaches and parameters to link two aptamers by hybridization. Aptamer domains can be linked by hybridization in which the 5′ end the first aptamer is extended and designed to hybridize and form a duplex with a 3′ extension on the second aptamer. The duplex length (DL) can vary between 3 and 35 nucleotides. The duplex may be separated from the aptamer by a nucleotidyl linker 0 to 25 nucleotides in length or a non-nucleotidyl linker.

FIG. 9: Depicts an exemplary bispecific aptamer composed of Aptamer 285 and Aptamer 269 linked by hybridization. Depicted here, aptamer 285 and 269 are synthesized separately bearing a short 8 nucleotide complementary extension. The extension is linked to each aptamer at the 3′ end by a hexaethylene glycol linker (S18). The 5′ end of aptamer 269 is PEGylated. mA, mC, mU and mG are 2′ OMe RNA, fG is 2′F RNA and sp3 is a 1,3 propanediol linker.

DETAILED DESCRIPTION
I. Definitions

The term “about” as used herein means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term “about” means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/−10% of the specified value. In embodiments, about means the specified value.

The terms “administering” or “administration” as used herein generally refer to introducing a therapeutic agent, composition, formulation, etc., to a desired site or location on or within the body of a subject, e.g., a site or location within the eye. Administration may be performed, e.g., by a health care provider. For purposes of convenience, the present specification refers generally to ophthalmologists. However, the methods described herein, including both the methods of the invention and other methods (e.g., methods for diagnosing and/or monitoring a retinal disorder) may be practiced by any qualified health care provider.

The term “affinity” as used herein refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an aptamer) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). As used herein the term “high affinity” means less than 500 nM.

The term “antigen” as used herein refers to the binding site or epitope recognized by an antigen-binding aptamer. The term “aptamer” as used herein refers to a peptide or nucleic acid molecule, such as RNA or DNA that is capable of binding to a specific molecule with high affinity and specificity. Exemplary ligands that bind to an aptamer include, without limitation, small molecules, such as drugs, metabolites, intermediates, cofactors, transition state analogs, ions, metals, nucleic acids, and toxins, such as endotoxins. Aptamers may also bind natural and synthetic polymers, including proteins, peptides, nucleic acids, polysaccharides, glycoproteins, hormones, receptors and cell surfaces such as cell walls and cell membranes. The binding of a ligand to an aptamer, causes a conformational change in the effector domain and alters its ability to interact with its target molecule. An aptamer will most typically have been obtained by in vitro selection for binding of a target molecule. However, in vivo selection of an aptamer is also possible. Aptamers have specific binding regions which are capable of forming complexes with an intended target molecule in an environment, wherein other substances in the same environment are not complexed to the nucleic acid. The specificity of the binding is defined in terms of the comparative dissociation constants (Kd) of the aptamer for its ligand as compared to the dissociation constant of the aptamer for other materials in the environment or unrelated molecules in general. A ligand is one which binds to the aptamer with greater affinity than to unrelated material. Typically, the Kd for the aptamer with respect to its ligand will be at least about 10-fold less than the Kd for the aptamer with unrelated material or accompanying material in the environment. Even more preferably, the Kd will be at least about 50-fold less, more preferably at least 50 about 100-fold less, and most preferably at least about 200-fold less. An aptamer will typically be between about and about 300 nucleotides in length. More commonly, an aptamer will be between about 30 and about 100 nucleotides in length.

The term “aptamer domain” as used herein refers to refers to a nucleic acid element or domain within a nucleic acid sequence or polynucleotide sequence that, at a biophysically effective amount, will bind or have an affinity for one or a plurality of target molecules.

The term “bispecific aptamer” as used herein refers to an aptamer that binds two distinct antigens or two distinct epitopes within the same antigen. The bispecific aptamer may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs).

The term “carrier” as used herein refers to compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

The term “co-administration” as used herein refers to administration of the bispecific aptamer described herein to a subject simultaneously or consecutively with one or more additional therapeutic agents. In a particular embodiment, the one or more additional therapeutic agents include steroids such as Illuvien® and Ozurdex®. In a particular embodiment, the one or more additional therapeutic agents include a Complement Factor 3 (C3) or Complement Factor 5 (C5) inhibitor for the treatment of geographic atrophy and the dry form of advanced macular degeneration. In one embodiment, the C3 inhibitor is APL-2 (Apellis Pharmaceuticals). In one embodiment, the C5 inhibitor is Zimura® (Iveric Bio).

The terms “complementary” or “complementarity” refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T is complementary to the sequence T-C-A. Complementarity may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.

The term “conjugation” as used herein refers to a chemical compound that is formed by joining two or more compounds with one or more chemical bonds or linkers. In an embodiment disclosed herein, a bispecific aptamer is conjugated to a lipid or high molecular weight compound (e.g., PEG), and/or another therapeutic agent.

The term “DNA” means deoxyribonucleic acid.

The terms “effective amount” and “therapeutically effective amount,” are used herein interchangeably to refer to a sufficient amount of an agent or a composition or combination of compositions being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic uses is the amount of the composition as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate “effective” amount in any individual case may be determined using techniques, such as a dose escalation study. The dose could be administered in one or more administrations. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process and type of treatment desired (e.g., aggressive vs. conventional treatment).

The term “epitope” as used herein refers to the part of an antigen (e.g., a substance that stimulates an immune system to generate an antibody against) that is specifically recognized by the antibody. In certain embodiments, the antigen is a protein or peptide and the epitope is a specific region of the protein or peptide that is recognized and bound by an antibody.

The term “hydrodynamic radius” or “Rh” as used herein refers to the radius of an equivalent hard-sphere diffusing at the same rate as the molecule under observation. In certain embodiments, the bispecific aptamers disclosed herein have a hydrodynamic radius that is about 50% greater than aptamers known in the art and more particularly, about 9, about 10, about 11, about 12, about 13, about 14 or about 15 Rh. In certain embodiments, a bispecific RNA aptamer is disclosed that has a hydrodynamic radius of between about 12 and about 14, and more particularly about 13, about 13.5 or about 14 Rh. In certain embodiments, this Rh is measured before pegylation of the bispecific aptamer, wherein pegylation would further increase the hydrodynamic radius, e.g., by about 1, about 2, about 3, about 4 or about 5 Rh or more over the non-pegylated bispecific aptamer.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

The term “isolated” as used herein with reference to a nucleic acid or protein, indicates that the nucleic acid or protein or peptide is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography (HPLC). A protein or peptide that is the predominant species present in a preparation is substantially purified.

The term “linker” as used herein refers to molecule positioned between two moieties. Typically, linkers are bifunctional, i.e., the linker includes a functional group at each end, wherein the functional groups are used to couple the linker to the two moieties.

The term “nucleic acid” as used herein refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof. The term “polynucleotide” refers to a linear sequence of nucleotides. The term “nucleotide” typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA (including siRNA), and hybrid molecules having mixtures of single and double stranded DNA and RNA. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

The term “nucleotide linker” as used herein refers to oligonucleotide that connects an aptamer to another aptamer. In contrast a “non-nucleotide linker” refers to a linker that does not include nucleotides or nucleotide analogs. Without limitations, the nucleotide linker can be single-stranded or a double-stranded oligonucleotide, e.g., a linker comprising a first oligonucleotide strand and second oligonucleotide strand, wherein the first and the second strands are sufficiently complementary to each other. Furthermore, the nucleotide linker can comprise one or more of the nucleotide modifications described herein. A nucleotide linker can be of any length, e.g., between 4-30 nucleotides in length.

The term “pegylated compound” as used herein refers to a compound (e.g., an aptamer) with one or more polyethylene glycol moieties. In certain embodiments disclosed herein, the aptamer or bispecific aptamer is a pegylated compound.

The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. A polypeptide can be any protein, peptide, protein fragment or component thereof. A polypeptide can be a protein naturally occurring in nature or a protein that is ordinarily not found in nature. A polypeptide can consist largely of the standard twenty protein-building amino acids or it can be modified to incorporate non-standard amino acids. A polypeptide can be modified, typically by the host cell, by e.g., adding any number of biochemical functional groups, including phosphorylation, acetylation, acylation, formylation, alkylation, methylation, lipid addition (e.g., palmitoylation, myristoylation, prenylation, etc.) and carbohydrate addition (e.g., N-linked and O-linked glycosylation, etc.). Polypeptides can undergo structural changes in the host cell such as the formation of disulfide bridges or proteolytic cleavage. The peptides described herein may be therapeutic peptides utilized for e.g., the treatment of a disease.

The term “pharmaceutical composition” as used herein refers to compositions that include an amount (for example, a unit dosage) of one or more of the disclosed bi-specific aptamers together with one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents, and/or adjuvants, and optionally other biologically active ingredients.

The term “pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

The term “purified” as used herein refers to a peptide that gives rise to essentially one band in an electrophoretic gel. In some embodiments, the peptide is at least 50% pure, optionally at least 65% pure, optionally at least 75% pure, optionally at least 85% pure, optionally at least 95% pure, and optionally at least 99% pure.

The term “reduces” or “inhibits” are used interchangeably herein to refer to a negative alteration of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 100% or more.

The term “retinal disease” and “retinal disorder” are used interchangeably herein and refers to any disease or disorder in which the retina is affected due to multiple and variant etiologies.

The term “RNA” refers to ribonucleic acid.

The term “SELEX” as used herein refers to Systematic evolution of ligands by exponential enrichment and is a combination of (1) the selection of aptamers that interact with a target molecule in a desirable manner, for example binding with high affinity to a protein, with (2) the amplification of those selected nucleic acids. The SELEX process can be used to identify aptamers with high affinity to a specific target or biomarker.

The term “specifically binds” as used herein refers to the ability of an aptamer to bind to an antigen with an Kd of at least about 1 micromolar down to 1 picomolar and/or bind to an antigen with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen. It shall be understood, however, that the bispecific aptamers disclosed herein are capable of specifically binding to two or more antigens which are related in sequence. For example, the bispecific aptamers disclosed herein can specifically bind to both a human antigen and a non-human ortholog of that antigen.

The terms “subject” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

The term “substantially homologous” or “substantially identical” in the context of two or more oligonucleotides, nucleic acids, or aptamers, generally refers to two or more sequences or subsequences that have at least 40%, 60%, 80%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide identity, when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms or by visual inspection.

The term “unit dosage form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated (e.g., for a single eye); each unit containing a predetermined quantity of an active agent selected to produce the desired therapeutic effect, optionally together with a pharmaceutically acceptable carrier, which may be provided in a predetermined amount. The unit dosage form may be, for example, a volume of liquid (e.g., a pharmaceutically acceptable carrier) containing a predetermined quantity of a therapeutic agent, a predetermined amount of a therapeutic agent in solid form, an ocular implant containing a predetermined amount of a therapeutic agent, a plurality of nanoparticles or microparticles that collectively contain a predetermined amount of a therapeutic agent, etc. It will be appreciated that a unit dosage form may contain a variety of components in addition to the therapeutic agent. For example, pharmaceutically acceptable carriers, diluents, stabilizers, buffers, preservatives, etc., may be included. In certain embodiments, the aptamer or bispecific aptamer disclosed herein is provided in a unit dosage form.

The term “target molecule” or “target” are used interchangeably herein to refer any molecule of interest. The term includes any minor variation of a particular molecule, such as, in the case of a protein, for example, minor variations in amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component, which does not substantially alter the identity of the molecule. A “target molecule”, “target”, or “analyte” refers to a set of copies of one type or species of molecule or multi-molecular structure. “Target molecules”, “targets”, and “analytes” refer to more than one type or species of molecule or multi-molecular structure. Exemplary target molecules include proteins, polypeptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, affibodies, antibody mimics, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, and any fragment or portion of any of the foregoing. In some embodiments, a target molecule is a protein, in which case the target molecule may be referred to as a “target protein.”

The term “treatment or treating” as used herein means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

The term “variant” as used herein with respect to a peptide, refers to a peptide in which an, insertion, deletion, addition and/or substitution has occurred in at least one amino acid residue relative to the reference peptide. The variant may be approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% sequence of the aptamer or aptamer domain.

The terms “vascular endothelial growth factor”, and “VEGF” as used herein refer to naturally-occurring VEGF, including isoforms and variants thereof. As used herein, VEGF includes all mammalian species of VEGF, including but not limited to human, canine, feline, murine, primate, equine, and bovine VEGF.

II. Bispecific Aptamer Compositions

In one aspect, a bispecific aptamer is disclosed comprising Formula A:

(aptamer1)-(linker)-(aptamer2) Formula A

In one embodiment, the bispecific aptamer is a DNA aptamer. In another embodiment, the bispecific aptamer is an RNA aptamer.

In a particular embodiment, the bispecific aptamer is an RNA aptamer wherein the sequence identities of (aptamer 1) and (aptamer 2) are indicated in Table 1.

In certain embodiments, the positions of (aptamer 1) and (aptamer 2) can be exchanged.

In certain embodiments, the linker is a nucleotide linker having between 0 and 20 nucleotides.

In certain embodiments, the linker is a non-nucleotide linker selected from the group consisting of 1,3-propanediol, 1,6 hexanediol, 1,12 dodecyldiol, triethylene glycol or hexaethylene glycol.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to vascular endothelial growth factor A (VEGF-A) selected from the group consisting of SEQ ID NOS: 1-46.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to interleukin 8 (IL8) selected from the group consisting of SEQ ID NOS: 47-48.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to angiopoietin 2 (ANG2) selected from the group consisting of SEQ ID NOS: 49-50.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to complement component 5 (C5) comprising SEQ ID NO: 51.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to platelet-derived growth factor (PDGF) comprising SEQ ID NO: 52.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to fibroblast growth factor (FGF) comprising SEQ ID NO: 53.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to Factor D comprising SEQ ID NO: 54.

In one aspect, a bispecific aptamer is disclosed comprising Formula II:

X
₁-(aptamer1)-X₂-(linker)-Y₁-(aptamer2)-Y₂-invdT Formula I

In certain embodiments, the sequence identities of (aptamer 1) and (aptamer 2) are indicated in Table 1.

In certain embodiments, the positions of (aptamer 1) and (aptamer 2) can be exchanged. In certain embodiments, X₁is 0-5 nucleotides in length that are designed to base pair with region X₂.

In certain embodiments, Y₁is 0-5 nucleotides in length that are designed to base pair with region Y₂.

In certain embodiments, the linker is a nucleotide linker having between 0 and 20 nucleotides.

In certain embodiments, the linker is a non-nucleotide linker selected from the group consisting of 1,3-propanediol, 1,6 hexanediol, 1,12 dodecyldiol, triethylene glycol or hexaethylene glycol.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to vascular endothelial growth factor A (VEGF-A) selected from the group consisting of SEQ ID NOS: 1-46.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to interleukin 8 (IL8) selected from the group consisting of SEQ ID NOS: 47-48.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to angiopoietin 2 (ANG2) selected from the group consisting of SEQ ID NOS: 49-50.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to complement component 5 (C5) comprising SEQ ID NO: 51.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to platelet-derived growth factor (PDGF) comprising SEQ ID NO: 52.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to fibroblast growth factor 2 (FGF2) comprising SEQ ID NO: 53.

In certain embodiments, aptamer 1 or aptamer 2 is an aptamer that binds to Factor D comprising SEQ ID NO: 54.

In another aspect, a bispecific aptamer is disclosed comprising Formula III:

5′-X₁-(aptamer1)-X₂-(linker)-(Hyb1) (Hyb2)-(linker)-Y₂-(aptamer2)-Y₁-5′ Formula II

wherein Hyb1 and Hyb2 are complementary.

In certain embodiments, the sequence identities of (aptamer 1) and (aptamer 2) are indicated in Table 1.

In certain embodiments, the positions of (aptamer 1) and (aptamer 2) can be exchanged.

In certain embodiments, X₁is 0-5 nucleotides in length that are designed to base pair with region X₂.