Multi-valent Anti-c5 molecules

Abstract

Provided herein are methods and compositions for the inhibition complement component (C5). The methods and compositions involve the use of aptamer-containing molecules, comprising one or more moieties that bind to and inhibit C5. The application further provides aptamer-containing molecules for the treatment of retinal diseases and / or hematological diseases.

Description

WSGR Docket No. 66711-706.601 PCT / IB2025 / 000622MULTI- VALENT ANTI-C5 MOLECULESCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application Serial Number 63 / 722,007 filed on November 18, 2024, and U.S. Provisional Application Serial Number 63 / 763,760 filed on February 26, 2025, each of which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Dysregulation of complement system function is implicated in many diseases, ranging from tissue specific (e.g., retinal) to systemic (e.g., hematological) with significant impact on function and overall survival. Complement is complex system comprising many biologically active proteins and fragments thereof, interconnected to many other pathways with and via complicated feedback and regulatory loops. For instance, the complement pathway includes proteins such as, but not limited to, Cl, Clq, C4, C4a, C4b, C3, C3a, C3aR, C3b, C5, C5a, C5aR, C5b, fB, Ba, Bb, fD, fH, and fl. However, the complement pathway is complex and interconnected with many other pathways via numerous negative, positive, and / or regulatory feedback loops.SUMMARY

[0003] Complement dysregulation is associated with various diseases, including ocular conditions, but the precise mechanisms and roles of specific complement proteins remain poorly understood. For example, abnormal levels of complement proteins are observed, but methods to identify specific proteins and their levels that predict differential responses to complementmodulating therapies are still lacking. This underscores the need for targeted approaches to better understand and address complement-related pathways in disease (e.g., ocular diseases) treatment. More specifically, it is unclear how to design and administer anti-C5 molecules to deliver consistent and robust clinical benefit, with first year treatment intervals of longer than 4 weeks, or without increased risk of CNV, for patients with retinal diseases, including AMD and GA.

[0004] Provided herein are methods and compositions to regulate complement pathway by inhibiting the biological function of C5 that addresses this unmet need. Provided herein is a multivalent C5 binding molecule comprising (a) a first aptamer moiety that selectively binds to a complement component C5; and (b) a second, a third, a fourth, a fifth, a sixth, a seventh, and / or a eighth aptamer moiety that selectively binds to the complement component C5, wherein the multivalent C5 binding molecule inhibits a biological function of a complement system. In some embodiments, the multivalent C5 binding molecule further comprises an intravitreal half-lifeWSGR Docket No. 66711-706.601extending moiety. In some embodiments, the first aptamer moiety blocks generation of C5a and / or C5b. In some embodiments, the first aptamer moiety blocks activity of C5a and / or C5b. In some embodiments, each aptamer moiety of the multivalent C5 binding molecule comprises no unmodified RNA or unmodified DNA nucleotide. In some embodiments, each aptamer moiety of the multivalent C5 binding molecule comprises or consists of a nucleotide sequence that is at least about 80% identical to a nucleotide sequence of SEQ ID NOs: 1-9. In some embodiments, each aptamer moiety of the multivalent C5 binding molecule comprises or consists of a nucleotide sequence that is at least about 80% identical to a nucleotide sequence of SEQ ID NO: 2-9. In some embodiments, the first aptamer moiety is avacincaptad. In some embodiments, the multivalent C5 binding molecule binds to C5 with a Kdof less than 20 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 500 pM, less than 100 pM, or less than 50 pM. In some embodiments, the multivalent C5 binding molecule binds simultaneously to multiple C5 molecules. In some embodiments, the multivalent C5 binding molecule inhibits the biological function of the complement system with an IC50of less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, or less than 500 pM as measured by a complement dependent assay. In some embodiments, the multivalent C5 binding molecule provides a greater inhibition of complement function than a monovalent C5 binding molecule or an avacincaptad pegol. In some embodiments, the multivalent C5 binding molecule inhibits the complement function by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70, at least about 80%, at least about 90%, at least about 100%, or greater than that of the monovalent C5 binding molecule or the avacincaptad pegol. In some embodiments, the multivalent C5 binding molecule remains at least about 70%, at least about 80%, or at least about 90% intact after exposure to serum or vitreous for about 48 hours, about 72 hours, or about 96 hours in vitro. In some embodiments, the multivalent C5 binding molecule inhibits at least about 90% of C5 mediated complement activity in human ocular fluid for at least about 90 days, at least about 100 days, at least about 110 days, or at least about 120 days after a single intravitreal injection. In some embodiments, each aptamer moiety of the multivalent C5 binding molecule is covalently attached to the intravitreal half-life extending moiety. In some embodiments, the first aptamer moiety and the second, the third, the fourth, the fifth, the sixth, the seventh, and / or the eighth aptamer moiety are covalently attachedvia a polyethylene glycol (PEG) linker. In some embodiments, the first aptamer moiety and the second, the third, the fourth, the fifth, the sixth, the seventh and / or the eighth aptamer moiety are attached via a molecular linker. In some embodiments, the intravitreal half-life extending moiety comprises a PEG polymer with a molecular weight of about 5 kDa, about 10 kDa, about 20 kDa, about 40 kDa, or about 60 kDa. In some embodiments, theWSGR Docket No. 66711-706.601intravitreal half-life extending moiety has hydrodynamic radius that is from about 6 nm to aboutlO nm. In some embodiments, the multivalent C5 binding molecule has an in vivo intravitreal half-life that is at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, or at least about 8 days. In some embodiments, the multivalent C5 binding molecule is a bivalent C5 binding molecule, and wherein the bivalent C5 binding molecule exhibits at least about 90% inhibition of classical complement pathway that persists for at least about 60 days, at least about 90 days, at least about 120 days, or more after a single intravitreal injection. In some embodiments, the multivalent C5 binding molecule has the in vivo intravitreal half-life or a serum half-life that is greater than that of a control molecule. In some embodiments, the in vivo intravitreal half-life or the serum half-life is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than that of the monovalent C5 binding molecule or the avacincaptad pegol. In some embodiments, the multivalent C5 binding molecule comprises or consists of a nucleotide sequence that is at least about 80% identical to a nucleotide sequence of SEQ IDNOs: 10-12. In some embodiments, the multivalent C5 binding molecule comprises a linking moiety of about 1 kDa to about 10 kDa which links the first aptamer moiety and the second, third, fourth, fifth, sixth, seventh, or eighth aptamer moiety. Provided herein is an aptamer comprising a nucleotide sequence that is at least about 80% identical to a nucleotide sequence of SEQ ID NOs: 2-9. Further provided herein is a pharmaceutical formulation comprising the multivalent C 5 binding molecule described herein, or the aptamer described herein, and a pharmaceutically acceptable excipient, carrier, and / or diluent. In some embodiments, the pharmaceutical formulation is for an intravitreal administration. In some embodiments, the pharmaceutical formulation is administered via a 32 gauge (G), a 31G, a 30G, or a 28G needle. In some embodiments, the pharmaceutical formulation is administered at a concentration of about 1 mg / 100 pL, about 2 mg / 100 pL, about 3 mg / 100 pL, or about 4 mg / 100 pL. In some embodiments, the pharmaceutical formulation is administered with a break force of less than 12 N. In some embodiments, the pharmaceutical formulation is formulated at about 0.1 mg, about 0.25 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 8 mg per 100 pL injection volume, calculated as an acid form of the first aptamer moiety of the multivalent C5 binding molecule described herein.

[0005] Provided herein is a method of inhibiting complement activity in a subject in need thereof, the method comprising: administering to the subject in need thereof a therapeutically effective amount of the multivalent C5 binding molecule described herein, the aptamer described herein, or the pharmaceutical formulation described herein. Further provided herein is a method of treating a subject in need thereof, the method comprising: administering to theWSGR Docket No. 66711-706.601subject in need thereof a therapeutically effective amount of the multivalent C5 binding molecule described herein, the aptamer described herein, or the pharmaceutical formulation described herein. In some embodiments, the therapeutically effective amount of the multivalent C5 binding molecule comprises a dose and frequency that does not exceed an average mass of PEG present in the multivalent C5 binding molecule or the pharmaceutical formulation. In some embodiments, the therapeutically effective amount of the multivalent C5 binding molecule comprises a dose and frequency that does not exceed about 5 mg per eye per month, about 4 mg per eye per month, about 3 mg per eye per month, or 2 mg per eye per month. In some embodiments, the subject in need thereof has, is suspected of having, or is at risk of having a retinal disease or disorder, an ocular disease or disorder, or one or more symptoms associated with the retinal disease or disorder or the ocular disease or disorder. In some embodiments, the retinal disease or disorder or the ocular disease or disorder is selected from the group consisting of: wet age-related macular degeneration, dry age-related macular degeneration, geographic atrophy, diabetic retinopathy, diabetic macular edema, proliferative diabetic retinopathy, retinal vein occlusion, Stargardt disease, retinitis pigmentosa, retinal detachment, and non -arteritic anterior ischemic optic neuropathy (NAION). In some embodiments, the administering comprises an intravitreal administration. In some embodiments, the therapeutically effective amount of the multivalent C5 binding molecule, the aptamer, or the pharmaceutical formulation is administered at every 4 weeks (q4w), every 8 weeks (q8w), every 12 weeks (ql2w), every 16 weeks (ql6w), every 20 weeks (q20w), every 24 weeks (q24w), every 36 weeks (q36w), or every 48 weeks (q48w) intervals.

[0006] Provided herein is a method of reducing a growth rate of Geographic Atrophy (GA) lesion or area, the method comprising: (a) identifying or having identified a subject as having or at risk of having GA; (b) identifying or having identified the subject not having hypoactive (ocular) complement pathway activity; and (c) administering to the subject, or recommending that the subject be treated with, a C5 inhibitor; (d) optionally, measuring the Geographic Atrophy lesion or area at least two different time points; and (e) optionally, calculating the growth rate of the Geographic Atrophy lesion or area growth between the at least two different time points. In some embodiments, the identifying of having identified of (b) comprises measuring the complement pathway activity by proteomic assay. In some embodiments, the hypoactive (ocular) complement pathway activity comprises having a Ba level that is about 10 ng / mL or less. In some embodiments, the hypoactive (ocular) complement pathway activity comprises having a Bb level that is about 40 ng / mL or less. In some embodiments, the Ba level ortheBb level is measured in a sample collected from the subject. In some embodiments, the sample comprises a sample from or processed from serum, aqueous humor, or vitreous humor.WSGR Docket No. 66711-706.601In some embodiments, the multivalent C5 binding molecule described herein, the aptamer described herein, or the pharmaceutical formulation described herein. In some embodiments, the administration to the subject the C5 inhibitor results in an average reduction in the growth rate of the GA lesion or area by at least about 30% as compared to that of a patient received no treatment. In some embodiments, the administration to the subject the C5 inhibitor results in significantly increase in visual function outcomes, when compared to that of a patient received no treatment. In some embodiments, the visual functional outcomes are measured by Best Corrected Visual Acuity (BCVA) or Low Luminance Visual Acuity (LLVA). In some embodiments, the C5 inhibitor is administered, oris recommended to administer, every 8 weeks (q8w), every 12 weeks (ql2w), every 16 weeks (ql6w), every 20 weeks (q20w), every 24 weeks (q24w), every 36 weeks (q36w), or every 48 weeks (q48w) intervals.

[0007] Further provided herein is a method of reducing the growth rate of Geographic Atrophy area or lesion, the method comprising: (a) identifying or having identified a subject as having Geographic Atrophy; (b) identifying or having identified a subject as having pathologic complement pathway activity relevant to C5; (c) administering to the subject, or recommending that the subject be treated with, a C5 inhibitor; and (d) optionally, measuring the Geographic Atrophy area or lesion at least two different time points and calculating the rate of Geographic Atrophy between time points. In some embodiments, the having pathologic complement pathway activity relevant to C5 comprise having at least one of: (a) above a threshold concentration of at least one ocular complement protein; (b) elevated retina flavoprotein fluorescence intensity; or (c) presence of at least one genetic polymorphism in a complement pathway protein. In some embodiments, the having pathologic complement pathway activity relevant to C5 comprises measuring complement pathway activity via a proteomic assay. In some embodiments, the complement pathway protein is measured in a sample collected from the subject. In some embodiments, the sample comprises a sample from or processed from serum, aqueous humor, or vitreous humor. In some embodiments, the having pathologic complement pathway activity relevant to C5 comprises having a Ba level that is at least 10 ng / mL, at least 15 ng / mL, at least 20 ng / mL, at least 25 ng / mL, or at least 30 ng / mL. In some embodiments, the having pathologic complement pathway activity relevant to C5 comprises having a Bb level that is at least 40 ng / mL, at least 80 ng / mL, at least 100 ng / mL, at least 120 ng / mL, or at least 150 ng / mL. In some embodiments, the having pathologic complement pathway activity relevant to C5 comprises having a C5a level that is at least 20 pg / mL, at least 25 pg / mL, at least 30 pg / mL, at least 35 pg / mL, or at least 40 pg / mL. In some embodiments, the having elevated retina flavoprotein fluorescence intensity comprises having an intensity value that is at least about 1 -fold, atleast about 1.25 -foldat least about 1.5-fold, at least about 1.75-fold, or at least about2-WSGR Docket No. 66711-706.601fold greater than that of an age-matched healthy patient group. In some embodiments, the having pathologic complement activity relevant to C5 comprises having a genetic test. In some embodiments, the genetic test comprises testing for a mutation of the Tyr at amino position 402 of the human complement factor H (CFH) protein. In some embodiments, the mutation is present in both alleles. In some embodiments, the genetic test comprises testing for a mutation in CD46, CD55, or CD59. In some embodiments, the mutation in the CD59 comprises having c,146delA (p.Asp49Valfs*31), c,123delC (p.Val42Serfs*38), c.266G>A (p.Cys89Tyr), c.238A>G (p.Arg80Gly), and / or c,146A>T (p.Asp49Val) mutation in a RPE cell. In some embodiments, the genetic test comprises measuring CD59 expression level in the RPE cell of the subject. In some embodiments, the CD59 expression level of the subject is reduced by at least about20%, at least about 30%, at least about 40%, or at least about 50% as compared to that of a healthy subject. In some embodiments, the C5 inhibitor comprises the multivalent C5 binding molecule described herein, the aptamer described herein, or the pharmaceutical formulation described herein. In some embodiments, the method results in an average reduction in the growth rate of the Geographic Atrophy area or lesion by at least about 40% or more when compared to no treatment. In some embodiments, the method results in a significantly increase in visual function outcomes when compared to that of a patient received no treatment. In some embodiments, the visual function outcomes are measured by Best Corrected Visual Acuity (BCVA) or Low Luminance Visual Acuity (LLVA). In some embodiments, the method results in at least about 50% likelihood that an individual eye has at least about 40% slower growth rate of the Geographic Atrophy area or lesion as compared to that of an individual eye prior to the administering. In some embodiments, the C5 inhibitor is administered, or is recommended for administration, beginning at treatment initiation, at q8w, ql2w, ql6w, or q24w intervals.INCORPORATION BY REFERENCE

[0008] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the invention are set forth with particularity in the appended claims. Abetter understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:WSGR Docket No. 66711-706.601

[0010] FIG. 1 depicts a non-limiting example of a role for the alternative complement pathway in the pathogenesis of geographic atrophy.

[0011] FIG. 2A depicts a non-limiting example of a modified anti-C5 aptamer and its target nucleotide(s) for modifications.

[0012] FIG.2B and FIG.2C depict non-limiting formulas for C5 binding aptamers that maybe comprised within a multivalent C5 binding molecule. FIG. 2B shows a C5 binding aptamer comprising stem A5’ and stem A3’. FIG. 2C shows a C5 binding aptamer further comprising Spl8.

[0013] FIGs. 3A-3F depict non-limiting examples of formulas, structures, and designs of multivalent C5 binding molecules described herein. FIG.3A depicts a non-limiting example of a general formula for chemical conjugation of multiple C5 binding aptamer moieties to generate a multivalent C5 binding molecule. FIG. 3B depicts three non-limiting examples of bivalent, trivalent, and tetravalent C5 binding molecules. FIG.3C depicts a non-limiting general formula for a bivalent C5 binding molecule. FIG. 3D depicts a non-limiting general formula for multivalent C5 binding molecules. FIG.3E depicts three non-limiting examples resulting from the general formula of FIG.3C used to generate bivalent, trivalent, and tetravalent C5 binding molecules. FIG. 3F depicts the structure and sequence of a bivalent C5 binding molecule.

[0014] FIG.4A depicts a non-limiting synthetic scheme for synthesis of a bivalent C5 binding molecule.

[0015] FIG.4B shows a non-limiting synthetic scheme for synthesis of a C5 binding molecule from reaction between NHS Ester on PEG and primary amine on Aptamer.

[0016] FIG.4C shows a non-limiting synthetic scheme for synthesis of a C5 binding molecule from reaction between NHS Ester on Aptamer and primary amine on PEG.

[0017] FIG. 5 depicts the complement activity of GA patients that are suitable for an anti-C5 treatment (e.g., a treatment with multivalent C5 binding molecule).

[0018] FIG. 6 depicts the complement activity of GA patients and related expected efficacy with an anti-C5 treatment (e.g., a treatment with multivalent C5 binding molecules).

[0019] FIG.7 depicts inhibition of classical complement pathway activity with exemplary anti-C5 bivalent molecules of the disclosure.

[0020] FIG. 8 depicts purity of synthesized compound by reversed -phase high-performance liquid chromatography (RP-HPLC).DETAILED DESCRIPTION

[0021] Provided herein are methods and compositions comprising a complement component C5 (C5) binding molecule (e.g., a multivalent binding molecule) that comprises multiple aptamerWSGR Docket No. 66711-706.601moieties that inhibit the biological function of the innate or adaptive immune system by selectively binding to and inhibiting the biological function of C5. In some embodiments, a binding molecule described herein is bivalent or multivalent (e.g., trivalent, tetravalent, or hexavalent) binding molecules. In some embodiments, a binding molecule comprises two or more aptamer binding moieties selectively bind to C5. In some embodiments, the one or more binding moieties described herein can be aptamers that contain no unmodified nucleotides.

[0022] In some embodiments, a binding molecule described herein can comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten C5 binding aptamer moieties. For example, in some embodiments, a binding molecule described herein can be a bivalent C5 aptamer binding molecule.

[0023] In some embodiments, the binding molecule described herein can inhibit the biological function of the innate or adaptive immune system by selectively binding to and inhibiting the biological function of C5. In some embodiments, the binding molecule described herein comprises at least two binding moieties that selectively binds to C5 thereby reducing, or inhibiting, either partially or fully, the activity or production of a C5 complement protein or a variant thereof. The binding molecule described herein can directly or indirectly reduce or inhibit the activity or production of a C5 complement protein or variant thereof. In some embodiment, at least one binding moiety can target C5 activation sites or a cleavage site by the C5 convertase. In some embodiments, at least one binding moiety can block generation of and / or activity of C5a and / or C5b, thereby decreasing membrane attack complex (MAC) formation. In some embodiments, the binding molecule described herein can directly or indirectly reduce or inhibit the activity or production of a C5 complement protein or variant thereof. In some embodiments, the binding molecule described herein can reduce or inhibit the conversion of C5 complement protein into its component polypeptides C5a and C5b . In some embodiments, the binding molecule described herein can inhibit C5 convertase. In some embodiments, the binding molecule described herein can reduce or inhibit the activity or production of C5a and / or C5b. In some embodiments, a binding molecule can block C5a and C5a Receptor (C5aR) interaction. In some embodiments, a binding molecule can block C5b-C9 complex formation or C5b and the other components (e.g., C6, C7 or C8).

[0024] The binding molecules described herein (e.g., multivalent C5 binding molecules) may comprise a first aptamer moiety that selectively binds to a complement component C5, and may further comprise a second, a third, a fourth, a fifth, a sixth, a seventh, or an eighth aptamer moiety that selectively binds to C5. For example, in various aspects, the binding molecules may comprise a first aptamer that selectively binds to and inhibits C5, and a second aptamer that selectively binds to and inhibits C5. In some embodiments, the binding molecules describedWSGR Docket No. 66711-706.601herein can simultaneously bind to multiple C5 complement proteins. The binding molecules described herein may inhibit C5 with an IC50 or affinity that is better than (e.g., lower than) a monospecific aptamer molecule that binds to C5.

[0025] The disclosure further provides methods of using C5 binding molecules (e.g.smultivalent C5 binding molecules) as described herein (e.g., to inhibit C5). In various aspects, the methods may involve administering the binding molecule described herein to a biological system, thereby inhibiting C5 production and / or activity. In various aspects, the methods and compositionsprovidedhereinmay be used for the treatment of a retinal disease or disorder. In some cases, the retinal disease or disorder may be macular degeneration, such as age-related macular degeneration. Age-related macular degeneration may be dry age-related macular degeneration, advanced dry age-related macular degeneration (e.g., geographic atrophy), or wet age-related macular degeneration. In some cases, the retinal disease or disorder is an inherited retinal dystrophy. The inherited retinal dystrophy may be Stargardt disease, retinitis pigmentosa, or other related disease. Stargardt disease may be STGD-1. In some cases, the retinal disease or disorder may be the result of an acute event, such as retinal detachment or non -arteritic anterior ischemic optic neuropathy (NAION).

[0026] In various aspects, a binding molecule described herein can be a multivalent binding molecule. In some embodiments, the multivalent binding molecule may be a bivalent C5-C5 molecule. The bivalent C5-C5 molecule may comprise a first anti-C5 aptamer moiety that binds to and inhibits C5, and a second anti-C5 aptamer moiety that binds to and inhibits C5. In some embodiments, the first anti-C5 moiety and the second anti-C5 moiety may be the same. In some embodiments, the first anti-C5 moiety and the second anti-C5 moiety can be different. In some embodiments, the first anti-C5 moiety or the second anti-C5 moiety can be a fully modified anti-C5 aptamer having no unmodified RNA or DNA nucleotides. In some embodiments, the first anti-C5 moiety and the second anti-C5 moiety may be conjugated to one another (e.g., such as by the use of linkers as described herein). In some embodiments, the first, the second, the third, the fourth, the fifth, the sixth, the seventh, and / or the eighth anti-C5 aptamer moieties may all be conjugated to one another. For example, in some embodiments, the first, the second, the third, the fourth, the fifth, the sixth, the seventh, and / or the eighth anti-C5 aptamer moieties be covalently attached via a polyethylene glycol (PEG) linker. In some embodiments, the first, the second, the third, the fourth, the fifth, the sixth, the seventh, and / or the eighth anti-C5 aptamer moieties be covalently attached via a molecular linker.

[0027] The practice of some embodiments disclosed herein employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of theWSGR Docket No. 66711-706.601art. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0028] In general, “sequence identity” refers to an exact nucleotide -to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively . Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the longer sequences and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version2.2.9, available from theNational Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, etal., J. Mol. Biol. 215:403-410(1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (generally nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program . The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton andFederhen, Computers and Chemistry 17:149-163 (1993). Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values therebetween. Typically, the percent identities between a disclosed sequence and a claimed sequence are at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0029] The term “aptamer” as used herein refers to an oligonucleotide and / or nucleic acid analogues that can bind to a specific target molecule. Aptamers can include RNA, DNA, modified RNA, modified DNA, any nucleic acid analogue, and / or combinations thereof. Aptamers can be single-stranded oligonucleotides. In some cases, aptamers may comprise more than one nucleic acid strand (e.g., two or more nucleic acid strands). Aptamers may bind to aWSGR Docket No. 66711-706.601target (e.g. , a protein) with high affinity and specificity through non -Watson-Crick base pairing interactions. Generally, the aptamers described herein are non-naturally occurring oligonucleotides (e.g., synthetically produced) that are isolated and used for the treatment of a disorder or a disease. Aptamers can bind to essentially any target molecule including, without limitation, proteins, oligonucleotides, carbohydrates, lipids, small molecules, and even bacterial cells. Aptamers may be monomeric (composed of a single unit) or multimeric (composed of multiple units). Multimeric aptamers can be homomeric (composed of multiple identical units) or heteromeric (composed of multiple non-identical units). Aptamers herein may be described by their primary structures, meaning the linear nucleotide sequence of the aptamer. Aptamer sequences herein are generally described from the 5’ end to the 3’ end, unless otherwise stated . Additionally or alternatively, aptamers herein may be described by their secondary structures which may refer to the combination of single -stranded regions and base-pairing interactions within the aptamer. Whereas many naturally occurring oligonucleotides, such as mRNA, encode information in their linear base sequences, aptamers generally do not encode information in their linear base sequences. Further, aptamers can be distinguished from naturally occurring oligonucleotides in that binding of aptamers to target molecules is dependent upon secondary and tertiary structures of the aptamer. Aptamers may be suitable as therapeutic agents and may be preferable to other therapeutic agents because: 1) aptamers may be fast and economical to produce because aptamers can be developed entirely by in vitro processes; 2) aptamers may have low toxicity and may lack an immunogenic response; 3) aptamers may have high specificity and affinity for their targets; 4) aptamers may have good solubility; 5) aptamers may have tunable pharmacokinetic properties; 6) aptamers may be amenable to site-specific conjugation of PEG and other carriers; and 7) aptamers may be stable at ambient temperatures.

[0030] The term “bivalent” as used herein refers to a molecule that can simultaneously bind to two separate and unique antigens (e.g., two separate amino acid sequences on C5), to two separate but identical antigens (e.g., the same amino acid sequence on two separate C5 proteins).

[0031] The term “monospecific” or “monovalent” as used herein refers to a molecule that can bind to a single antigen.

[0032] The term “multivalent” as used herein refers to a molecule that comprises more than one binding site such that said molecule can bind more than one antigen; bivalent molecules (comprising two binding sites) are a subset of multivalent molecules. Multivalent molecules, while comprising multiple binding sites, bind to epitopes of a single protein (e.g., a multivalent molecule might bind to at least two different C5 molecules simultaneously).

[0033] The term “epitope” as used herein refers to the part of an antigen that is specifically recognized by an antibody or an aptamer. In some cases, the antigen is a protein or peptide andWSGR Docket No. 66711-706.601the epitope is a specific region of the protein or peptide that is recognized and bound by an antibody or an aptamer. In some cases, the binding molecules described herein (e.g., bivalent binding molecules) may bind to a region of a target protein that is an epitope for an antibody or antibody fragment thereof, or an aptamer, wherein the antibody or aptamer inhibits a function associated with the target protein. In some cases, the binding molecule (e.g., bivalent binding molecules) binding region of the target protein overlaps with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the epitope for an antibody or an aptamer or the binding site of another molecule that inhibits the target protein.

[0034] The term “half-life extending moiety” refers to a molecular entity that does not inhibit the biological function of a retinal protein and when linked to or conjugated to a therapeutic, results in a longer half-life of elimination compared to said therapeutic without said half-life extending moiety. In some cases, the half-life extending moiety may be a polymer. In some cases, the half-life extending moiety may be PEG, PLGA, or hyaluronan. In some cases, the half-life extending moiety may have a molecular weight of 5 kDa, 10 kDa, 20kDa, 30 kDa, 40 kDa, 60 kDa, or 80 kDa.The Complement System and the Alternative Complement Pathway

[0035] The complement system is a part of the immune system that enhances (complements) the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promote inflammation, and attack the pathogen's cell membrane.

[0036] The complement system consists of a number of small proteins found in the blood, and generally synthesized by the liver. Significant amounts are also produced by tissue macrophages, blood monocytes, and epithelial cells. When stimulated by one of several triggers, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end result of this complement activation or complement fixation cascade is massive amplification of the response and activation of the cell-killing membrane attack complex. Over 30 proteins and protein fragments make up the complement system, including serum proteins, serosal proteins, and cell membrane receptors.

[0037] The complement system generally includes three pathways: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). The classical complement pathway typically requires antigen-antibody complexes for activation, the alternative pathway can be activated by foreign material, pathogens, or damaged cells, and the lectin pathway can be activated by mannose or other residues on the pathogen surface antigens.

[0038] Generally, each pathway has an activation, amplification, and terminal function, with different proteins and complexes involved in each function. The CP is activated by Cl-complexes, the APby C3 hydrolysis and surface-bound C3b, and the LP is homologous to theWSGR Docket No. 66711-706.601CP, but with the opsonin, mannose-binding lectin (MBL), and ficolins, instead of Clq. In all three pathways, the amplification loop is based on C3 -containing convertases that cause a cascade of further cleavage and activation events. The terminal function includes anaphylatoxin activity (e.g., C3a, C5a), inflammation, opsonization, and the formation of the membrane attack complex (with associated target cell lysis).

[0039] The classical complement pathway is activated when Clq binds to IgM or IgG complexed with antigens. This also occurs when Clq binds directly to the surface of the pathogen. Such binding leads to conformational changes in the Clq molecule, which leads to the activation of two Clrmolecules. Clris a serine protease. They then cleave Cis (another serine protease). The Clr2s2 component now splits C4 and then C2, producing C4a, C4b, C2a, and C2b (historically, the larger fragment of C2 was called C2abut is now referred to as C2b). C4b and C2a bind to form the classical pathway C3 -convertase (C4b2a complex), which promotes cleavage of C3 into C3a and C3b. C3b later joins with C4b2ato make C5 convertase (C4b2a3b complex)

[0040] The alternative complement pathway is a rapid, antibody -independent route for complement system activation and amplification. Activation of the alternative pathway occurs when C3b, a proteolytic cleavage form of C3, is bound to an activating surface agent such as a bacterium. fB is then bound to C3b, and cleaved to yield the C3 convertase C3bBb .Amplification of C3 convertase activity occurs as additional C3b is produced and deposited . The amplification response is negatively regulated (e.g. , partially inhibited) by complement factor H (CFH) and complement factor I (CFI).

[0041] The C3 convertase further assembles into a C5 convertase (C3b3bBb). This complex subsequently cleaves complement component C5 into two components: the C5a polypeptide (9 kDa) and the C5b polypeptide (170 kDa). The C5 a polypeptide binds to a 7 transmembrane G-protein coupled receptor, which was originally associated with leukocytes and is now known to be expressed on a variety of tissues including hepatocytes and neurons. The C5a molecule is the primary chemotactic component of the human complement system and can trigger a variety of biological responses including leukocyte chemotaxis, smooth muscle contraction, activation of intracellular signal transduction pathways, neutrophil -endothelial adhesion, cytokine and lipid mediator release and oxidant formation. The C5 convertase is also critical to the formation of the Membrane Attack Complex (MAC), which leads to cell death / apoptosis. C5a and the MAC have been implicated in mitochondrial stress and dysfunction, with studies showing calcium channel dysregulation and loss of cellular energy and function due to mitochondrial stress.

[0042] The lectin pathway is homologous to the classical pathway, but with the opsonin, mannose-binding lectin (MBL), and ficolins, instead of Clq. This pathway is activated byWSGR Docket No. 66711-706.601binding of MBL to mannose residues on the pathogen surface, which activates the MBL-associated serine proteases, MASP-1, and MASP-2 (very similar to Clr and Cis, respectively), which can then split C4 into C4a and C4b and C2 into C2a and C2b . C4b and C2b then bind together to form the classical C3 -convertase, as in the classical pathway.

[0043] Altered activity of the complement system is believed to play a role in the pathogenesis and modulation of a variety of ischemic, inflammatory and autoimmune diseases including age-related macular degeneration, geographic atrophy, Stargardt disease, systemic lupus erythematosus, rheumatoid arthritis, asthma, paroxysmal nocturnal hemoglobinuria (PNH), generalized Myasthenia Gravis (gMG), and atypical hemolytic uremic syndrome (aHUS). Cellular, pre-clinical, and clinical evidence indicate the potential for proteins of the complement system to be important targets for the treatment of these diseases. In systemic rare diseases complement pathway inhibition has demonstrated transformative clinical outcomes and is the standard of care. In PNH, systemic administration of anti-C5 antibodies (e.g., eculizumab, ravalizumab, crovalizumab) significantly reduce hemolysis in patients and form part of the standard of care. Inhibition of C5, with anti-C5 antibodies, has been clinically proven to improve outcomes in patients with PNH, aHUS, and gMG and antibody -based therapies are approved for the treatment of those diseases. However, anti-C5 therapy still leaves a large number of patients with unsatisfactory clinical outcomes. For example, in a study of 123 PNH patients receiving anti-C5 therapy over a median of 4.5 years, 72% were anemic (Impact of eculizumab treatment on paroxysmal nocturnal hemoglobinuria: a treatment versus no-treatment study, Loschi et al, Am J Hematol. 2016). The clinical experience with therapies targeting C5 alone demonstrates the need for therapeutics with improved ability to inhibit complement activity.

[0044] There have been many reports and studies of associations of complement related biomarkers (e.g., proteins, genetics) with disease incidence and progression, clinical outcomes, and treatment. In some cases, there is highly significant genetic association of complement pathway mutations with disease, as in the case of CFH with risk of developing AMD. However, at least for retinal indications, there is a lack of prospective methods that enable selection of patients for differential, or superior, outcomes with anti-C5 therapeutics.

[0045] A potential challenge for studies analyzing associations between a range of complement components and clinical outcomes is statistical; in generating exploratory, post hoc, or retrospective associative analysis of proteomics / genomics with clinical outcomes, the number of possible associations between different proteins associated with the complement pathway and clinical endpoints becomes so large that correlations are likely to be found / occur with observed statistical significance irrespective of the underlying truth of the correlation simply due to the high number of potential relationships (e.g., a multifactorial regression analysis between 100WSGR Docket No. 66711-706.601independent randomly distributed variables each with multiple data points will likely return an apparently statistically significant association between at least two variables). As a result, post hoc or retrospective correlations reported in small sample size studies or Phase 2 trials may not be replicated prospectively. As an example, lampalizumab in the Phase 2 MAHALO demonstrated a -20% slowing of growth rate of GA in the primary endpoint for all enrolled patients, but a -44% slowing of rate of GA (with nominal / post hoc p=0.004) in patients with mutations in CFI, presumed to reduce expression or reduce function in CFI, a negative regulator of the alternative complement pathway amplification loop (i.e., presumed to be associated increased alternative complement pathway activity). On the basis of this Phase 2, lampalizumab was advanced into two large Phase 3 programs, that included prospective stratification by CFI mutation status. Both large Phase 3 trials failed to show a benefit on the primary endpoint of change in GA area from baseline at one year with either monthly or every 6 week IVT lampalizumab, and neither showed a benefit regardless of CFI mutation status of patients.

[0046] Molecules, such as those described herein, that provide multivalent inhibition of C5 function have the potential to improve clinical outcomes where immune response plays a pathologic role and to be a superior therapeutic approach compared to monovalent C5 inhibitors.

[0047] Furthermore, methods described herein use a framework to establish complement pathway activity relevant to C5 in order to select and guide treatment to provide patients affected with retinal diseases superior outcomes with anti-C5 therapeutics. These frameworks involve prospectively defining specific thresholds of protein levels related to amplification loop (alternative, classical, mannose-lectin binding) activity, C5 convertase activity, and specific features of retinal cell biology that inform and predict response to anti-C5 therapeutics and have the potential to provide superior clinical benefit compared to current methods of administering C5 inhibitors.Age-related macular degeneration

[0048] Age-related macular degeneration (“AMD”) is a chronic and progressive eye disease that is the leading cause of irreparable vision loss in the United States, Europe, and Japan . AMD is characterized by the progressive deterioration of the central portion of the retina referred to as the macula. The clearest indicator of progression to AMD is the appearance of drusen, yellowwhite deposits under the retina, which are plaques of material that are derived from the metabolic waste products of retinal cells. The appearance of drusen is an important component of both forms of AMD: exudative (“wet”) and non-exudative (“dry”). The presence of numerous, interm ediate-to-large drusen is associated with the greatest risk of progression to latestage disease, characterized by geographic atrophy and / or neovascularization. The majority of patients with wet AMD experience severe vision loss in the affected eye within months to twoWSGR Docket No. 66711-706.601years after diagnosis of the disease, although vision loss can occur within hours or days. Dry AMD is more gradual and occurs when light-sensitive cells in the macula slowly atrophy, gradually blurring central vision in the affected eye. Vision loss is exacerbated by the formation and accumulation of drusen and sometimes the deterioration of the retina, although without abnormal blood vessel growth and bleeding. Geographic atrophy is a term used to refer to advanced dry AMD. Geographic atrophy is characterized by an “island” of atrophied photoreceptors cells. It is believed that the alternative complement pathway may play a role in the pathogenesis of AMD.

[0049] For example, FIG. 1 depicts a potential role for the complement pathway in the pathogenesis of geographic atrophy . In this example, multiple factors may lead to activation of the complement pathway, including the appearance of drusen in the eye, immune dysfunction, and genetic differences that predispose patients to activation of the complement pathway . As described above, amplification of C3 convertase activity may occur as additional C3b is produced and deposited. C3 convertase activity may lead to inflammation and opsonization. The C3 convertase may further assemble into a C5 convertase (C3b3bBb) which may lead to cell death through formation of the Membrane Attack Complex.

[0050] In some aspects, the binding molecules of the disclosure (e.g. , the multivalent C5 binding molecules) may be used to treat AMD. In some cases, the binding molecules of the disclosure may be used to treat geographic atrophy . In some cases, the binding molecules of the disclosure may be used to treat wet AMD. In some cases, the binding molecules of the disclosure may be used to stop, slow, or reverse the progression of wet AMD or geographic atrophy . In some cases, the binding molecules of the disclosure may be used to treat symptoms associated with geographic atrophy or wet AMD.C5 Binding Molecule

[0051] In various aspects, the methods and compositions described herein comprises binding molecules, wherein the binding molecules are C5 binding molecules. In some embodiments, the use of a C5 binding molecule for modulating an activity associated with C5, and, in some cases, activity associated with another complement pathway protein are described herein.

[0052] In some embodiments, a C5 binding molecule described here can be a multivalent C5 binding molecule. In some embodiments, the multivalent C 5 binding molecule described herein may be a multivalent binding molecule comprising at least two, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight C5 binding aptamer moieties. In some embodiments, one or more C5 binding aptamer moieties of the multivalent C5 binding molecules can be the same or different. In some embodiments, at least three aptamer moieties of the C5 binding molecule can be anti-C5 aptamers. In some embodiments, at least four aptamerWSGR Docket No. 66711-706.601moieties of the C5 binding molecule can be anti-C5 aptamers. In some embodiments, at least five aptamer moieties of the C5 binding molecule can be anti-C5 aptamers. In some embodiments, at least six aptamer moieties of the C5 binding molecule can be anti-C5 aptamers. In some embodiments, at least seven aptamer moieties of the C5 binding molecule can be anti-C5 aptamers. In some embodiments, at least eight aptamer moieties of the C5 binding molecule can be anti-C5 aptamers. For example, in some embodiments, a bivalent C5 binding molecule comprises a first C5 binding aptamer moiety and a second C5 binding aptamer moiety.

[0053] In some embodiments, the multivalent C5 binding molecule provides a greater inhibition of complement function than a monovalent C5 binding molecule (e.g., an avacincaptad pegol). In some embodiments, the multivalent C5 binding molecule can inhibit the complement function by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70, at least about 80%, at least about 90%, or at least about 100% more than that of a monovalent C5 binding molecule (e.g. , an avacincaptad pegol).

[0054] Aptamers as described herein may include any number of modifications that can affect the function or affinity of the aptamer. For example, aptamers may be unmodified or they may contain modified nucleotides to improve stability, nuclease resistance, or delivery characteristics. Examples of such modifications may include, without limitation, chemical substitutions at the sugar and / or phosphate and / or base positions, for example, at the 2’ position of ribose, the 5 position of pyrimidines, and the 8 position of purines, various 2’-modified pyrimidines and modifications with 2’-amino (2’-NH2), 2’-fluoro (2’-F), and / or 2’-O-methyl (2’-0me) substituents. In some cases, aptamers described herein comprise a 2’-0me and / or a 2’F modification to increase in vivo stability. In some cases, the aptamers described herein contain modified nucleotides to improve the affinity and specificity of the aptamers for a specific epitope. Examples of modified nucleotides include those modified with guanidine, indole, amine, phenol, hydroxymethyl, or boronic acid. In other cases, pyrimidine nucleotide triphosphate analogs or CE-phosphoramidites may be modified at the 5 position to generate, for example, 5 -benzylaminocarbonyl-2 ’-deoxyuridine (BndU); 5-[N-(phenyl-3-propyl)carboxamide]-2’-deoxyuridine (PpdU); 5-(N-thiophenylmethylcarboxyamide)-2’-deoxyuridine (ThdU); 5-(N-4-fluorobenzylcarboxyamide)-2’-deoxyuridine (FbndU); 5-(N-(l-naphthylmethyl)carboxamide)-2 '-deoxyuridine (NapdU); 5 — (N-2-naphthylmethylcarboxyamide)-2’ -deoxyuridine (2NapdU); 5-(N-l-naphthylethylcarboxyamide)-2 ’-deoxyuridine (NedU); 5-(N-2-naphthylethylcarboxyamide)-2’-deoxyuridine (2NedU); 5-(N-tryptaminocarboxyamide)-2’-deoxyuridine(TrpdU); 5-isobutylaminocarbonyl-2’-deoxyuridine (IbdU); 5-(N-tyrosylcarboxyamide)-2'-deoxyuridine (TyrdU); 5-(N-isobutylaminocarbonyl-2’-deoxyuridine (iBudU); 5-(N-benzylcarboxyamide)-2’-O-methyluridine, 5-(N-WSGR Docket No. 66711-706.601benzylcarboxyamide)-2’ -fluorouridine, 5 -(N-phenethylcarboxyamide)-2’ -deoxyuridine (PedU), 5-(N-3,4-methylenedioxybenzylcarboxyamide)-2’-deoxyuridine (MbndU), 5-(N-imidizolylethylcarboxyamide)-2’-deoxyuridine (ImdU), 5-(N-isobutylcarboxyamide)-2’-O-methyluridine, 5 -(N-isobutylcarboxyamide)-2’ -fluorouridine, 5-(N — R-threoninylcarboxyamide)-2’ -deoxyuridine (ThrdU), 5-(N-tryptaminocarboxyamide)-2’-O-methyluridine, 5 -(N-tryptaminocarboxyamide)-2’ -fluorouridine, 5-(N-[l-(3-trimethylamonium)propyl]carboxyamide)-2’-deoxyuridine chloride, 5-(N-naphthylmethylcarboxyamide)-2’-O-m ethyluridine, 5-(N-naphthylmethylcarboxyamide)-2’-fluorouridine, 5-(N-[l-(2,3-dihydroxypropyl)]carboxyamide)-2’-deoxyuridine), 5-(N-2-naphthylmethylcarboxyamide)-2’-O-methyluridine, 5-(N-2-naphthylmethylcarboxyamide)-2’-fluorouridine, 5-(N-l-naphthylethylcarboxyamide)-2’-O-methyluridine, 5-(N-l-naphthylethylcarboxyamide)-2’ -fluorouridine, 5-(N-2-naphthylethylcarboxyamide)-2’-O-methyluridine, 5-(N-2-naphthylethylcarboxyamide)-2’-fluorouridine, 5-(N-3-benzofuranylethylcarboxyamide)-2’-deoxyuridine (BfdU), 5-(N-3-benzofuranylethylcarboxyamide)-2’-O-m ethyluridine, 5-(N-3-benzofuranylethylcarboxyamide)-2 ’-fluorouridine, 5-(N-3-benzothiophenylethylcarboxyamide)-2’-deoxyuridine (BtdU), 5-(N-3-benzothiophenylethylcarboxyamide)-2'-O-methyluridine, 5-(N-3-benzothiophenylethylcarboxyamide)-2'-fluorouridine; 5-[N-(l-morpholino-2-ethyl)carboxamide]-2'-deoxyuridine (MOEdu); R-tetrahydrofuranylmethyl-2’ -deoxyuridine (RTMdU); 3 -methoxyb enzyl-2’ -deoxyuridine (3MBndU); 4 -methoxyb enzyl-2’ -deoxyuridine (4MBndU); 3, 4-dimethoxybenzyl -2 ’-deoxyuridine (3,4DMBndU); S-tetrahydrofuranylmethyl-2 ’-deoxyuridine (STMdU); 3,4-methylenedioxyphenyl-2-ethyl-2’-deoxyuridine (MPEdU); 4-pyridinylmethyl-2’-deoxyuridine(PyrdU); or l-benzimidazol-2-ethyl-2’-deoxyuridine (BidU); 5-(amino-l-propenyl)-2'-deoxyuridine; 5 -(indole-3-acetamido-l-propenyl)-2 '-deoxyuridine; or 5-(4-pivaloylbenzamido-l-propenyl)-2 '-deoxyuridine.

[0055] Modifications of the aptamers contemplated in this disclosure include, without limitation, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and functionality to the nucleic acid aptamer bases or to the nucleic acid aptamer as a whole. Modifications to generate oligonucleotide populations that are resistant to nucleases can also include one or more substitute internucleotide linkages, altered sugars, altered bases, or combinations thereof. Such modifications include, but are not limited to, 2' -position sugar modifications, 5 -position pyrimidine modifications, 8-position purine modifications, substitution of 4 -thiouridine, substitution of 5-bromo or 5 -iodo-uracil; backbone modifications, phosphorothioate, phosphorodithioate, or alkyl phosphate modifications, methylations, and unusual base-pairingWSGR Docket No. 66711-706.601combinations such as the isobases isocytidine and isoguanosine. Modifications can also include 3' and 5' modifications such as capping, e.g. , addition of a 3'-3'-dT cap to increase exonuclease resistance. In some cases, an aptamer as disclosed herein has fewer than 2 non -modified nucleotides or ribonucleotides. For example, in a binding molecule described herein (e.g., a bivalent C5 binding molecule) comprising a first aptamer and a second aptamer, either one or both of the first aptamer moiety and the second aptamer moiety may have fewer than 2 nonmodified nucleotides or ribonucleotides. In some embodiments, a binding molecule described herein (e.g., a bivalent C5 binding molecule) comprising a first aptamer and a second aptamer can have either one or both of the first aptamer moiety and the second aptamer moiety having fewer than 1 non-modified nucleotides or ribonucleotides. In some embodiments, a binding molecule described herein (e.g. , a bivalent C5 binding molecule) comprising a first aptamer and a second aptamer can have either one or both of the first aptamer moiety and the second aptamer moiety having no non-modified nucleotides or ribonucleotides.

[0056] Aptamers of the disclosure may generally comprise nucleotides having ribose in the P-D-ribofuranose configuration. In some cases, 100% of the nucleotides present in the aptamer have ribose in the P-D-ribofuranose configuration. In some cases, at least 50% of the nucleotides present in the aptamer have ribose in the P-D-ribofuranose configuration. In some cases, at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the nucleotides present in the aptamer have ribose in the P-D-ribofuranose configuration.

[0057] The length of the aptamer can be variable. In some cases, the length of the aptamer is less than 100 nucleotides. In some cases, the length of the aptamer is greater than 10 nucleotides. In some cases, the length of the aptamer is between 10 and 90 nucleotides. The aptamer can be, without limitation, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, or about 90 nucleotides in length.

[0058] In some embodiments, the C5 binding molecule described herein (e.g., the multivalent C5 binding molecule) may further comprise a half-life extending moiety. In some embodiments, the C5 binding molecule described herein (e.g., the multivalent C5 binding molecule) may further comprise an intravitreal half-life extending moiety. In some instances, a polyethylene glycol (PEG) polymer chain is covalently bound to the aptamer, referred to herein as PEGylation. Without wishing to be bound by theory, PEGylation may increase the half-life and stability of the aptamer in physiological conditions. In some cases, the PEG polymer is covalently bound to the 5’ end of the aptamer. In some cases, the PEG polymer is covalently bound to the 3’ end of the aptamer. In some cases, the PEG polymer is covalently bound to aWSGR Docket No. 66711-706.601specific site on a nucleobase within the aptamer, including the 5 -position of a pyrimidine opposition of a purine. In some cases, the PEG polymer is covalently bound to an abasic site within the aptamer. In some embodiments, the PEG polymer may be a linear configuration. In some embodiments, at least one binding moiety (e.g., anti-C5 aptamer) of the binding molecule described herein can be conjugated to the PEG polymer. In some embodiments, the PEG polymer can be a branched PEG. In some embodiments, the PEG polymer can be Y-shaped. In some embodiments, at least two binding moieties (e.g, two aptamers) can be conjugated to the branched PEG polymer or the Y-shaped PEG polymer. In some embodiments, a PEG polymer may be a multi-armed PEG polymer. In some embodiments, a PEG polymer may be a multiarmed PEG polymer with multiple reactive groups, each of which is used to attach a single C5 binding aptamer moiety. In some embodiments, a plurality of binding moieties (e.g, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten binding moieties) can be conjugated to each arm of the multi-armed PEG polymer.

[0059] In some cases, an aptamer described herein may be conjugated to a PEG having the general formula, H-(O-CH2-CH2)n-OH. In some cases, an aptamer described herein may be conjugated to a methoxy -PEG (mPEG) of the general formula, CH3O-(CH2-CH2-O)n-H. In some cases, the aptamer is conjugated to a linear chain PEG or mPEG. The linear chain PEG or mPEG may have an average molecular weight of up to about 30 kD. Multiple linear chain PEGs or mPEGs can be linked to a common reactive group to form multi-arm or branched PEGs or mPEGs. For example, more than one PEG or mPEG can be linked together through an amino acid linker (e.g., lysine) or another linker, such as glycerin. In some cases, the aptamer is conjugated to a branched PEG or branched mPEG. Branched PEGs or mPEGs may be referred to by their total mass (e.g., two linked 20 kD mPEGs have a total molecular weight of 40 kD). Branched PEGs or mPEGs may have more than two arms. Multi-arm branched PEGs or mPEGs may be referred to by their total mass (e.g. four linked 10 kD mPEGs have a total molecular weight of 40 kD). In some cases, an aptamer of the present disclosure is conjugated to a PEG polymer having a total molecular weight from about 5 kD to about 200 kD, for example, about 5 kD, about 10 kD, about20 kD, about30 kD, about40 kD, about 50 kD, about 60 kD, about 70 kD, about 80 kD, about 90 kD, about 100 kD, about 110 kD, about 120kD, about 130 kD, about 140 kD, about 150 kD, about 160 kD, about 170 kD, about 180kD, about 190 kD, or about 200 kD. In one non-limiting example, the aptamer is conjugated to a PEG having a total molecular weight of about 40 kD.

[0060] In some cases, the reagent that may be used to generate PEGylated aptamers is a branched PEGN-Hydroxysuccinimide (mPEG-NHS) having the general formula:WSGR Docket No. 66711-706.601<with a 20 kD, 40 kD or 60 kD total molecular weight (e.g., where eachmPEGis about lOkD, 20 kD or about 30 kD). As described above, the branched PEGs can be linked through any appropriate reagent, such as an amino acid (e.g., lysine or glycine residues).

[0061] In one non-limiting example, the reagent used to generate PEGylated aptamers is [N2-(monomethoxy 20K polyethylene glycol carbamoyl)-N6-(monomethoxy 20K polyethylene glycol carbamoyl)]-lysine N-hydroxysuccinimide having the formula:

[0062] In yet another non-limiting example, the reagent used to generate PEGylated aptamers has the formula:

[0063] where X is N-hydroxysuccinimide and the PEG arms are of approximately equivalent molecular weight. Such PEG architecture may provide a compound with reduced viscosity compared to a similar aptamer conjugated to a two-armed or single-arm linear PEG.

[0064] In some examples, the reagent used to generate PEGylated aptamers has the formula:WSGR Docket No. 66711-706.601where X is N-hydroxysuccinimide and the PEG arms are of different molecular weights, for example, a 40 kD PEG of this architecture may be composed of 2 arms of 5 kD and 4 arms of 7.5 kD. Such PEG architecture may provide a compound with reduced viscosity compared to a similar aptamer conjugated to a two-armed PEG or a single-arm linear PEG.

[0065] In some cases, the reagent that may be used to generate PEGylated aptamers is a non-branched mPEG-Succinimidyl Propionate (mPEG-SPA), having the general formula:where mPEG is about 20 kD or about 30 kD. In one example, the reactive ester may be -O-CH2- CH2-CO2-NHS.

[0066] In some instances, the reagent that may be used to generate PEGylated aptamers may include a branched PEG linked through glycerol, such as the Sunbright™ series from NOF Corporation, Japan. Non-limiting examples of these reagents include:

[0067] In another example, the reagents may include a non-branched mPEG Succinimidyl alpha-methylbutanoate (mPEG-SMB) having the general formula:WSGR Docket No. 66711-706.601where mPEGis between 10 and 30 kD. In one example, the reactive ester may be -O-CH2.CH2. CH(CH3)-CO2-NHS.

[0068] In other instances, the PEG reagents may include nitrophenyl carb onate -linked PEGs, having the general formula:

[0069] Compounds including nitrophenyl carbonate can be conjugated to primary amine containing linkers.

[0070] In some cases, the reagents used to generate PEGylated aptamers may include PEG with thiol-reactive groups that can be used with a thiol-modified linker. One non-limiting example may include reagents having the following general structure:where mPEG is about 10 kD, about 20 kD or about 30 kD. Another non -limiting example may include reagents having the following general structure:where each mPEGis about 10 kD, about 20 kD, or about 30 kD and the total molecular weight is about 20 kD, about 40 kD, or about 60 kD, respectively. Branched PEGs with thiol reactive groups that can be used with a thiol-modified linker, as described above, may include reagents in which the branched PEG has a total molecular weight of about 40 kD or about 60 kD (e.g., where each mPEG is about 20 kD or about 30 kD).WSGR Docket No. 66711-706.601

[0071] In some cases, the reagents used to generated PEGylated aptamers may include reagents having the following structure:In some cases, the reaction is carried out between about pH 6 and about pH 10, or between about pH 7 and pH 9 or about pH 8.

[0072] In some cases, the reagents used to generate PEGylated aptamers may include reagents having the following structure:><

[0073] In some cases, the reagents used to generate PEGylated aptamers may include reagents having the following structure:

[0074] In some cases, the aptamer is associated with a single PEG molecule. In other cases, the aptamer is associated with two or more PEG molecules.

[0075] In some cases, a multivalent C5-binding molecule described herein (e.g., a bivalent C5 binding molecule) can comprise at least the at least first binding moiety and the at least a second binding moiety conjugated to one another. For example, a binding molecule can have a first aptamer conjugated to a second aptamer. In some cases, a binding molecule can comprise at least a first moiety and at least a second moiety covalently attached to one another. In a nonlimiting example, the first moiety and the second moiety maybe covalently attached by the use of a linker moiety (e.g., a PEG linker). FIG. 3A depicts an example of bimolecular assembly achieved by chemical conjugation using bioconjugation techniques. In this example, a PEGWSGR Docket No. 66711-706.601linker may be used to covalently conjugate two moieties to generate a bivalent binding molecule (e.g., FIG.3B). In such cases, the binding moieties may be coupled to each other or coupled to the PEG polymer via a linker (e.g., a PEG linker). In some embodiments, the length of the linker may be altered to increase or decrease the distance between the two moieties. In some cases, altering the length of the linker may increase the efficacy of the binding molecule (e.g., by increasing the length of the linker, the two moieties may be less constrained and more capable of simultaneous binding to two epitopes).

[0076] In some cases, to enable conjugation of a C5 binding aptamer moiety, a linker with a reactive moiety can be attached to the aptamer to provide a specific site for conjugation. Various linkers and attachment chemistries are known in the art. In a non-limiting example, 6-(trifluoroacetamido) hexanol (2-cyanoethyl-N,N-diisopropyl) phosphoramidite can be used to add a hexylamino linker to the 5' end of the synthesized aptamer. This linker, as with the other amino linkers provided herein, once the group protecting the amine has been removed, can be reacted with PEG-NHS esters to produce covalently linked PEG-aptamers. Other non-limiting examples of linker phosphoramidites may include: TFA-amino C4 CED phosphoramidite having the structure:5'-amino modifier C3 TFA having the structure:MT amino modifier C6 CED phosphoramidite having the structure:WSGR Docket No. 66711-706.6015'-amino modifier 5 having the structure:MMT : 4-Monomethoxytrityl5'-amino modifier C12 having the structure:MMT: 4-Monomethoxytrityland 5' thiol-modifier C6 having the structure:

[0077] The 5'-thiol modified linker may be used, for example, with PEG-maleimides, PEG-vinylsulfone, PEG-iodoacetamide and PEG-orthopyridyl-disulfide. In one example, the aptamer may be bonded to the 5'-thiol through a maleimide or vinyl sulfone functionality.

[0078] Bivalent C5 binding aptamer compositions consisting of a C5 binding Aptamer 1 covalently bonded to a C5 binding Aptamer 2 can be produced by solid phase oligonucleotide synthesis using standard phosphoramidite chemistry. In one example, the C5 binding aptamer 1 is synthesized on the solid support in a 3’ to 5’ direction, followed by a joining linker (L) andWSGR Docket No. 66711-706.601the C5 binding aptamer 2, also in the 3 ’to 5 ’ direction. This strategy produces a bivalent aptamer of the geometry 5 ’-(Aptamer l)-L-( Aptamer 2)-3 ’ . Alternatively, the C5 binding aptamer 2 is first synthesized on the solid support in the 3’ to 5’ direction, followed by a joining linker and the C5 binding aptamer 1, also in the 3’ to 5’ direction. This strategy produces a bivalent aptamer of the geometry 5 ’-(Aptamer l)-L-(Aptamer 2)-3 ’. Linkers of the disclosure are generally inert linkers composed of carbon atoms or hexaethylene glycol repeats of < 5,000 Da. In one non-limiting example, the linker used to join the C5 binding aptamer 1 and C5 binding aptamer 2 is 3 -(4,4 '-Dimethoxy trityloxy )propyl-l-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosporamidite of the formula:which yields a 3 -carbon linker upon incorporation into the bivalent aptamer.

[0079] In another non-limiting example, the linker used to join the C5 binding aptamer 1 andC5 binding aptamer 2 is 6-(4,4'-Dimethoxytrityloxy)hexanediol-l-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosporamidite of the formula:which yields a 6-carbon linker upon incorporation into the bivalent aptamer.

[0080] In another non-limiting example, the linker used to join the C5 binding aptamer 1 andC5 binding aptamer 2 is 8-(4,4'-Dimethoxytrityloxy)triethyleneoxide-l-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosporamidite of the formula:which yields a triethylene glycol linker upon incorporation into the bivalent aptamer.

[0081] In another non-limiting example, the linker used to join the C5 binding aptamer 1 andC5 binding aptamer 2 is 17-(4,4'-Dimethoxytrityloxy)hexaethyleneoxide-l-[(2-cyanoethyl)-(N,N-WSGR Docket No. 66711-706.601diisopropyl)]-phosporamidite of the formulawhich yields a hexaethylene glycol linker upon incorporation into the bivalent aptamer.

[0082] In some embodiments, the bivalent C5 binding molecule may comprise or consist of a C5 binding aptamer 1 covalently bonded to a C5 binding aptamer 2, which can be produced by site-specific conjugation using a bifunctional linker. In one example, a reactive moiety such a thiol is incorporated at the 5’ end of the C5 binding aptamer 1 and a different reactive moiety such as a primary amine is incorporated at the 5’ end of the C5 binding aptamer 2. Conjugation of two such aptamers using a bifunctional linker produces a bivalent aptamer of the geometry 3’-(C5 binding aptamer 2)-L-(C5 binding aptamer 1) -3’. In another example, a reactive moiety such as a thiol is incorporated at the 5’ end of the C5 binding aptamer 1 and a different reactive moiety such as a primary amine is incorporated at the 3’ end of the C5 binding aptamer 2. Conjugation of two such aptamers using a bifunctional linker produces a bivalent aptamer of the geometry 5’-(C5 binding aptamer B)-L-(C5 binding aptamer A)-3’. In yet another example, a reactive moiety such as a thiol is incorporated at the 3’ end of the C5 binding aptamer 1 and a different reactive moiety such as a primary amine is incorporated at the 3’ end of the C5 binding aptamer 2. Conjugation of two such aptamers using a bifunctional linker produces a bivalent aptamer of the geometry 5’-(C5 binding aptamer 2)-L-(C5 binding aptamer 1 )-5 ’ . Such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may be utilized as needed.

[0083] In some instances, the bivalent C5 binding molecule may comprise a C5 binding aptamer 1 that can be produced with a C6-disulfide linker, and a C5 binding aptamer 2 that can be produced with a C6-amino linker and conjugated as follows. The C5 binding aptamer 2 with the C6-amino linker may be conjugated to a linker consisting of the general formula NHS (N-hydroxysuccinimide)-[PEG]n-maleimide, leaving groups and unreacted linker may be removed by dialysis or a similar method, and then the disulfide of the linker on the vitreous binding aptamer may be reduced, and reacted with the (C5 binding aptamer 2)-[PEG]n-MAL to produce the (C5 binding aptamer 2)-[PEG]n-( C5 binding aptamer 1) bivalent aptamer, which may be subsequently purified by chromatography and desalting. In some cases, this reaction is carriedWSGR Docket No. 66711-706.601out between about pH 6 and about pH 10, or between pH 7 and 9 or about pH 8 to promote reaction of the NHS ester with the primary amine on the aptamer while maintaining the integrity of the maleimide. Alternatively, the C6-amino linker can be incorporated into the C5 binding aptamer 1 and the C6-disulfide linker into the C5 binding aptamer 2 and the conjugation performed in a similar manner.

[0084] In one non-limiting example, the linker used to conjugate the C5 binding aptamer 1 and C5 binding aptamer 2 is maleimide-PEG2-succinimidyl ester of the formula:which yields a diethylene glycol linker upon formation of the bivalent aptamer.

[0085] In another non-limiting example, the linker used to conjugate the C5 binding aptamer 1 and C5 binding aptamer 2 is maleimide-PEG4-succinimidyl ester of the formula:which yields a tetraethylene glycol linker upon formation of the bivalent aptamer.

[0086] In another non-limiting example, the linker used to conjugate the C5 binding aptamer 1 and C5 binding aptamer 2 is maleimide-PEGs-succinimidyl ester of the formula:which yields an octaethylene glycol linker upon formation of the bivalent aptamer.

[0087] In another non-limiting example, the linker used to conjugate the C5 binding aptamer 1 and C5 binding aptamer 2 is maleimide-PEGn-succinimidyl ester of the formula:which yields a dodecaethylene glycol linker upon formation of the bivalent aptamer.

[0088] In another non-limiting example, the linker used to conjugate the C5 binding aptamer 1 and C5 binding aptamer 2 is maleimide-PEG24-succinimidyl ester of the formula:WSGR Docket No. 66711-706.601which yields a tetracosaethylene glycol linker upon formation of the bivalent aptamer. Such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may be utilized as needed.

[0089] Provided herein is an aptamer comprising a nucleotide sequence that is at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a nucleotide sequence of SEQ ID NOs: 2-9.

[0090] Further provided herein are a multivalent C5 binding molecule comprising a plurality of aptamer moieties comprising at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a nucleotide sequence of SEQ ID NOs: 2-9.

[0091] In some cases, the multivalent C5 binding molecules described herein can comprise at least two aptamer moieties that bind to and inhibit a function associated with C5. Non-limiting examples of anti-C5 aptamers that may be used in the binding molecules of the disclosure have been described, for example, in PCT Publication Nos. W02006088888A2, W02007103549A2, WO2022094364A1, and W02021091718A1, the disclosures of which are herein incorporated by reference in their entireties. In a particular example, at least one anti-C5 aptamer can be an avacincaptad (e.g., SEQ ID NO: 1), as described in Table 1.

[0092] In some embodiments, at least one of the anti-C5 aptamers can be further modified (e.g., as compared to SEQ ID NO: 1 ). In some embodiments, at least one anti-C5 aptamer can have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least nine, or at least ten modified nucleotides when compared to SEQ ID NO: 1. In some embodiments, the one or more modifications may include, without limitation, chemical substitutions at the sugar and / or phosphate and / or base positions, for example, at the 2’ position of ribose, the 5 position of pyrimidines, and the 8 position of purines, various 2'-modified pyrimidines and modifications with 2' -amino (2'-NH2), 2'-fluoro (2'-F), and / or 2'-O-methyl (2'-OMe) substituents. In some embodiments, at least one anti-C5 aptamer described herein can have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, atleastten modified nucleotides with 2’-amino (2'-NH2) substituents when compared to a control anti-C5 aptamer (e.g., a control anti-C5 aptamer comprising SEQ ID NO:1). In some embodiments, at least one anti-C5 aptamer described herein can have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten modified nucleotides with 2 ’-fluoro (2’-F) substituents when compared to SEQ IDNO:1. In some embodiments, at least one anti-C5 aptamer described herein can haveWSGR Docket No. 66711-706.601at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten modified nucleotides with 2'-O-methyl (2'-0Me) substituents when compared to SEQ ID NO:1 . In some embodiments, at least one anti-C5 aptamer moiety described herein can have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten modified nucleotides with a combination of nucleotide modification comprising 2' -O-methyl (2'-0Me), 2 ’-fluoro (2’-F), or 2'-amino (2'-NH2) substituents when compared SEQ ID NO:1.

[0093] In some embodiments, the multivalent C5 binding molecules can comprises a first aptamer moiety that selectively binds to a complement component C5, and a second, a third, a fourth, a fifth, a sixth, a seventh, and / or a eighth aptamer moiety that selectively binds to the complement component C5, wherein each aptamer moiety of the multivalent C5 binding molecule may comprise or consist of a nucleotide sequence of SEQ ID NOs: 1-9. In some embodiments, each aptamer moiety of the multivalent C5 binding molecule may comprise or consist of a nucleotide sequence of SEQ ID NOs: 2-9.

[0094] In some embodiments, each aptamer moiety of the multivalent C5 binding molecule can comprise or consist of a nucleotide sequence that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a nucleotide sequence of SEQ ID NOs: 1-9.

[0095] In some embodiments, at least one anti-C5 aptamer described herein can have an aptamer that contains no unmodified RNA or DNA nucleotides. In some embodiments, at least one or more nucleotides can be modified to improve stability, nuclease resistance, or delivery characteristics. In some embodiments, C5 binding aptamer moieties incorporated into the multivalent C5 binding molecule may include complementary pairing 2 ’-OMe or 2’-F modified nucleotide sequences of 1, 2, 3, or 4 nucleotides in length at each of the 5’ and 3’ ends of SEQ ID: 1-9 (e.g., FIG. 2A). FIG. 2A (left) depicts avacincaptad (SEQ ID NO: 1). FIG. 2A (right) depicts modified avacincaptad (SEQ ID NO: 2), differentially modified nucleotides between SEQ ID NO: 1 and 2 are indicated by the arrows and are bolded in Table 1 below. In some embodiments, C5 binding aptamer moieties incorporated into the multivalent C5 binding molecule may also include “spacing” moieties of between 1 kDa to 5 kDa on either or both of the 5’ or 3’ ends of the complementary pairing nucleotide sequences of 1, 2, 3, or 4 nucleotides in length at each of the 5’ and 3’ ends of SEQ ID: 1 -9. In some embodiments, the “spacing” moieties are stem A5’ & stem A3’ (FIG. 2B) which comprise complementary modified (2’-WSGR Docket No. 66711-706.601fluoro, 2’-O-methyl) nucleotide sequences of 1 to 4 nucleotides each (e.g., A5’ = 5 ’AU A3’ and A3’ = 5’UAU3’).

[0096] In some embodiments, the multivalent C5 binding molecule described herein can have at least one linker. In some embodiments, a linker can be a plurality of Sp 18 spacer (e.g., spacer phosphoramidite 18; Glen 10-1918; FIG.2C) having a total molecular weight of about 1 kDA, 2 kDA, 3 kDA, 4 kDA, 5 kDA, 6 kDA, 7 kDA, 8 kDA, 9 kDA, or 10 kDA. In some embodiments, Spl8 is between 1 to 14 units of Spl8 covalently linked or a linear PEG of similar molecular weight. (FIG. 2C; x represents a number between 1 to 14).

[0097] In some embodiments, a monovalent C5 binding aptamer can be covalently linked to a half-life extending moiety (e.g., a PEG polymer) comprising one or more reactive sites (R’) that can form a covalentbond with the reactive site (R) on the monovalent C5 binding aptamer (FIG.3A). In some embodiments, R’ & R are reactive pairs that form a covalent bond under appropriate conditions, e.g., primary amine +NHS ester. In some embodiments, a half-life extending moiety can covalently link a plurality of monovalent C5 binding aptamer moieties to form a multivalent C5 binding aptamer molecule, wherein the half-life extending moiety contains 2 or more reactive sites (R’).

[0098] FIG.3B depicts non limiting examples of bivalent, trivalent, and tetravalent C5 binding molecules. FIG. 3B (top) depicts a bivalent C5 binding molecule comprising a half-life extending moiety with 2 reactive sites (linking sites 1 and linking site 2) covalently linked through optional linkers to 2 aptamer moieties (aptamer moiety 1 and aptamer moiety 2). FIG.3B (middle) depicts a trivalent C5 binding molecule comprising a half-life extending moiety with 3 reactive sites (linking sites 1, linking site 2, and linking site 3) covalently linked through optional linkers to 3 aptamer moieties (aptamer moiety 1 , aptamer moiety 2, and aptamer moiety 3). FIG.3B (bottom) depicts a tetravalent C5 binding molecule comprising a half -life extending moiety with 4 reactive sites (linking sites 1, linking site 2, linking site 3, and linking site 4) covalently linked through optional linkers to 4 aptamer moieties (aptamer moiety 1, aptamer moiety 2, aptamer moiety 3, and aptamer moiety 4).

[0099] In some embodiments, the multivalent C5 binding compound comprises a bivalent C5 binding aptamer moiety. In some embodiments the bivalent C5 binding aptamer moiety comprises a linker moiety (e.g., a PEG linker) which may further comprise a reactive site (R) for conjugation to other moieties (e.g., half-life extending moiety; FIG.3C). In some embodiments, the linker moiety is a molecule covalently linked to the 5’ end of both C5 binding aptamers (SEQ ID: 1-9), or the 5’ end of Spl8x of both C5 binding aptamer moiety A and C5 binding aptamer moiety B (FIG. 3C).WSGR Docket No. 66711-706.601

[0100] In some embodiments, a bivalent C5 binding aptamer moiety can be covalently linked to a half-life extending moiety (e.g. a PEG polymer) comprising a reactive site (R’) that can form a covalent bond with the reactive site (R) on the linker moiety (FIG. 3D). The half-life extending moiety may comprise a one or more reactive sites (R’n), where n is greater than or equal to 1 (FIG 3D). In some embodiments, R’ & R are reactive pairs that form a covalent bond under appropriate conditions, e.g., primary amine +NHS ester. In some embodiments the halflife extending molecule can covalently link one or more C5 monovalent binding aptamers and / or one or more C5 binding bivalent aptamers. In some embodiments the half-life extending molecule can covalently link a plurality of bivalent C5 binding aptamers.

[0101] FIG.3E depicts non limiting examples of bivalent, trivalent, and tetravalent C5 binding molecules resulting from covalently linking a half-life extending molecule with one or more C5 monovalent binding aptamers and / or one or more C5 binding bivalent aptamers. FIG. 3E (top) depicts a bivalent C5 binding molecule comprising a half -life extending moiety with 1 reactive site (reaction site) covalently linked to a bivalent C5 binding aptamer moiety which further comprises a linker moiety, aptamer moiety 1, and aptamer moiety 2. FIG.3E (middle) depicts a trivalent C5 binding molecule comprising a half-life extending moiety with 2 reactive sites (linking site 1 and linking site 2) covalently linked to a bivalent C5 binding aptamer moiety, which further comprises a linker moiety, 2 optional linkers, aptamer moiety 1, and aptamer moiety 2, and covalently linked through an optional linker to a monovalent C5 binding aptamer moiety, which further comprises aptamer moiety 3, respectively. FIG. 3E (bottom) depicts a tetravalent C5 binding molecule comprising a half life extending moiety comprising 2 reactive sites (linking site 1 and linking site 2) covalently linked to 2 bivalent C5 binding aptamer moieties. Each bivalent C5 binding aptamer moiety comprises a linker moiety, 2 optional linkers, and 2 aptamer moieties (aptamer moieties 1 and 2 or aptamer moieties 3 and 4, respectively).

[0102] FIG. 3F illustrates an exemplary multivalent C5 binding compound with 2 aptamer moieties. The aptamers are covalently bound at the 5’ end and comprises a nucleotide sequence of any one of SEQ ID NOs: 1-9. The aptamers comprise the following structure:(Sp 18)(Sp 18)(Sp 18)(Sp 18)(Sp 18)(Sp 18)(Sp 18)(Sp 18 )(Sp 18)(Sp 18)(Sp 18)(Sp 18)(Sp 18)(Sp 18)(f CmGfC fCfGfC mGmGfU fCfUfC m AmGmGfCfGfC fUmGmA mGfUfC fUmGmA mGfUfU fUfAfC fCfUmG fCmG(Inv-dT), wherein Spl8 is Spacer Phosphoramidite 18, A is adenine, C is cytosine, Gis guanine, U is uracil, r is unmodified RNA, f(X)is 2’ fluoro A / C / G / U and m(X) is 2’ methyl A / C / G / U.

[0103] FIG.4 A illustrates an exemplary synthesis process for making a multivalent C5 binding compound. The aptamers comprise the following structure:WSGR Docket No. 66711-706.601(Sp 18 )(Sp 18)(Sp 18)(Sp 18)(Sp 18)(Sp 18)(Sp 18 )(Sp 18 )(Sp 18)(Sp 18)(Sp 18)(Sp 18)(Sp 18)(Sp 18)(f CmGfC fCfGfC mGmGfU fCfUfC m AmGmGfCfGfC fUmGmA mGfUfC fUmGmA mGfUfU fUfAfC fCfUmG fCmG(Inv-dT), wherein Spl8 is Spacer Phosphoramidite 18, A is adenine, C is cytosine, G is guanine, U is uracil, ris unmodified RNA, f(X)is2’ fluoro A / C / G / U and m(X) is 2’ methyl A / C / G / U. SPOS represents solid phase oligo synthesis.

[0104] In some embodiments, the NHS ester is located on PEG (R) and the primary amine is on the aptamer (P; FIG. 4B), wherein R comprises 5 kDa PEG, 10 kDa PEG, 20 kDa PEG, or 40 kDa PEG (e.g., NHS Ester on PEG is SUNBRIGHT PTE2-400GS3) and P comprises any one of SEQ IDs: 1 -9 with primary amine at the 5 ’ end. The coupling reaction results in the formation of an amide and release of an NHS molecule. Purification results in enrichment for multivalent C5 binding compounds. In some embodiments the NHS ester is located on the Aptamer (R) and the primary amine is located on PEG (P; FIG. 4C), wherein R comprises any one of SEQ IDs: 1-9 with a NHS ester at the 5 ’ end and P comprises 5 kDa PEG, 10 kDa PEG, 20 kDa PEG, or 40 kDa PEG (e.g, NHS Ester on PEG is SUNBRIGHT PTE2-400EA). The coupling reaction results in the formation of an amide bond and release of an NHS. Purification results in enrichment for multivalent C5 binding compounds.

[0105] In some embodiments, the multivalent C5 binding molecule comprising anti-C5 aptamers described herein (e.g., SEQ ID NOs: 2-9) has a longer intravitreal half-life in a mammal compared to a control monovalent C5 binding molecule. In some embodiments, a control molecule can be a binding molecule lacking the anti-C5 aptamer variant (e.g., SEQ ID NOs: 2-9), a binding molecule comprising an unmodified anti-C5 aptamer, a binding molecule comprising SEQ ID NO: 1 with or without a half-life extending moiety (e.g., avacincaptad pegol), or a binding molecule comprising an anti-C5 aptamer that have at least one unmodified RNA or DNA nucleotide. In some aspects, the multivalent C5 binding molecules comprising at least one anti-C5 aptamer variant (e.g., SEQ ID NO: 2-9) disclosed herein may have an improved intravitreal half-life in a mammal as compared to an antibody or a control molecule described herein. In some cases, the multivalent C5 binding molecules comprising the anti-C5 aptamer variants described herein have an improved half-life in a biological fluid or solution as compared to an antibody or a control molecule described herein. In some cases, the multivalent C5 binding molecules have an improved half-life in vivo as compared to an antibody or a control molecule described herein. In one example, the multivalent C5 binding molecules comprising the anti-C5 aptamers described herein has an improved half-life when injected into the eye (intraocular half-life) as compared to an antibody or a control molecule. In some cases, the multivalent C5 binding molecules may have an improved intraocular half-life when injected intoWSGR Docket No. 66711-706.601the eye of a human. In some cases, the multivalent C5 binding molecules may demonstrate improved stability over antibodies or control molecules under physiological conditions.

[0106] In some cases, the multivalent C5 binding molecules comprising the anti-C5 aptamers described herein (e.g., SEQ ID NO: 2-9) have an intraocular half-life of at least 7 days in a human. In some cases, the multivalent C5 binding molecules described herein have an intraocular half-life of at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 20 days or greater in a human. In some cases, the multivalent C5 binding molecules described herein have an intraocular or intravitreal half-life of at least 1 day in a non-human animal (e.g., rodent / rabbit / monkey). In some cases, the multivalent C5 binding molecules described herein have an intraocular or intravitreal half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days or greater in a non-human animal such as a rodent, rabbit or monkey.

[0107] In some embodiments, the multivalent C5 binding molecules exhibits hydrodynamic radius (e.g. , as measured by dynamic light scattering) that is at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, or at least about 15 nm. In some embodiments, the multivalent C5 binding molecules exhibits hydrodynamic radius (e.g., as measured by dynamic light scattering) that is from about 3 nm to about 15 nm. In some embodiments, the multivalent C5 binding molecules exhibits hydrodynamic radius (e.g., as measured by dynamic light scattering) that is from about 6 nm to about 10 nm.

[0108] In some cases, the multivalent C5 binding molecule may comprise two aptamers, each of which bind to and inhibit C5 (e.g., the first binding moiety comprising SEQ ID NO:1 and the second binding moiety comprising SEQ ID NO: 1, the first binding moiety comprising SEQ ID NO: 1 and the second binding moiety comprising any one of SEQ ID NOs: 2-9, or the first binding moiety comprising any one of SEQ ID NOs: 2-9 and the second binding moiety comprising any one of SEQ ID NOs: 2-9)Table 1. Anti-C5 Aptamer SequencesWSGR Docket No. 66711-706.601

[0109] Exemplary aptamer moiety of the multivalent C5 binding molecules is described in Table 2. In some embodiments, a multivalent C5 binding molecule can be a bivalent binding molecule. In some embodiments, a bivalent-C5 molecule described herein can comprise a first anti-C5 aptamer variant (e.g., SEQ ID NOs: 1-9) and a second anti-C5 aptamer variant (e.g., SEQ ID Nos: 1-9). In some embodiments, the multivalent C5 binding molecule described herein can comprise at least one PEG polymer. For example, in some embodiments, the at least one PEG polymer can be 5 kDa, 10 kDa, 20 kDa, 30 kDa, or 40 kDA PEG. In some embodiments, the at least one PEG polymer can be a linear PEG. In some embodiments, the at least one PEG polymer can have at least two arms, at least three arms, at least four arms, at least five arms, at least six arms, at least seven arms, at least eight arms, at least nine arms, wherein each arm comprises at least one domain for conjugation. In some embodiments, the multivalent C5 binding molecule described herein (e.g., abivalent-C5 molecule) can have at least one linker. In some embodiments, a linker can be a plurality of Sp 18 spacer (e.g., spacer phosphoramidite 18; Glen 10-1918) having a molecular weight of about 1 kDA, 2 kDA, 3 kDA, 4 kDA, 5 kDA, 6 kDA, 7 kDA, 8 kDA, 9 kDA, or 10 kDA. In some embodiments, a linker can be at least about 1 kDA, at least about 2 kDA, at least about 3 kDA, at least about 4 kDA, at least about 5 kDA, at least about 6 kDA, at least about 7 kDA, at least about 8 kDA, at least about 9 kDA, or at least about 10 kDA. In some embodiments, a linker can be a single PEG of equivalent or similarWSGR Docket No. 66711-706.601molecular weight. In some embodiments, a linker can be a molecule with at least two reactive sites that serve to link a half-life extending moiety (e.g., 40 kDa PEG) with an aptamer moiety. In some embodiments, the multivalent C5 binding molecule may comprise molecules with the general structure or formulas illustrated in, for example, FIGs 3A-3F.Table 2. Exemplary Bivalent C5 binding molecules

[0110] In some embodiments, the multivalent C5 binding molecule can be a bivalent C5 binding molecule. In some embodiments, the bivalent C5 binding molecule can comprise or consist of a nucleotide sequence that is at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a nucleotide sequence of SEQ ID NOs: 10-12.

[0111] The multivalent C5 binding molecules disclosed herein (e.g., bivalent C5 binding molecules) may comprise one or more moieties that can bind to specific regions of C5 with high specificity and affinity.

[0112] The dissociation constant (Kd) can be used to describe the affinity of a molecule for a target (or to describe how tightly the molecule binds to the target) or to describe the affinity of a molecule for a specific epitope of a target (e.g., exosite, catalytic cleft, etc.). The dissociation constant is defined as the molar concentration at which half of the binding sites of a target are occupied by the binding moiety. Thus, the smaller the Kd, the tighter the binding of the moleculeWSGR Docket No. 66711-706.601to its target. In various aspects, the multivalent C5 binding molecule are capable of simultaneously binding to at least two epitopes. For example, the multivalent C5 binding molecule may be capable of simultaneously binding to an epitope of C5 and at least one epitope of another C5. In some embodiments, multivalent C 5 binding molecule can simultaneously binds to multiple C5 molecules. In some cases, the multivalent C5 binding molecule described herein binds to each of the two epitopes with a dissociation constant (Kd) of less than 1 mM, less than 100 pM, less than 10 pM, less than 1 pM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 500 pM, or less than 100 pM. In some cases, a multivalent C5 binding molecule described herein simultaneously binds to each of the two epitopes with a dissociation constant (Kd) for each of the two epitopes of less than 1 mM, less than 100 pM, less than 10 pM, less than 1 pM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 500 pM, or less than 100 pM. In some cases, the multivalent C5 binding molecule described herein binds to each of the two epitopes with a dissociation constant (Kd) of less than 25 nM. In some cases, the multivalent C5 binding molecule described herein binds to each of the two epitopes with a dissociation constant (Kd) of less than 10 nM. In some cases, the multivalent C5binding molecule described herein binds to each of the two epitopes with a dissociation constant (Kd) of less than 5 nM. In some cases, the multivalent C5binding molecule described herein binds to each of the two epitopes with a dissociation constant (Kd) of less than 2.5 nM. In some cases, the multivalent C5 binding molecule described herein binds to each of the two epitopes with a dissociation constant (Kd) of less than 500 pM. In some cases, the multivalent C5 binding molecule described herein binds to each of the two epitopes with a dissociation constant (Kd) of less than 100 pM. In some cases, the multivalent C5 binding molecule described herein binds to each of the two epitopes with a dissociation constant (Kd) of less than 5 pM. In some cases, the Kdis determined by a flow cytometry assay as described herein.

[0113] The multivalent C5 binding molecules disclosed herein (e.g., bivalent C5 binding molecules) may bind to each epitope of a plurality of epitopes (e.g., two epitopes) with a Kdof less than 50 nM and may have an IC50of less than 50 nM as measured by a complement dependent assay. The multivalent C5 binding molecules disclosed herein may bind to each of the two epitopes with a Kdof less than 50 nM and may have an IC50 of less than 10 nM as measured by a complement dependent assay. The multivalent C5 binding molecules disclosed herein may bind to each epitope with a Kdof less than 50 nM and may have an IC50 of less than 5 nM as measured by a complement dependent assay. The multivalent C5 binding molecules disclosed herein may bind to each epitope with a Kdof less than 10 nMand may have an IC50of less than 50 nM as measured by a complement dependent assay . The multivalent C5 binding moleculesWSGR Docket No. 66711-706.601disclosed herein may bind to each epitope with a Kdof less than 10 nM and may have an IC50of less than 10 nM as measured by a complement dependent assay. The multivalent C5 binding molecules disclosed herein may bind to each epitope with a Kdof less than 10 nM and may have an IC50of less than 5 nM as measured by a complement dependent assay . The multivalent C5 binding molecules disclosed herein may bind to each epitope with a Kdof less than 5 nM and may have an IC50of less than 50 nM as measured by a complement dependent assay. The multivalent C5 binding molecules disclosed herein may bind to each epitope with a Kdof less than 5 nM and may have an IC50of less than 10 nM as measured by a complement dependent assay. The multivalent C5 binding molecules disclosed herein may bind to each epitope with a Kdof less than 5 nM and may have an IC50of less than 5 nM as measured by a complement dependent assay. The multivalent C5 binding molecules disclosed herein may bind to each epitope with a Kdof less than 1 nM and may have an IC50 of less than 50 nM as measured by a complement dependent assay. The multivalent C5 binding molecules disclosed herein may bind to each epitope with a Kdof less than 1 nM and may have an IC50of less than 10 nM as measured by a complement dependent assay. The multivalent C5 binding molecules disclosed herein may bind to each epitope with a Kdof less than 1 nM and may have an IC50of less than 5 nM as measured by a complement dependent assay. The multivalent C5 binding molecules disclosed herein may bind to epitope with a Kdof 500 pM or greater and may have an IC50of less than 50 nM as measured by a complement dependent assay. The multivalent C5 binding molecules disclosed herein may bind to each epitope with a Kdof 500 pM or greater and may have an IC50of less than 10 nM as measured by a complement dependent assay . The multivalent C5 binding molecules disclosed herein may bind to each epitope with a Kdof 500 pM or greater and may have an IC50of less than 5 nM as measured by a complement dependent assay .

[0114] In some aspects, the multivalent C5 binding molecules disclosed herein may have an increased avidity as compared to monospecific molecules. For example, a bivalent aptamer comprising a first moiety that binds to C5 and a second moiety that binds to C5 demonstrates an increased avidity for both C5 proteins as compared to a monospecific anti-C5 aptamer. In some cases, the linker length and / or geometry may increase or decrease the avidity of a binding molecule of the disclosure for a target epitope. For example, a longer linker may enable greater flexibility and may allow for simultaneous binding of the binding molecule to a plurality of epitopes (e.g., two binding epitopes, three binding epitopes), whereas a shorter linker may prevent simultaneous binding of the binding molecule to the plurality of epitopes.

[0115] In some aspects, the multivalent C5 binding molecules disclosed herein may have an improved half-life as compared to other therapeutics, such antibodies. In some cases, the multivalent C5 binding molecules have an improved half-life in a biological fluid or solution asWSGR Docket No. 66711-706.601compared to an antibody. In some cases, the multivalent C5 binding molecules have an improved half-life in vivo as compared to an antibody. In one example, the multivalent C5 binding molecules have an improved half-life when injected into the eye (intraocular half-life) as compared to an antibody. In some cases, the multivalent C5 binding molecules may have an improved intraocular half-life when injected into the eye of a human. In some cases, the multivalent C5 binding molecules may demonstrate improved intraocular half-life over intravitreally administered antibodies or fusion proteins (e.g., faricimab or aflibercept) that are FDA approved for treatment of retinal diseases with dosing schedules of every 16 weeks in at least some patients.

[0116] In some cases, the multivalent C5 binding molecules described herein have an in vivo or in vitro intraocular half-life of at least 7 days in a human. In some cases, the multivalent C5 binding molecules described herein have an intraocular half-life of at least 8 days, at least 9 days, atleast 10 days, atleast 11 days, atleast 12 days, atleast 13 days, at least 14 days, at least 15 days, at least 20 days or greater in a human.

[0117] In some cases, the multivalent C5 binding molecules described herein have an intraocular or intravitreal half-life of at least 1 day in a non-human animal (e.g., rodent / rabbit / monkey). In some cases, the multivalent C5 binding molecules described herein have an intraocular or intravitreal half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days or greater in a non-human animal such as a rodent, rabbit or monkey. In some cases, the multivalent C5 binding molecules described herein exhibit at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95 % or greater complement pathway inhibition for at least about 30 days, at least about 45 days, at least about 60 days, at least about 90 days, at least about 100 days, at least about 110 days, or at least about 120 days after a single intravitreal injection in a rabbit, a mini-pig, a non-human primate, or a human.

[0118] For example, in some embodiments, the C5 binding molecules can be a bivalent C5 binding molecule, and can exhibit at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95% or greater complement pathway inhibition for at least about 30 days, at least about 45 days, at least about 60 days, atleast about 90 days, atleast about 100 days, at least about 110 days, or at least about 120 days after a single intravitreal injection in a rabbit, a mini-pig, a non-human primate, or a human.

[0119] The activity of a therapeutic agent can be characterized by the half maximal inhibitory concentration (IC50). The IC50 is calculated as the concentration of therapeutic agent in nM at which half of the maximum inhibitory effect of the therapeutic agent is achieved. The IC50is dependent upon the assay utilized to calculate the value. In some examples, the IC50of a bindingWSGR Docket No. 66711-706.601molecule described herein is less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM or less than 0.01 nM as measured by an alternative complement dependent hemolysis assay (Pangbum, 1988, Methods in Enzymology, and Katschke, 2009, Journal of Biological Chemistry). In various aspects, a multivalent C5 binding molecule described herein may exhibit an IC50 that is lower than the IC50 of a monospecific molecule. For example, a bivalent C5-C5 aptamer as described herein may exhibit an IC50in an complement dependent hemolysis assay that is lower than the IC50of an anti-C5 monospecific aptamer or an anti-C5 monospecific aptamer containing molecule (e.g, avacincaptad pegol). In various aspects, a multivalent C5 binding molecule described herein requires an excess of binding molecules to target protein that is less than required for a monospecific molecule. For example, a bivalent C5-C5 aptamer may require an excess of aptamer to target protein that is less than required for either a monospecific anti-C5 aptamer or a monospecific anti-C5 aptamer containing molecule (e.g., avacincaptad pegol).

[0120] Aptamers generally have high stability at ambient temperatures for extended periods of time. In some cases, the multivalent C5 binding molecule described herein demonstrate at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, atleast about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% activity in solution under physiological conditions at 30 days or later.

[0121] In some embodiments, the multivalent C5 binding molecule can remain at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% intact after exposure to serum or vitreous. In some embodiments, the multivalent C5 binding molecule can remain at least about 70%, at least about 75%, atleast about 80%, atleast about 85%, at least about 90%, at least about 95%, or at least about 98% intact after exposure to serum or vitreous for at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 60 hours, or at least about 70 hours in vitro.

[0122] In some cases, a composition of the disclosure comprises the multivalent C5 binding molecules, wherein essentially 100% of the multivalent C 5 binding molecules comprise nucleotides having ribose in the P-D-ribofuranose configuration. In other examples, a composition of the disclosure may comprise bivalent aptamer-containing molecules, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90% of the bivalent aptamer-containing molecules have ribose in the P-D-ribofuranose configuration.

[0123] In some embodiments, multivalent C5 binding molecules described herein can have at least three C5 binding aptamer moieties, each aptamer moiety being conjugated to the same PEGWSGR Docket No. 66711-706.601polymer. In some embodiments, the binding molecule can have at least three binding moieties covalently conjugated to a PEG.

[0124] In some embodiments, multivalent C 5 binding molecules described herein can have at least four binding moieties, wherein at least one binding moiety of the binding molecules comprises an aptamer coupled to a PEG polymer. In some embodiments, the binding molecule can have at least four binding moieties covalently conjugated to a PEG.Indications

[0125] In some aspects, the methods and compositions provided herein are used for the treatment of retinal diseases or disorders. In some cases, the retinal disease or disorder is macular degeneration. In some cases, macular degeneration is age-related macular degeneration. In some cases, the methods and compositions can be utilized to treat non -exudative (“dry”) age-related macular degeneration. In some cases, advanced forms of dry age-related macular degeneration can be treated, including geographic atrophy. In other cases, the methods and compositions can be utilized to treat neovascular or exudative (“wet”) age-related macular degeneration. In other cases, the methods and compositions can be utilized to treat both or cooccurring geographic atrophy and neovascular age-related macular degeneration. In some cases, the methods and compositions herein can be utilized to prevent age-related macular degeneration and associated diseases thereof. In other cases, the methods and compositions herein can be utilized to slow or halt the progression of age-related macular degeneration and associated diseases thereof.

[0126] In some aspects, the methods and compositions provided herein are suitable for the treatment of Stargardt disease, for example, STGD-1. In some cases, the methods and compositions herein can be utilized to prevent age-related Stargardt disease. In other cases, the methods and compositions herein can be utilized to slow or halt the progression of Stargardt disease.

[0127] In some aspects, the methods and compositions provided herein are suitable for the treatment of diseases causing ocular symptoms. Examples of symptoms which may be amenable to treatment with the methods disclosed herein include: increased drusen volume, reduced reading speed, reduced color vision, retinal thickening, increase in central retinal volume and / or, macular sensitivity, loss of retinal cells, increase in area of retinal atrophy, reduced best corrected visual acuity such as measured by Snellen or ETDRS scales, Best Corrected Visual Acuity under low luminance conditions, impaired night vision, impaired light sensitivity, impaired dark adaptation, contrast sensitivity, and patient reported outcomes.

[0128] In some cases, the methods and compositions provided herein may alleviate or reduce a symptom of a disease. In some cases, treatment with a binding molecule provided herein mayWSGR Docket No. 66711-706.601result in a reduction in the severity of any of the symptoms described herein. In some cases, treatment with multivalent C5 binding molecules described herein may slow, halt or reverse the progression of any of the symptoms described herein. In some cases, treatment with a multivalent C5 binding molecule described herein may prevent the development of any of the symptoms described herein. In some cases, treatment with a multivalent C5 binding molecule described herein may slow, halt or reverse the progression of a disease, as measured by the number and severity of symptoms experienced. Examples of symptoms and relevant endpoints where the binding molecule may have a therapeutic effect include increased drusen volume, reduced reading speed, reduced color vision, retinal thickening, increase in central retinal volume and / or, macular sensitivity, loss of retinal cells, increase in area of retinal atrophy, reduced best corrected visual acuity such as measured by Snellen or ETDRS scales, Best Corrected Visual Acuity under low luminance conditions, impaired night vision, impaired light sensitivity, impaired dark adaptation, contrast sensitivity, and patient reported outcomes. In some instances, treatment with a multivalent C5 binding molecule described herein may have beneficial effects as measured by clinical endpoints including drusen volume, reading speed, retinal thickness as measured by Optical Coherence Tomography or other techniques, central retinal volume, number and density of retinal cells, area of retinal atrophy as measured by Fundus Photography or Fundus Auto fluorescence or other techniques, best corrected visual acuity such as measured by Snellen or ETDRS scales, Best Corrected Visual Acuity under low luminance conditions, light sensitivity, dark adaptation, contrast sensitivity, and patient reported outcomes as measured by such tools as the National Eye Institute Visual Function Questionnaire and Health Related Quality of Life Questionnaires.Subjects

[0129] 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, research animals, farm animals, sport animals, and pets. In some cases, the methods described herein may be used on tissues or cells derived from a subject and the progeny of such tissues or cells. For example, multivalent C5 binding molecules described herein may be used to effect some function in tissues or cells of a subject. The tissues or cells may be obtained from a subject in vivo. In some cases, the tissues or cells are cultured in vitro and contacted with a composition provided herein (e.g., a bivalent binding molecule).

[0130] In some aspects, the methods and compositions provided herein are used to treat a subject in need thereof. In some cases, the subject suffers from a retinal disease or disorder. In some cases, the subject is a human. In some cases, the human is a patient at a hospital or a clinic. In some cases, the subject is a non-human animal, for example, a non -human primate, aWSGR Docket No. 66711-706.601livestock animal, a domestic pet, or a laboratory animal. For example, a non-human animal can be an ape (e.g., a chimpanzee, a baboon, a gorilla, or an orangutan), an old world monkey (e.g., a rhesus monkey), a new world monkey, a dog, a cat, a bison, a camel, a cow, a deer, a pig, a donkey, a horse, a mule, a lama, a sheep, a goat, a buffalo, a reindeer, a yak, a mouse, a rat, a rabbit, or any other non-human animal.

[0131] In cases where the subject is a human, the subjectmay be of any age. In some cases, the subject has an age-related retinal disease or disorder (e.g., age-related macular degeneration, Stargardt disease). In some cases, the subject is about 50 years or older. In some cases, the subject is about 55 years or older. In some cases, the subject is about 60 years or older. In some cases, the subject is about 65 years or older. In some cases, the subject is about 70 years or older. In some cases, the subject is about 75 years or older. In some cases, the subject is about 80 years or older. In some cases, the subject is about 85 years or older. In some cases, the subject is about 90 years or older. In some cases, the subject is about 95 years or older. In some cases, the subject is about 100 years or older. In some cases, the subject is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or greater than 100 years old. In some cases, the subject is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater than 20 years old.

[0132] In cases where the subject is a human, the subject may have any genetic profile. In some cases, the subject may have mutations in complement Factor H (CFH), complement component 3 (C3), complement component 2 (C2), complement Factor B, complement Factor I (CFI), ABC4A, ELOVL4, or any combination thereof.

[0133] In some aspects, the methods and compositions provided herein are utilized to treat a subject suffering from symptoms as described herein. In some aspects, the methods and compositions provided herein are used to treat a subject having, suspected of having, or at risk of developing a retinal disease as provided herein. In some cases, the methods and compositions provided herein are used to treat a subject having, suspected of having, or at risk of developing wet AMD. In some cases, the methods and compositions provided herein are used to treat a subject having, suspected of having, or at risk of developing dry AMD or geographic atrophy. In some cases, the methods and compositions provided herein are used to treat a subject having, suspected of having, or at risk of developing from Stargardt disease. In some aspects, the methods and compositions provided herein are used to treat a subject having, suspected of having, or at risk of developing a hematological disease or disorder. In some embodiments, the methods and compositions provided herein comprises identifying or having identified the subject meeting one or more patient selection criteria. In some embodiments, provided hereinWSGR Docket No. 66711-706.601are methods of reducing a growth rate of Geographic Atrophy (GA) lesion or area. In some embodiments, the method comprises identifying or having identified a subject as having or at risk of having GA, identifying or having identified the subject not having hypoactive (ocular) complement pathway activity; and administering to the subject, or recommending that the subject be treated with, a C5 inhibitor; optionally, measuring the Geographic Atrophy lesion or area at least two different time points; and optionally, calculating the growth rate of the Geographic Atrophy lesion or area growth between the at least two different time points.

[0134] In some embodiments, the administration to the subject the C5 inhibitor results in an average reduction in the growth rate of the GA lesion or area by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% as compared to that of a patient received no treatment. In some embodiments, the administration to the subject the C5 inhibitor results in an increase in visual function outcomes as measured using, for example, Best Corrected Visual Acuity (BCVA) or Low Luminance Visual Acuity (LLVA). In some embodiments, visual function outcomes are increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% as compared to that of a patient received no treatment.

[0135] In some embodiments, the methods and compositions provided herein comprise identifying or having identified the subject not having hypoactive ocular complement pathway activity . For example, in some embodiments, hypoactive ocular complement pathway activity can be identified by measuring or assessing level(s) of Ba or Bb. In some embodiments, hypoactive ocular complement pathway activity comprises having a Ba level that is less than about 1 ng / mL, less than about 2 ng / mL, less than about 3 ng / mL, less than about 4 ng / mL, less than about 5 ng / mL, less than about 6 ng / mL, less than about 7 ng / mL, less than about 8 ng / mL, less than about 9 ng / mL, or less than about 10 ng / mL. In some embodiments, hypoactive ocular complement pathway activity comprises having Bb level that is less than about 20 ng / mL, less than about 21 ng / mL, less than about 22 ng / mL, less than about 23 ng / mL, less than about 24 ng / mL, less than about 25 ng / mL, less than about 26 ng / mL, less than about 27 ng / mL, less than about 28 ng / mL, less than about 29 ng / mL, less than about 30 ng / mL, less than about 31 ng / mL, less than about 32 ng / mL, less than about 33 ng / mL, less than about 34 ng / mL, less than about 35 ng / mL, less than about 36 ng / mL, less than about 37 ng / mL, less than about 38 ng / mL, less than about 39 ng / mL, or less than about 40 ng / mL. In some embodiments Ba or Bb level can be measured by collecting samples of a subject. In some embodiments, samples can be obtained from aqueous humor from the anterior chamber of the eye. In some embodiments, samples canWSGR Docket No. 66711-706.601be obtained from serum. In some embodiments, Ba orBb level can be measured using enzyme -linked immunosorbent assay (ELISA), western blot, or immunohistochemistry.

[0136] In some embodiments, the method described herein comprises identifying or having identified a subject as having ocular complement amplification loop activity within a range defined as homeostatic or having ocular complement amplification loop activity that is up to at mostl-fold, at most about 1 ,5-fold, at most about 2-fold, at most about 2.5-fold, at most about 3-fold, at most about 3.5-fold, at most about 4-fold, at most about 4.5 -fold, at most about 5 -fold, at most about 5.5-fold, at most about 6-fold, at most about 6.5-fold, at most about 7-fold, at most about 7.5-fold, atmost about 8-fold, at most about 8.5 -fold, at most about 9-fold, at most about 9.5-fold, at most about 10-fold homeostatic level of the homeostatic level. In some embodiments, homeostatic level of the ocular complement amplification loop activity can comprise having a Ba level that is about 10 ng / mL to about 80 ng / mL. In some embodiments, homeostatic level of the ocular complement amplification loop activity can comprise having a Ba level that is about 10 ng / mL to about 20 ng / mL, about 10 ng / mL to about 30 ng / mL, about 10 ng / mL to about 40 ng / mL, about 10 ng / mL to about 50 ng / mL, about 10 ng / mL to about 60 ng / mL, about 10 ng / mL to about 70 ng / mL, about 10 ng / mL to about 80 ng / mL, about 20 ng / mL to about 30 ng / mL, about 20 ng / mL to about 40 ng / mL, about 20 ng / mL to about 50 ng / mL, about 20 ng / mL to about 60 ng / mL, about 20 ng / mL to about 70 ng / mL, about 20 ng / mL to about 80 ng / mL, about30 ng / mL to about40 ng / mL, about30 ng / mL to about 50 ng / mL, about 30 ng / mL to about 60 ng / mL, about 30 ng / mL to about 70 ng / mL, about 30 ng / mL to about 80 ng / mL, about 40 ng / mL to about 50 ng / mL, about 40 ng / mL to about 60 ng / mL, about 40 ng / mL to about 70 ng / mL, about 40 ng / mL to about 80 ng / mL, about 50 ng / mL to about 60 ng / mL, about 50 ng / mL to about 70 ng / mL, about 50 ng / mL to about 80 ng / mL, about 60 ng / mL to about 70 ng / mL, about 60 ng / mL to about 80 ng / mL, or about 70 ng / mL to about 80 ng / mL. In some embodiments, homeostatic level of the ocular complement amplification loop activity can comprise having a Ba level that is about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, or about 80 ng / mL. In some embodiments, homeostatic level of the ocular complement amplification loop activity can comprise having a Ba level that is at least about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, or about 70 ng / mL. In some embodiments, homeostatic level of the ocular complement amplification loop activity can comprise having a Ba level that is at most about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, or about 80 ng / mL. In some embodiments, homeostatic level of the ocular complement amplification loop activity can comprise having a Bb level that is about 40 ng / mL to about 120 ng / mL. In some embodiments, homeostatic level of the ocularWSGR Docket No. 66711-706.601complement amplification loop activity can comprise having a Bb level that is about 40 ng / mL to about 50 ng / mL, about 40 ng / mL to about 60 ng / mL, about 40 ng / mL to about 70 ng / mL, about 40 ng / mL to about 80 ng / mL, about 40 ng / mL to about 90 ng / mL, about 40 ng / mL to about 100 ng / mL, about 40 ng / mL to about 110 ng / mL, about 40 ng / mL to about 120 ng / mL, about 50 ng / mL to about 60 ng / mL, about 50 ng / mL to about 70 ng / mL, about 50 ng / mL to about 80 ng / mL, about 50 ng / mL to about 90 ng / mL, about 50 ng / mL to about 100 ng / mL, about 50 ng / mL to about 110 ng / mL, about 50 ng / mL to about 120 ng / mL, about 60 ng / mL to about 70 ng / mL, about 60 ng / mL to about 80 ng / mL, about 60 ng / mL to about 90 ng / mL, about 60 ng / mL to about 100 ng / mL, about 60 ng / mL to about 110 ng / mL, about 60 ng / mL to about 120 ng / mL, about 70 ng / mL to about 80 ng / mL, about 70 ng / mL to about 90 ng / mL, about 70 ng / mL to about 100 ng / mL, about 70 ng / mL to about 110 ng / mL, about 70 ng / mL to about 120 ng / mL, about 80 ng / mL to about 90 ng / mL, about 80 ng / mL to about 100 ng / mL, about 80 ng / mL to about 110 ng / mL, about 80 ng / mL to about 120 ng / mL, about 90 ng / mL to about 100 ng / mL, about 90 ng / mL to about 110 ng / mL, about 90 ng / mL to about 120 ng / mL, about 100 ng / mL to about 110 ng / mL, about 100 ng / mL to about 120 ng / mL, or about 110 ng / mL to about 120 ng / mL. In some embodiments, homeostatic level of the ocular complement amplification loop activity can comprise having a Bb level that is about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 110 ng / mL, or about 120 ng / mL. In some embodiments, homeostatic level of the ocular complement amplification loop activity can comprise having a Bb level that is at least about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, or about 110 ng / mL. In some embodiments, homeostatic level of the ocular complement amplification loop activity can comprise having a Bb level that is at most about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 110 ng / mL, or about 120 ng / mL. In some embodiments Ba or Bb level can be measured by collecting samples of a subject. In some embodiments, samples can be obtained from aqueous humor from the anterior chamber of the eye. In some embodiments, samples can be obtained from vitreous humor. In some embodiments, samples can be obtained from serum. In some embodiments, Ba or Bb level can be measured using enzyme-linked immunosorbent assay (ELISA), western blot, or immunohistochemistry.

[0137] Further provided herein are methods of reducing the growth rate of Geographic Atrophy area or lesion, the method comprising identifying or having identified a subject as having Geographic Atrophy, identifying or having identified a subject as having pathologic complement pathway activity relevant to C5, administering to the subject, or recommending that the subject be treated with, a C5 inhibitor; and, optionally, measuring the Geographic Atrophy area or lesionWSGR Docket No. 66711-706.601at least two different time points and calculating the rate of Geographic Atrophy between time points. In some embodiments, a subject having pathologic complement pathway activity relevant to C5 may comprise a subject having at least one of above a threshold concentration of at least one ocular complement protein, elevated retina flavoprotein fluorescence intensity or presence of at least one genetic polymorphism in a complement pathway protein.

[0138] In some embodiments, the methods and compositions provided herein comprise identifying or having identified a subject having pathologic complement pathway activity relevant to C5. For example, in some embodiments, pathologic complement pathway activity relevantto C5 can be identified by measuring or assessing level(s) of Ba, Bb, or C5a. In some embodiments, pathologic complement pathway activity relevantto C5 may compromise having a Ba level that is at least about 5 ng / mL, at least about 10 ng / mL, at least about 15 ng / mL, at least about 20 ng / mL, at least about 25 ng / mL, at least about 30 ng / mL, at least about 35 ng / mL, at least about 40 ng / mL, at least about 45 ng / mL, or at least about 50 ng / mL. In some embodiments, pathologic complement pathway activity relevantto C5 may compromise having Bb level that is at least about 20 ng / mL, at least about 30 ng / mL, at least about 40 ng / mL, at least about 50 ng / mL, at least about 60 ng / mL, at least about 70 ng / mL, at least about 80 ng / mL, at least about 90 ng / mL, at least about 100 ng / mL, at least about 110 ng / mL, at least about 120 ng / mL, at least about 130 ng / mL, at least about 140 ng / mL, at least about 150 ng / mL, at least about 160 ng / mL, atleast about 170 ng / mL, atleast about 180 ng / mL, atleast about 190 ng / mL, or at least about 200 ng / mL. In some embodiments, pathologic complement pathway activity relevantto C5 may compromise having C5 a level that is at least about 10 pg / mL, at least about 15 pg / mL, at least about 20 pg / mL, at least about 25 pg / mL, at least about 30 pg / mL, at least about 35 pg / mL, at least about 40 pg / mL, at least about 45 pg / mL, or at least about 50 pg / mL.

[0139] In some embodiments Ba, Bb, or C5a level can be measured by collecting samples of a subject. In some embodiments, samples can be obtained from aqueous humor from the anterior chamber of the eye. In some embodiments, samples can be obtained from serum. In some embodiments, Ba or Bb level can be measured using enzyme-linked immunosorbent assay (ELISA), western blot, or immunohistochemistry. In some embodiments, samples can be obtained from aqueous humor from the anterior chamber of the eye. In some embodiments, samples can be obtained from vitreous humor. In some embodiments, samples can be obtained from serum.

[0140] In some embodiments, the method described herein comprises identifying or having identified a subject as having elevated retina flavoprotein fluorescence intensity may compromise having an intensity value that is at least 1-fold, at least about 1.25-fold, at least about 1.5-fold, atleast about 1.75-fold, at least about 2-fold, at least about 2.25-fold, atleastWSGR Docket No. 66711-706.601about 2.5-fold, at least about 2.75-fold, or at least about 3-fold higher than that of an age-matched healthy patient group.

[0141] In some embodiments, the method described herein comprises identifying or having identified a subject as having pathologic complement activity relevant to C5 comprises having a genetic test. In some embodiments, the genetic test can comprise testing for a mutation of the Tyr at amino position 402 of the human complement factor H (CFH) protein. In some embodiments, the genetic test comprises testing for a mutation in CD46, CD55, or CD59. For example, in some embodiments, the methods and compositions provided herein comprise identifying or having identified the subject having a loss of function or loss of function mutation in CD59. In some embodiments, a loss of function or loss of function mutation of CD59 comprises a decreased expression of CD 59 in cells (e.g., RPE cells). For example, in some embodiments, the subject can have at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 90% reduction in CD59 expression compared to that of a healthy control subject (e.g., a control subject that does not have or diagnosed for having an ocular disease). In some embodiments, a loss of function or loss of function mutation in CD59 comprises cells (e.g., RPE cells) having one or mutations including, but not limited to, of c,146delA (p.Asp49Valfs*31), c,123delC (p.Val42Serfs*38), c.266G>A (p.Cys89Tyr), c.238A>G (p.Arg80Gly), and / or c,146A>T (p.Asp49Val).

[0142] In some embodiments, the methods described herein may result in more than a 50% likelihood (e.g., more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%) that an individual eye has at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% slower growth rate of Geographic Atrophy area or lesion after treatment with the methods and compositions described herein, as compared to that of an individual eye in a prior period without treatment.Pharmaceutical compositions or medicaments

[0143] Disclosed herein are pharmaceutical compositions or medicaments, used interchangeably, for use in a method of therapy, or for use in a method of medical treatment. Such use may be for the treatment of retinal diseases. In some cases, the pharmaceutical compositions can beused to treat AMD. In some cases, the pharmaceutical compositions can be used to treat non-exudative (dry) AMD. In some cases, the pharmaceutical compositions can be used to treat geographic atrophy (advanced dry AMD). In some cases, the pharmaceuticalWSGR Docket No. 66711-706.601compositions can be used to treat wet AMD. In some cases, the pharmaceutical compositions can be used to treat Stargardt disease.

[0144] Pharmaceutical compositions described herein may include one or more multivalent C5 binding molecules (e.g. bivalent binding molecule) for the treatment of dry AMD.Pharmaceutical compositions described herein may include one or more multivalent C5 binding molecules for the treatment of Stargardt disease. In some cases, the one or more binding molecules bind to and inhibit C5. In some cases, the one or more binding molecules simultaneously bind to two different epitopes. In some cases, the one or more multivalent C5 binding molecules comprise at least two aptamer moieties that bind to and inhibit C5.

[0145] In some cases, the compositions include an effective amount of the multivalent C5 binding molecule, alone or in combination with one or more vehicles (e.g., pharmaceutically acceptable compositions or e.g., pharmaceutically acceptable carriers). In some cases, the compositions described herein are administered with one or more additional pharmaceutical treatments (e.g., co-administered, sequentially administered or co-formulated).Formulations

[0146] Compositions as described herein may comprise a liquid formulation, a solid formulation or a combination thereof. Non-limiting examples of formulations may include a tablet, a capsule, a gel, a paste, a liquid solution and a cream. The compositions of the present disclosure may further comprise any number of excipients. Excipients may include any and all solvents, coatings, flavorings, colorings, lubricants, disintegrants, preservatives, sweeteners, binders, diluents, and vehicles (or carriers). Generally, the excipient is compatible with the therapeutic compositions of the present disclosure. The pharmaceutical composition or formulation may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as, for example, sodium acetate, and triethanolamine oleate.

[0147] In some cases, a pharmaceutical formulation provided herein comprises a multivalent C5 binding molecule as described herein (e.g., a bivalent C5 binding molecule) and a pharmaceutically acceptable excipient, carrier, and / or diluent. In some cases, the pharmaceutical formulation is formulated for intravitreal administration. In some cases, the pharmaceutical formulation is formulated for intravitreal administration via a syringe and needle. In some cases, the needle is a 32-gauge, a 31 -gauge, a 30-gauge, or a 28-gauge needle. In some cases, the pharmaceutical formulation is formulated at a concentration of the multivalent C5 binding molecule of greater than 1 mg / 100 pL, 2 mg / 100 pL, 3 mg / 100 pL, 4 mg / 100 pL, or 5 mg / 100 pL with a break force of less than 12 N.WSGR Docket No. 66711-706.601

[0148] In some embodiments, the pharmaceutical formulation comprises about 0.1 mg to about 10 mg of the multivalent C5 binding molecules described herein (e.g. bivalent C5 binding molecules) per eye. In some embodiments, the pharmaceutical formulation comprises about 0.1 mg to about 0.5 mg, about 0.1 mg to about 1 mg, about 0.1 mg to about 2 mg, about 0.1 mg to about 3 mg, about 0.1 mg to about 4 mg, about 0.1 mg to about 5 mg, about 0.1 mg to about 6 mg, aboutO.l mgto about? mg, aboutO.l mgto about8 mg, aboutO.l mgto about9 mg, about 0.1 mgto about 10 mg, about0.5 mgto about 1 mg, about 0.5 mg to about 2 mg, about 0.5 mg to about 3 mg, about 0.5 mgto about4 mg, about 0.5 mgto about 5 mg, about 0.5 mgto about 6 mg, about 0.5 mgto about? mg, about 0.5 mgto about 8 mg, about 0.5 mgto about 9 mg, about 0.5 mg to about 10 mg, about 1 mg to about 2 mg, about 1 mg to about 3 mg, about 1 mg to about 4 mg, about 1 mg to about 5 mg, about 1 mg to about 6 mg, about 1 mg to about 7 mg, about 1 mgto about 8 mg, about 1 mgto about 9 mg, about 1 mg to about 10 mg, about 2 mg to about 3 mg, about 2 mg to about 4 mg, about 2 mg to about 5 mg, about 2 mg to about 6 mg, about 2 mgto about 7 mg, about 2 mg to about 8 mg, about 2 mg to about 9 mg, about 2 mg to about 10 mg, about 3 mg to about 4 mg, about 3 mg to about 5 mg, about 3 mg to about 6 mg, about 3 mgto about 7 mg, about 3 mg to about 8 mg, about 3 mg to about 9 mg, about 3 mg to about 10 mg, about 4 mg to about 5 mg, about 4 mg to about 6 mg, about 4 mg to about 7 mg, about 4 mgto about 8 mg, about 4 mgto about 9 mg, about 4 mg to about 10 mg, about 5 mg to about 6 mg, about 5 mg to about 7 mg, about 5 mg to about 8 mg, about 5 mg to about 9 mg, about 5 mgto about 10 mg, about 6 mgto about? mg, about 6 mgto about 8 mg, about 6 mgto about 9 mg, about 6 mg to about 10 mg, about 7 mg to about 8 mg, about 7 mg to about 9 mg, about 7 mg to about 10 mg, about 8 mg to about 9 mg, about 8 mg to about 10 mg, or about 9 mg to about 10 mg to about 10 mg of the binding molecules (e.g. bivalent C5 binding molecules) per eye. In some embodiments, the pharmaceutical formulation comprises about 0.1 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or about 10 mg of the binding molecules (e.g. bivalent C5 binding molecules) per eye. In some embodiments, the pharmaceutical formulation comprises an effective amount of at least about 0.1 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, or about 9 mg of the binding molecules (e.g. bivalent C5 binding molecules) per eye. In some embodiments, the pharmaceutical formulation comprises an effective amount of at most about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or about 10 mg of the binding molecules (e.g. bivalent C5 binding molecules) per eye. Such dosages may be administered ocularly, for example by intravitreal injection monthly, bimonthly, quarterly, every four months, semi-annually, or optionally by a sustained release device orWSGR Docket No. 66711-706.601formulation. The dosage may be administered as a single dose or divided into multiple doses. In general, the desired dosage should be administered at set intervals for a prolonged period, usually at least over months, although longer periods of administration of several months or years or more may be needed. In some embodiments, the therapeutically effective amount of the multivalent C5 binding molecule may comprise a dose and frequency that does not exceed an average mass of PEG present in the multivalent C5 binding molecule or the pharmaceutical formulation. For example, in some embodiments, the therapeutically effective amount of the multivalent C5 binding molecule may comprise a dose and frequency that does not exceed about 5 mg per eye per month, about 4 mg per eye per month, about 3 mg per eye per month, or 2 mg per eye per month.Dosage and Routes of Administration

[0149] Therapeutic doses of formulations of the disclosure can be administered to a subject in need thereof. In some cases, a formulation is administered to the eye of a subject to treat, for example, dry AMD, geographic atrophy, wet AMD or Stargardt disease. Administration to the eye can be a) topical; b) local ocular delivery; orc) systemic. In some cases, a formulation of the disclosure is administered by local ocular delivery. Non-limiting examples of local ocular delivery include intravitreal (IVT), intracam arel, subconjunctival, sub tenon, retrobulbar, posterior juxtascleral, and peribulbar. In some cases, a formulation of the disclosure is delivered by intravitreal administration (IVT). Local ocular delivery may generally involve injection of a liquid formulation. Other formulations suitable for delivery of the pharmaceutical compositions described herein may include a sustained release gel or polymer formulations by surgical implantation of a biodegradable microsize polymer system, e.g., microdevice, microparticle, or sponge, or other slow release transscleral devices, implanted during the treatment of an ophthalmic disease, or by an ocular delivery device, e.g. polymer contact lens sustained delivery device. In some cases, the formulation is a polymer gel, a self-assembling gel, a durable implant, an eluting implant, a biodegradable matrix or biodegradable polymers. In some cases, the formulation may be administered by iontophoresis using electric current to drive the composition from the surface to the posterior of the eye. In some cases, the formulation may be administered by a surgically implanted port with an intravitreal reservoir, an extra-vitreal reservoir or a combination thereof. Examples of implantable ocular devices can include, without limitation, the Durasert™ technology developed by Bausch & Lomb, the ODTx device developed by On Demand Therapeutics, the Port Delivery System developed by ForSight VISION4 and the Replenish MicroPump™ System developed by Replenish, Inc.

[0150] The binding molecules as described herein (e.g., bivalent aptamer-containing molecules) may be particularly advantageous over monospecific aptamers as they may sustainWSGR Docket No. 66711-706.601therapeutic intravitreal concentrations of drug for longer periods of time, thus requiring less frequent administration. For example, a monovalent anti-C5 aptamer (e.g., avacincaptad pegol) may show clinical efficacy for the treatment of geographic atrophy or STGD-1 with 1 to 2 mg when dosed every 4 weeks (q4w). The multivalent C5 binding molecules as described herein (e.g., bivalent C5 binding molecules) may have a longer intraocular half-life and greater ability to inhibit complement activity at equimolar concentrations vs. a monovalent anti-C5 aptamer, and / or sustain therapeutic intravitreal concentrations of drug for longer periods of time, than an anti-C5 aptamer. In some cases, the binding molecules as described herein (e.g., bivalent C5 binding molecules) are dosed, from treatment initiation, at least every 8 weeks (q8w), every 12 weeks (ql2w), every 16 weeks (ql6w) or greater than ql6w. In some cases, the multivalent C5 binding molecule will have non-inferior ocular complement inhibition throughout the dosing interval with every 12 weeks (ql2w) or every 16 weeks (ql6w) dosing when compared to a

[0151] In some aspects, a therapeutically effective amount of the multivalent C5 binding molecule is administered. A “therapeutically effective amount” or “therapeutically effective dose” are used interchangeably herein and refer to an amount of a therapeutic agent (e.g., multivalent C5 binding molecule) that provokes a therapeutic or desired response in a subject. The therapeutically effective amount of the composition may be dependent on the route of administration.

[0152] In some aspects, a therapeutically effective amount is administered with a dosage and frequency that minimizes or mitigates the formation of PEG mediated development of choroidal neovascularization (CNV). In some cases, the dose and frequency of administration is selected such that the mass of PEG, comprised as part of the C5 binding molecule, that is administered on average per month per eye is less than 3.1 mg, less than 2.5 mg, or less than 2 mg..Methods

[0153] Disclosed herein are methods of inhibiting complement activity in a subject in need thereof. Further disclosed herein are methods for the treatment of retinal diseases disease. In some cases, the retinal disease is wet age-related macular degeneration, dry age-related macular degeneration, geographic atrophy, diabetic retinopathy, diabetic macular edema, proliferative diabetic retinopathy, retinal vein occlusion, Stargardt disease, retinitis pigmentosa, retinal detachment, and non-arteritic anterior ischemic optic neuropathy (NAION).

[0154] In some aspects, patient complement activity is assessed prior to or after treatment administration. In some cases, assays are used to measure complement pathway protein concentrations in samples from patients. In some cases, the samples are from aqueous humor. In some cases, the assays used may be ELISA based chemiluminescent assays for complement system analytes (e.g., Quidel Complement Assays, Millipore MILLIPLEX Assays). In someWSGR Docket No. 66711-706.601cases, the assays used may use Proximity Extension Assay technology (e.g., OLink Target 96 Assay).

[0155] In some aspects, complement activity is stratified into pathologic or non-pathologic based on measurements from complement pathway protein assays, retina flavoprotein fluorescence hyperactivity, and / or genetic mutations (FIG. 5). In some embodiments, the complement pathway proteins measured are one or more of Ba, Bb, C5a, C4a. In some embodiments, assessing complement activity comprises determining retinal flavoprotein fluorescence hyperactivity and / or genetic mutations in CD59. In some embodiments, pathological complement activity is defined by measurements of at least one of Ba, Bb, C5a, C4a, retinal flavoprotein fluorescence hyperactivity, or one or more genetic mutations in CD59. In some embodiments, complement activity is stratified into pathologic or non-pathologic based on measurement results relative to a threshold. In some embodiments, Ba greater than or equal to 10 ng / mL is pathologic. In some embodiments Bb, greater than or equal to 40 ng / mL is pathologic. In some embodiments, C5 a greater than or equal to 40 pg / mL is pathologic. In some embodiments C4a, greater than or equal to 1.2 ng / mL is pathologic. In some embodiments, having retina flavoprotein hyperactivity is pathologic. In some embodiments, genetic mutations in CD59 are pathologic. In some embodiments, if a subject is classified as having pathological complement activity, then it is therapeutically appropriate to treat the subject with one or more multivalent C5 binding molecules as described herein. In some embodiments, if a subject is classified as having non-pathological complement activity, then it is inappropriate to treat the subject with one or more multivalent C5 binding molecules as described herein.

[0156] In some cases, the method involves administering a therapeutically effective amount of a composition to a subject to treat the disease. In some cases, the composition includes one or more multivalent C5 binding molecules as described herein (e.g., bivalent C5 binding molecules). The multivalent C5 binding molecules may inhibit a function associated with C5 as described herein. The methods can be performed at a hospital or a clinic, for example, the pharmaceutical compositionscan be administered by a health-care professional. Treatment may commence with the diagnosis of a subject with a retinal disease (e.g., AMD). In the event that further treatments are necessary, follow-up appointments may be scheduled for the administration of subsequent doses of the composition, for example, administration every 8 weeks, every 12 weeks, or every 16 weeks.

[0157] Treatment efficacy can be determined by changes in complement activity after administration of a therapeutically amount of a composition (FIG. 6). In some cases, the composition includes one or more multivalent C5 binding molecules as described herein (e.g., bivalent C5 binding molecules). In some embodiments, significant efficacy (A) occurs when aWSGR Docket No. 66711-706.601treatment causes a change in status from pathologic to non -pathologic and for the level of complement activity to decrease by greater than or equal to 50% from complement activity prior to treatment. In some embodiments, some efficacy (B) occurs when a treatment causes a decrease in the level of complement activity by greater than or equal to 40% but does not cause a change in pathologic status or when a treatment causes a decrease in the level of complement activity by 35% to 49 % and a change in status from pathologic to non-pathologic. In some embodiments, nonsignificant efficacy (C) occurs when a treatment does not cause a change in the complement activity of a pathologic subject, causes an increase in the complement activity of a pathologic subject, causes a decrease in the complement activity of a non -pathological subject, does not cause a change in the complement activity of a non -pathological subject, or causes an increase in the complement activity of a non -pathological subject.EXAMPLES

[0158] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion . The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.Example 1. Manufacturing Bivalent C5 binding molecules that include linker moiety / moieties

[0159] Table 2 and FIGs 3C-3F illustrate exemplary multivalent C5 binding aptamer molecules constructs that are synthesized and evaluated.

[0160] The general scheme of synthesis of the bivalent aptamer molecule described herein is shown in FIG. 4A-4C. The process of manufacturing bivalent aptamer molecules starts with serinol azide CPG (Glen 20-2999), a substance that is pre-functionalized with azide for later coupling with a second aptamer. An asymmetric doubler (Glen 10-1981) is installed initially. Following this, the first aptamer is assembled on one branch of the doubler on the solid support. It is to be noted that multiple Spacer Phosphoramidite 18 (Spl8; Glen 10-1918) can be incorporated before the first aptamer in order to precisely adjust the linker length.

[0161] Upon the completion of the solid phase synthesis, the oligo undergoes cleavage from the solid support and is subsequently deprotected. Through this process, the primary amine functional group on the second branch of the doubler is uncovered for future modification. Subsequently, the second aptamer, is synthesized separately. This aptamer, which is terminatedWSGR Docket No. 66711-706.601with multiple Spl8 and a DBCO functional group, is then coupled to the product generated earlier. The final stage of the manufacturing process involves a 40 kDa PEG NHS ester reacting with the product from the previous reaction. In some cases, the 40 kDa PEG can be replaced with a 10 or 20 kDa PEG. In some embodiments, any total 10, 20, or 40 kDa PEG can be of architecture that is linear, 2 arm, 3 arm, 4 arm, 6 arm, or 8 arm design or structure. This reaction results in the creation of the final bivalent aptamer product. This robust and systematic process allows for the efficient production of bivalent aptamer molecules.

[0162] By utilizing the method described herein, the core structure is easier to make using all commercially available materials, and the process is likely as scalable, or amenable for large scale synthesis. It allows flexibility which allows the attaching terminal of aptamer 1 or aptamer 2 (or other moieties like aptamer 1 and antibody 2) to be switchable from 5’ to 3’ or 3’ to 5’. In some cases, the location of Aptamer 1, Aptamer 2, or PEG can be easily changed or switched.Examples 2. Manufacture of multivalent C5 binding molecules

[0163] FIG. 3A illustrates a general formula for multivalent C5 binding aptamer molecules constructs that are synthesized structures illustrates illustrated in FIG. 3B.

[0164] The general scheme of synthesis of the bivalent aptamer molecule described herein is shown in FIGs. 4A-4C.

[0165] Aptamers selected from SEQ ID: 1 to SEQ ID: 9, with 5’ NHS ester functionality are reacted with a range of PEG reagents of 20kDa to 40 kDa size with multiple primary amine reactive sites, and purified to isolate multivalent C5 binding molecules of the disclosure.

[0166] By utilizing the method described herein, the core structure is easier to make using all commercially available materials, and the process is likely as scalable, or amenable for large scale synthesis. As illustrated in FIG. 3A, it allows flexibility which allows the attaching terminal of Aptamer Moiety 1 or Aptamer Moiety 2 to be switchable from 5 ’ to 3 ’ or 3 ’ to 5 ’ . In some cases, the identity of Aptamer Moiety 1, Aptamer Moiety 2, or PEG can be easily changed or switched.Example 3. Increased stoichiometry of binding with multivalent C5 binding molecules

[0167] To test whether monospecific orbi-specific aptamers can bind to multiple C5 proteins simultaneously, Isothermal Calorimetry (ITC) is used.

[0168] In brief, standard methods and validated instruments (e.g., Malvern MicroCai PEAQ-ITC) are used, with multivalent C5 binding molecules and aptamers of the disclosure prepared in appropriate solutions. Human complement C5 is obtained. Other molecules with establishedWSGR Docket No. 66711-706.601binding properties to C5 are included as reference standards / as experimental methodology controls.

[0169] Surprisingly, multivalent C5 binding molecules of the disclosure show favorable stoichiometry of C5 binding compared to avacincaptad pegol and monovalent C5 binding aptamers. Surprisingly, the size and molecular dimensions of the linkers in the multivalent C5 binding molecule affect the stoichiometry of binding, with some multivalent C5 binding molecules of the disclosure demonstrating significantly improved stoichiometry of binding vs. other multivalent C5 binding molecules that comprise the same number of C5 binding aptamer moieties.Example 4. Assessment of binding affinity of C5 binding aptamers and multivalent C5 binding molecules to C5 by Surface Plasmon Resonance (SPR)

[0170] To measure binding affinity of PEGylated multivalent C5 binding molecules SEQ IDs 1-12 (Table 1 and Table 2) surface plasmon resonance (SPR) experiments were conducted against C5 as compared to a previously described C5 binding aptamer, avacincaptad pegol (40 kDaPEG conjugated to 5’ end of SEQ ID NO: 1 (Table 1) (Ruckman et al., 1998). Briefly, in the SPR studies, 1.0-2.5 pg / mL of glycan biotinylated C5 was diluted in HEPES running buffer (10 mM HEPES, 137.5 mMNaCl, 5.7 mMKCl, 1 mMMgCl2, 1 mM CaCl2, and 0.05% Tween20; pH 7.4) and were immobilized to a streptavidin dextran chip (GE Healthcare Life Sciences) for 1-2 minutes. The chip was then blocked with 0.2 mg / mL biotin in running buffer, followed by three cycles of buffer blanks and regeneration with 50 mMNaOH to equilibrate the chip. This resulted in 200-3000 RIU immobilized. After coating and blocking, samples were screened at 100 nM in duplicate injections at a flow rate of 25 pL / min, with an association time of 3 minutes 20 seconds and a dissociation time of 10 minutes. Regeneration conditions of chips were optimized based on the protein immobilized and were effected with either 50 mMNaOH for 30 seconds or 2M guanidine HC1 for 2 x 40 seconds. To determine binding affinities, multivalent C5 binding molecules were subsequently ran in an 11 -point, 2-fold dose response starting at a top concentration of 1.0 mM. Data was fit to a 1 : 1 kinetic binding model.

[0171] The results show therapeutically advantageous affinity characteristics for multivalent C5 binding molecules compared to biotinylated aptamer sequences (as per Example 2), and for different configurations of bivalent PEGylated anti-C5 aptamers (SEQ ID NOs: 10-13) compared to a monovalent PEGylated anti-C5 aptamer (avacincaptad pegol). These results demonstrate multivalent PEGylated anti-C5 aptamers with affinities of 10 nM or less to C5.WSGR Docket No. 66711-706.601Example 5. Assessment of inhibition of classical and alternative complement activation by monovalent and multivalent C5 binding aptamers

[0172] In this example, both classical and alternative complement-dependent hemolysis assays are performed to assess the ability of bi-specific aptamers to inhibit human complement activation. For the classic complement-dependent hemolysis assay, 1 mL of antibody -sensitized sheep red blood cells (sRBCs) (Colorado Serum Company) in 0.1 M sucrose in gelatin veronal buffer (GVB) (Complement Technology, Inc.) is washed twice with 10 mL GVB and spun down at 1000 gfor 10 minutes. The supernatant is removed with a transfer pipette and the remaining sRBCs are reconstituted in 10 mL of GVB. The sRBC concentration is then calculated by using a hemocytometer. The preparation of antibody-sensitized sheep erythrocytes is done with an anti-sheep red blood cell stroma antibody produced in rabbit (Sigma -Aldrich, catalog #S1389) according to the manufacturer’s protocol.

[0173] To determine the CH50 of normal human serum (NHS), 100 pL of NHS at increasing concentrations from 0.2% to 2% (diluted in GVB + 0.3 mM CaCl2+ 1 mM MgCl2) is mixed with 100 pL of GVB-washed sRBCs at a concentration of 2 x 108sRBCs / mL in a single well of a 96-well plate. The total volume of each well is 200 pL and samples are tested in duplicate. The final concentration of NHS, CaCl2, and MgCl2in the assay is 0.1% to 1% NHS in GVB, 0.15 mM CaCl2, and 0.5 mMMgCl2. The NHS and sRBC mixture are incubated at 37°C for 30 minutes and the reaction is quenched by adding 20 pL of 500 mM EDTA. The 96-well plate is spun at 600 gfor 3 minutes. 100 pLofthe supernatant is then diluted with 100 pL of GVB and the absorbance is read at 405 nm. The results are plotted on GraphPad Prism 8.0 (Boston, MA, USA) to determine the concentration of human serum required to achieve 50% lysis of sRBC.

[0174] To determine the IC50 of a complement inhibiting compound, 10 pL of each compound at 20x the desired final concentration is mixed with 90 pL of NHS at a concentration of 1.111 multiplied by the AP50 value calculated above and diluted in GVB + 0.3 mM CaCl2+ 1 mM MgCl2. The mixture of the compound andNHS are mixed with 100 pL of GVB-washed sRBCs at a concentration of 2 x 108sRBCs / mL in a single well of a 96-well plate. As mentioned above, the total volume of each well is 200 pL and samples are tested in duplicate. The NHS and sRBC mixture are incubated at 37°C for 30 minutes and the reaction is quenched by adding 20 pL of 500 mMEDTA. The 96-well plate is spun at 600 g for 3 minutes. 100 pL of the supernatant is then diluted with 100 pL of GVB and the absorbance is read at 405 nm. The results are plotted on GraphPad Prism 8.0 (Boston, MA, USA) to the IC50 of the complement inhibiting compound.

[0175] For the alternative complement-dependent hemolysis assay, 1 mL of rabbit red blood cells (rRBCs) in Alsevers solution (Colorado Serum Company) is washed twice with 10 mLWSGR Docket No. 66711-706.601gelatin veronal buffer (GVB) (Complement Technology, Inc.) and spun down at 1000 gfor 10 minutes. The supernatant is removed with a transfer pipette and the remaining rRBCs are reconstituted in 10 mL of GVB. The rRBC concentration is then calculated by using a hemocytometer.

[0176] To determine the AP50 of normal human serum (NHS), 100 pL of NHS at increasing concentrations from 2% to 30% (diluted in GVB + 20 mM EGTA + 10 mM MgCl2) are mixed with 100 pL of GVB-washed rRBCs at a concentration of 2 x 108rRBCs / mL in a single well of a 96-well plate. The total volume of each well is 200 pL and samples are tested in duplicate. The final concentration of NHS, EGTA, and MgCl2in the assay is 1% to 15% NHS in GVB, 10 mM EGTA, and 5 mM MgCl2. The NHS and rRBC mixture is incubated at 37°C for 30 minutes and the reaction is quenched by adding20 pL of 500 mMEDTA. The 96-well plate is spun at 600 g for 3 minutes. 100 pL of the supernatant is then diluted with 100 pL of GVB and the absorbance is read at 405 nm. The results are plotted on GraphPad Prism 8.0 (Boston, MA, USA) to determine the concentration of human serum required to achieve 50% lysis of rRBC.

[0177] To determine the IC50 of a complement inhibiting compound, 10 pL of each compound at 20x the desired final concentration is mixed with 90 pL of NHS at a concentration of 1.111 multiplied by the AP50 value calculated above and diluted in GVB + 20 mM EGTA + 10 mM MgCl2. The mixture of the compound andNHS is mixed with 100 pL of GVB-washed rRBCs at a concentration of 2 x 108rRBCs / mL in a single well of a 96-well plate. As mentioned above, the total volume of each well is 200 pL and samples are testedin duplicate. The NHS and rRBC mixture are incubated at 37°C for 30 minutes and the reaction is quenched by adding 20 pL of 500 mMEDTA. The 96-well plate is spun at 600 g for 3 minutes. 100 pL of the supernatant is then diluted with 100 pL of GVB and the absorbance is read at 405 nm. The results are plotted on GraphPad Prism 8.0 (Boston, MA, USA) to determine the IC50 of the complement inhibiting compound.

[0178] Molecules in FIGs.2A-2C (SEQ IDs 1-9) and Example 1 (Table 2) are incubated with C 1 q-depleted human serum to allow binding to complement components present in the serum, then assayed for the ability to inhibit complement-dependent lysis of rabbit red blood cells. Exemplary IC50 of the control molecule (monospecific molecules) is shown in Table 3. These results show that monovalent, fully substituted anti-C5 aptamers (e.g., SEQ ID: 2) have reduced ability to inhibit C5 mediated complement activity compared to avacincaptad (SEQ ID: 1) and avacincaptad pegol.WSGR Docket No. 66711-706.601Table 3. IC 50 of the aptamer molecules

[0179] In subsequent experiments (Example 12), it is demonstrated that multivalent anti-C5 molecules (SEQ ID: 10 - 12) incorporating the fully substituted anti-C5 aptamer (SEQ ID: 2) have increased potency and ability to inhibit C5 mediated complement activity compared to avacincaptad pegol.Example 6. Measurement of inhibition of mitochondrial damage / dysfunction

[0180] Cultured blood outgrowth endothelial cells (BOECs) are derived from healthy normal donors and patients with AMD, and cultured in monolayers on coverslips as previously described. Cell surface complement activation is facilitated by sensitization with an anti-CD59 antibody (e.g., BRIC229, International Blood Group Reference Laboratory, NHS Blood and Transplant, Bristol, UK) 12 at 5 pg / mL prior to exposure to NHS as a source of complement factors. Heat inactivated serum (HIS) may serve as a complement-inactive control, or C5-depleted serum is used as an anaphylatoxin-competent but C5b-9-inactive control. Changes in membrane integrity / mitochondrial function are detected by measuring Ca2+-influx and calcium-efflux, assessing cell survival via live / dead staining, testing for apoptotic and necrotic markers, measuring mitochondrial membrane potential (using the fluorescent probe JC-1), and confocal microscopy. Varying amounts of multi-valent C5 binding aptamers of the disclosure and other reference compounds are added to the NHS to assess ability of the molecules to inhibit effect on mitochondrial function.

[0181] Surprisingly, multivalent C5 binding aptamers of the disclosure demonstrate superior efficacy in terms of mitigating negative changes in mitochondrial membrane potential and function compared to FDA approved intravitreally administered complement inhibitors.Example 7. Determination of hydrodynamic radius of multivalent C5 binding aptamers

[0182] Dynamic light scattering is used to determine the hydrodynamic radius of multivalent C5 binding molecules of the disclosure. In these experiments, established methods for validatedWSGR Docket No. 66711-706.601instruments, such as the Zetasizer Nano or Zetasizer Ultra, are used to assess molecules in solution (e.g., PBS).

[0183] Surprisingly, the multivalent C5 binding aptamers display therapeutically favorable HDR values compared to a control molecule for treatment of advanced AMD, including faricimab (VABYSMO) and aflibercept (EYLEA HD, EYLEA) that are indicated for every 8 week, 12 week or 16 week dosing in at least some patients, from avacincaptad pegol (IZERVAY) indicated for initial every 4 week dosing, and from pegcetacoplan (SYFOVRE) indicated for every 4 week or every 8 week dosing.Example 8. Determination of viscosity and injection break force

[0184] Rheometry is used to determine the viscosity and ability to inject multivalent C5 binding molecules with syringe and needle configurations appropriate for intravitreal administration. To perform an intravitreal injection, an injection break force of ION or less is preferred by the majority of retina specialists / ophthalmologists.

[0185] In brief, multivalent C5 binding molecules of the disclosure are evaluated using established methodologies with validated viscometers (e.g., Rheosense mVROC II).

[0186] Multivalent C5 binding molecules of the disclosure demonstrate acceptable viscosity parameters to support injection break force of ION or less with commercially available syringes to administer volumes of 50 uL to 100 uL with 30G needles.Example 9. Determination of stability of multivalent C5 binding aptamers in vitreous fluid

[0187] C5 binding aptamers are tested in rat, rabbit, and cynomolgus macaque plasma (Charles River Labs, Wilmington, MA) vitreous fluid in order to assess their stability, rate kinetics, and pathways of degradation. Testing is performed using 5’ end-radiolabeled (32P) aptamer incubated at 37° C in 95% pooled plasma (citrated) / vitreous fluid over the course of 50 hours. At selected time points, aliquots of aptamer-containing plasma / vitreous fluid are withdrawn, immediately flash frozen in liquid nitrogen, and stored at -80 °C. Detection and analysis of the aptamer and its metabolites in plasma is accomplished using liquid -liquid (phenol-chloroform) extraction followed by gel electrophoresis (on a 10% denaturing polyacrylamide sequencing gel) and high-resolution autoradiography. The oligo-only portion e.g. , non-pegylated) of an aptamer approved for retinal use (e.g., Macugen) is used as a control. These experiments demonstrate that the multivalent C5 binding aptamers comprising aptamer moieties of SEQ IDs: 2-9 of the disclosure exhibit properties in serum and / or vitreous fluid that are at least as good as an FDA approved intravitreally administered aptamer therapeutic (e.g., pegaptanib, avacincaptad pegol) that contains one or more unmodified nucleotides.WSGR Docket No. 66711-706.601Example 10: Determination of duration on intraocular complement inhibition in a rabbit IVT model

[0188] To assess the extent and duration of intraocular complement activity following IVT administration of a single dose of multivalent anti-C5 therapeutics, rabbit PK / PD studies may be conducted. In brief, cohorts of rabbits are constructed such that the aqueous and vitreous humor at various timepoints before and after IVT injection maybe be sampled. The aqueous and vitreous samples at each timepoint are then assessed for presence of active therapeutic that functionally inhibits human complement activity in (a suitably adjusted for ocular fluid matrix) ex vivo human serum hemolysis assay (e.g., as described in Example 6, above). The results of these intraocular complement inhibition IVT rabbit studies show that multivalent anti-C5 therapeutics are able to maintain active drug concentrations sufficient to inhibit >95% of classical complement activity in the eyes of rabbits for 60 days following a single initial injection. This is in contrast to the FDA approved 30 day dosing interval following initial injection for IZERVAY (a monovalent anti-C5 PEGylated aptamer).

[0189] Additionally, a multivalent C5 binding molecule may be compared to faricimab / aflibercept administered intravitreally to cohorts of rabbits. Ocular fluid is sampled from the rabbit cohorts and ocular drug concentrations (for the multivalent C5 binding molecule and faricimab / aflibercept) and intravitreal half-lives calculated. Surprisingly, the multivalent C5 binding molecule demonstrates a longer IVT half-life compared to the 3 - 5 day half -lives of faricimab / aflibercept.Example 11: Demonstrating 40% slowing of rate of Geographic Atrophy growth compared to no (sham) treatment

[0190] To demonstrate superior efficacy of multivalent C5 binding molecules and formulations thereof, patients diagnosed with Geographic Atrophy are screened for anatomic and demographic inclusion and exclusion criteria typical for a Phase 2 / 3 clinical trial (e.g., area of Geographic Atrophy >2.5 mm2and <17.5 mm2, non-concomitant neovascular AMD in the study eye, no immune reactions to excipients in compositions, no ocular contradictions, etc.). A paracentesis is performed to collect aqueous humor, the level of Ba / Bb is determined, and the eye is assessed as having therapeutically relevant ocular complement activity (e.g., Ba > 20 ng / mL, or Bb > 150 ng / mL) or not via an appropriately validated ELISA or other protein quantification assay. Only patients with one eye meeting general clinical trial criteria, baseline GA area of between 2.5 mm2to 17.5 mm2, and therapeutically relevant ocular complement activity (e.g., AH Ba > 20 ng / mL, AH Bb >150 ng / mL)) are enrolled in the clinical trial. Enrolled patients are randomized to one of three cohorts, Cohort Treatment q!2w, CohortWSGR Docket No. 66711-706.601Treatment ql6w, and Cohort Sham. Each study eye in Cohort Treatment ql2w and Cohort Treatment ql6w receives treatment with a 100 uL intravitreal injection of multivalent C5 binding molecule in an appropriate formulation using a syringe with 30G needle (e.g., 2 mg based on aptamer only sequence ofMOL ID: 001, formulated in Water for Injection with 0.2 mg dibasic sodium phosphate heptahydrate, 0.025 mg monobasic sodium phosphate monohydrate, and 0.8 mg sodium chloride and a pH of approximately 7.3) once every 12 weeks (ql2w) and 16 weeks (ql6w) respectively. Cohort Sham receives no treatment and a capped syringe is pressed to the eye to simulate an injection in half of eyes on a ql2w schedule and in the other half on a ql6w schedule. After 48 weeks, patients are then assessed for area of Geographic Atrophy. The mean change (and standard deviation) in area of Geographic Atrophy from baseline to 48 weeks is calculated for each Cohort, and the means are compared using appropriate statistical tests for superiority. The surprising result is observed wherein the mean rate of Geographic Atrophy growth at 48 weeks is statistically significantly and at least 40% slower in at least one of the Cohort Treatment compared to Cohort Sham.Example 12: Classical pathway complement inhibition in ex vivo human serum hemolysis assay

[0191] Bivalent C5-binding molecules, SEQ ID NOS: 10-12 (as described in Table 2), along with avacincaptad pegol and pegcetacoplan, were evaluated for their ability to inhibit classical complement pathway activity. The classical pathway and complement hemolytic 50 (CH50) were determined using the human serum & sheep erythrocyte (sRBC) method described in Example 5. Stock solutions of the bivalent C5-binding molecules (SEQ ID NOS: 10-12), avacincaptad pegol, and pegcetacoplan were prepared in gelatin veronal buffer (GVB), and further diluted such that the final concentration of test article in the assay well containing a total of 200 pL (10 pL of test article in GVB, 90 pL human serum, and 100 pL sRBCs) ranged from 0.5 nMto 50 nM. FIG.7 depicts the results of this study. FIG. 7 demonstrates that the “linked’ spacing of bivalent C5 -binding molecules affects potency of inhibition. FIG. 7 shows that bivalent C5-binding molecules having longer linkers (SEQ ID NOS: 10 and 11, with ~5 & ~3 kDaPEG spacers via Sp 18 units, respectively) enable higher potency vs. a bivalent C5 -binding molecule having a shorter linker (SEQ ID NO: 12). The data presented herein demonstrate that spacer length can play a role in improving potency of inhibition compared to bivalent C5-binding molecules with shorter linkers and compared to monovalent anti-C5 aptamer molecules.Example 13: Affinity of bivalent C5-binding molecules vs. avacincaptad pegol

[0192] Biotinylated human complement C5 protein (C05-H82E9-200 pg) was purchased from Acros Biosystems as a lyophilized powder and solubilized in 500 pL of water. The final bufferWSGR Docket No. 66711-706.601after solubilization was PBS (pH 7.4) with 10 % Trehalose. The test articles, avacincaptad pegol & bivalent C5 -binding molecules (SEQ ID NOS: 10-12 as describedin Table 2) were solubilized in PBS that had been prepared in DEPC-treated water to the concentrations indicated in Table 1.Immediately before use, the test articles were heated to 95 °C for 5 minutes and allowed to cool to room temperature prior to experimental setup.

[0193] Eight concentrations, ranging from 0.229 nMto 500 nM (3-fold dilutions), were used for each test article. The final buffer composition was PBS (pH 7.4) supplemented with 1 mM MgC12 in DEPC-treated water.

[0194] SPR measurements were taken using a Biacore 8K instrument with SAHC200M -Streptavidin derivatized HC poly carboxylate (SAHC) from Xantec, with biotinylated human C5 immobilized usingbindingbuffer of lx PBS, supplemented with 1 mMMgC12 in DEPC treated water. Assay conditions were single cycle kinetics with eight concentrations, association time of 204 s and dissociation time of 600 s, flowrate of 25 pL / min and temperature of 25 °C.

[0195] The results of this experiment are provided in Table 4 below and demonstrate that bivalent anti-C5 aptamer molecules show superior affinity for C5 compared to a monovalent anti-C5 aptamer.Table 4. Affinity parameters of bivalent C5-binding moleculesWSGR Docket No. 66711-706.601Example 14: Determination of hydrodynamic radius of bivalent C5 binding aptamers by dynamic light scattering

[0196] SEQ ID NOS: 10-12 (as described in Table 2), along with avacincaptad pegol and pegcetacoplan, were solubilized in PBS prepared with DEPC-treated water. 100 pL aliquots of were prepared at 0.25 mg / mL to 1 mg / mL. The aliquots were transferred into prepared 0.1 pm Ultra-free centrifugal filter units and centrifuged for 1 minute at max speed in a tabletop microfuge at room temperature. The filtered samples were transferred to the cuvette and read using Zetasizer Nano S, Malvern instruments (ZEN1600), using a quartz cuvette ZEN 2112. For each sample replicate, the system measures the sample three times. Two replicates were measured for each dose listed. The mean main peak for each compound is listed in Table 5 below. These results indicate that the bivalent anti-C5 aptamers have significantly increased hydrodynamic radius compared to FDA-approved intravitreally administered anti-complement molecules, including a monovalent anti-C5 aptamer. Hydrodynamic radius has been shown to be linearly correlated with intravitreal half-life. Thus, the bivalent anti-C5 aptamers tested here are expected to have a longer intravitreal half-life as compared to monovalent anti-C5 aptamers.Table 5. Hydrodynamic diameter for main peak by dynamic light scatteringExample 15. Synthesis of SEQ ID NO:1Q at 150 mg scale

[0197] SEQ ID NO: 10 was synthesized according to the process depicted in FIG. 4A. The aptamer sequence was synthesized using industry standard solid phase protocols following the standard phosphoramidite chemistry procedures developed by Carruthers in 1981. The solid phase phosphoramidite procedure involves a cycle of detritylation, coupling, capping, and oxidation to add one nucleotide at a time.

[0198] The Aptamer- Azide (AA) sequence was constructed on a solid support from the 3’ to 5’. When the last nucleotide was added to the growing chain, short pegylated units (also referred to as Sp 18) were added employing the same phosphoramidite chemistry . The individual PEG units contain 6 ethylene glycol units (molecular weight 784.9 Da, formula weight 344.3 Da).WSGR Docket No. 66711-706.601Consecutive additions were made to give a PEG spacer with an approximate molecular weight of 4.5 kDa). The final coupling introduces a C6 alkyl amine using phosphoramidite chemistry. At this point the aptamer-PEG-C6 amine sequence was cleaved from the resin using standard cleavage and deprotection conditions and purified using preparative HPLC. The free amine of the sequence was conjugated with an NHS ester C4 azide to give the Aptamer Azide sequence.

[0199] The Aptamer-DBCO (AD) sequence was initially synthesized in the same way as the Aptamer Azide sequence using solid phase phosphoramidite chemistry and thirteen short PEG units to generate the desired aptamer sequence with PEG spacer. The sequence was extended to introduce a branched C4 amino and a protected alcohol to support further addition using solid phase chemistry of a 5’-DBCON-TEG unit via phosphoramidite chemistry (molecular weight 708.9 Da, formula weight 344.3 Da). The sequence then underwent standard cleave and deprotection and HPLC purification. The AA and AD sequences were then combined in equimolar amounts in a copper-free Strain-Promoted Alkyne- Azide Cycloaddition (SPAAC). The Amine Functionalized Bis-Aptamer (AFBA) product was purified via HPLC. Identity of the AFBA was confirmed by Electrospray Ionization Mass Spectroscopy (ESI -MS), by comparing the observed molecular weight to the predicted theoretical molecular weight (36,130 g / mol). In one synthesis of SEQ ID NOTO, the observed molecular weight of AFBA product was 36,133 g / mol).

[0200] The final construct was synth esizedby utilizing an NHS ester amine coupling to form a stable amide bond between the N-hydroxy succinimide (NHS) ester of a branched 40 kDa PEG and the primary amine of the AFBA product typically at physiological pH (7 -9). The final product was purified by HPLC, desalted, concentrated and filtered before lyophilization to isolate the product as an amorphous solid. Further testing for endotoxin level (EU / mg) was performed.

[0201] In one synthesis, the final purity of SEQ ID: 10 was 91.5%, shown in FIG. 8, with an endotoxin level of <0.003 EU / mg.Example 16. Assessment of inhibition of classical complement activation by vitreous fluid containing monovalent and multivalent C5 binding aptamers

[0202] In this example, rabbit vitreous humor containing avacincaptad pegol or SEQ ID NO: 10 (from Example 15) was assessed for ability to inhibit classical complement-dependent hemolysis in human serum. These assays were performed to assess if the anti-C5 molecules retain activity following intravitreal administration to a rabbit eye. For the classic complementdependent hemolysis assay, 1 mL of antibody -sensitized sheep red blood cells (sRBCs) (Colorado Serum Company) in 0.1 M sucrose in gelatin veronal buffer (GVB) (ComplementWSGR Docket No. 66711-706.601Technology, Inc.) was washed twice with 10 mLGVB and spun down at 1000 g for 10 minutes. The supernatant was removed with a transfer pipette and the remaining sRBCs were reconstituted in 10 mL of GVB. The sRBC concentration was then calculated by using a hemocytometer.

[0203] To determine the CH50 of normal human serum (NHS), 100 pL of NHS at increasing concentrations from 0.2% to 2% (diluted in GVB + 0.3 mM CaCl2+ 1 mM MgCl2) was mixed with 100 pL of GVB-washed sRBCs at a concentration of 2 x 108sRBCs / mL in a single well of a 96-well plate. The total volume of each well was 200 pL and samples were tested in duplicate. The final concentration of NHS, CaCl2, and MgCl2in the assay ranged from 0.1% to 1% NHS in GVB, 0.15 mM CaCl2, and 0.5 mM MgCl2. The NHS and sRBC mixture were incubated at 37°C for 30 minutes and the reaction was quenched by adding 20 pL of 500 mM EDTA. The 96-well plate was spun at 600 g for approximately 3 minutes. 100 pL of the supernatant was then diluted with 100 pL of GVB and the absorbance is read at 405 nm. The results were plotted on GraphPad Prism 8.0 (Boston, MA, USA) to determine the concentration of human serum required to achieve 50% lysis of sRBC.

[0204] The IC50 of avancincaptad pegol and SEQ ID NO: 10 were assessed by serial dilution curve such that between 400 nMto 0.1 nM of each was present in the final 200 uL assay well, and each well contained 5 uL of rabbit vitreous and either avacincaptad pegol or SEQ ID NOTO, 5 uL of GVB, 90 uL of NHS at 1.111 x CH50 value obtained previously; the resulting 100 uL was then mixed with 100 pL of GVB-washed sRBCs ata concentration of2x 108sRBCs / mL in a single well of a 96-well plate. As mentioned above, the total volume of each well was 200 pL and samples were tested in duplicate. The NHS and sRBC mixture were incubated at 37°C for 30 minutes and the reaction was quenched by adding 20 pL of 500 mM EDTA. The 96-well plate was spun at 600 g for 3 minutes. 100 pL of the supernatant was then diluted with 100 pL of GVB and the absorbance is read at 405 nm. The results are plotted on GraphPad Prism 8.0 (Boston, MA, USA) to calculate the IC50 of the complement inhibiting compound. Results are shown in Table 6 below. The IC50 was calculated to be 5.27 nMfor SEQ ID NO: 10 compared to 13.58 nM for avacincaptad pegol, demonstrating significant improvement in classical pathway inhibition with multivalent C5 binding aptamer compared to a. monovalent anti-C5 aptamer.WSGR Docket No. 66711-706.601Table 6:Example 17. Measurement of viscosity and calculation of injection breakforce with a syringe and 30G needle

[0205] In this example, a stock solution was prepared along with two subsequent dilutions. The stock solution (Sample A) was made with 25.6 g of SEQ ID NO: 10 in powder form dissolved in 313 pL (0.313 m ) of IX RNase-free PBS, resulting in a concentration equivalent to 4 mg per 50 pL. Sample B was prepared with 150 pL of Sample A with 150 pL of IX PBS, yielding a solution equivalent to 2 mg per 50 pL. Sample C was prepared by diluting 150 pL of Sample B with 150 pL of IX PBS, resulting in a concentration equivalent to 1 mg per 50 pL.

[0206] The viscosity measurement was done on the m-VROC II® using the B05 chip (depth ~ 50 pm, Pmax ~ 42 kPa) at 20°C. Measurements were conducted at different shear rates using the level generator measurement protocol on the m-VROC II. The cleaning solvent order on the CCS included IX PBS + 1% Aquet as the primary, DI water as the secondary, and acetone as the enhancer. The results are shown in table 7 below.

[0207] In summary, SEQ ID NOTO at 4 mg per 50 uL of PBS demonstrated a shear rateindependent viscosity of approximately 15 cP. Using the viscosity and modified Hagen-Pouiseuille equation for resistive force:Zviscous resistive force = nlnQ —nWSGR Docket No. 66711-706.601

[0208] Along with the barrel and syringe bore diameter of an Ompi 0.5 mL syringe with 30G needle, the breakforce is calculated to be comparable to that of 0.3 mgMacugen (pegaptanib) in 90 uL in its FDA approved prefilled syringe with recommended 30G needle.Table 7:WSGR Docket No. 66711-706.601Example 18: Demonstrating 40% slowing of rate of Geographic Atrophy growth compared to no (sham) treatment

[0209] To demonstrate superior efficacy of multivalent C5 binding molecules and formulations thereof, patients diagnosed with Geographic Atrophy are screened for anatomic and demographic inclusion and exclusion criteria typical for a Phase 2 / 3 clinical trial (e.g., area of Geographic Atrophy >2.5 mm2 and <17.5 mm2, non -concomitant neovascular AMD in the study eye, no immune reactions to excipients in compositions, no ocular contradictions, etc.). A paracentesis is performed to collect aqueous humor, and the level of Ba / Bb is determined and the eye is assessed as having therapeutically relevant ocular complement activity (e.g., Ba > 20 ng / mL, or Bb > 150 ng / mL) or not via an appropriately validated ELISA or other protein quantification assay. Only patients with one eye meeting general clinical trial criteria, baseline GA area of between 2.5 mm2 to 17.5 mm2, and therapeutically relevant ocular complement activity (e.g., AH Ba > 20 ng / mL, AH Bb >150 ng / mL)) are enrolled in the clinical trial. Enrolled patients are randomized to one of four cohorts, Cohort Treatment q8w, Cohort Treatment ql2w, Cohort Treatment ql6w, and Cohort Sham. Each study eye in Cohort Treatment q8w, Cohort Treatment ql2w, and Cohort Treatment ql6w receives treatment with a 50 uL intravitreal injection of multivalent C5 binding molecule in an appropriate formulation using a syringe with 30G needle e.g., 4 mg of MOL ID: 001, formulated in Water for Injection with 0.2 mg dibasic sodium phosphate heptahydrate, 0.025 mg monobasic sodium phosphate monohydrate, and 0.8 mg sodium chloride and a pH of approximately 7.3) once every 8 weeks (q8w), every 12 weeks (ql2w), and 16 weeks (ql6w) respectively. Cohort Sham receives no treatment and a capped syringe is pressed to the eye to simulate an injection in half of eyes on a ql2w schedule and in the other half on a ql6w schedule. During and after a period of 48 weeks, patients are then assessed for area of Geographic Atrophy. The mean change (and standardWSGR Docket No. 66711-706.601deviation) in area of Geographic Atrophy from baseline to 48 weeks is calculated for each Cohort, and the means are compared using appropriate statistical tests for superiority. The surprising result is observed wherein the mean rate of Geographic Atrophy growth at 48 weeks is statistically significantly and at least 40% slower in at least one of the Cohort Treatment compared to Cohort Sham.

[0210] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. WSGR Docket No. 66711-706.6012.PCT / IB2025 / 000622 CLAIMS WHAT IS CLAIMED IS:

1. A multivalent C5 binding molecule comprising:4.(a) a first aptamer moiety that selectively binds to a complement component C5; and (b) a second, a third, a fourth, a fifth, a sixth, a seventh, and / or a eighth aptamer moiety that selectively binds to the complement component C5,5.wherein the multivalent C5 binding molecule inhibits a biological function of a complement system.

2. The multivalent C5 binding molecule of claim 1, wherein the multivalent C5 binding molecule further comprises an intravitreal half-life extending moiety.

3. The multivalent C5 binding molecule of claim 1 or 2, wherein the first aptamer moiety blocks generation of C5a and / or C5b.

4. The multivalent C 5 binding molecule of any one of claims 1-3, wherein the first aptamer moiety blocks activity of C5a and / or C5b.

5. The multivalent C5 binding molecule of any one of claim 1-4, wherein each aptamer moiety of the multivalent C5 binding molecule comprises no unmodified RNA or unmodified DNA nucleotide.

6. The multivalent C5 binding molecule of any one of claims 1 -5, wherein each aptamer moiety of the multivalent C 5 binding molecule comprises or consists of a nucleotide sequence that is at least about 80% identical to a nucleotide sequence of SEQ ID NOs: 1-9.

7. The multivalent C5 binding molecule of any one of claims 1-6, wherein each aptamer moiety of the multivalent C5 binding molecule comprises or consists of a nucleotide sequence that is at least about 80% identical to a nucleotide sequence of SEQ ID NO: 2-9.

8. The multivalent C5 binding molecule of any one of claims 1 -7, wherein the first aptamer moiety is avacincaptad.

9. The multivalent C5 binding molecule of any one of claims 1-8, wherein the multivalent C5 binding molecule binds to C5 with a Kdof less than 20 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 500 pM, less than 100 pM, or less than 50 pM.

10. The multivalent C5 binding molecule of any one of claims 1 -9, wherein the multivalent C5 binding molecule binds simultaneously to multiple C5 molecules.

11. The multivalent C5 binding molecule of any one of claims 1-10, wherein the multivalent C5 binding molecule inhibits the biological function of the complement system with an IC50 of less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, or less than 500 pM as measured by a complement dependent assay.WSGR Docket No. 66711-706.60112. The multivalent C5 binding molecule of any one of claims 1-11, wherein the multivalent C5 binding molecule provides a greater inhibition of complement function than a monovalent C5 binding molecule or an avacincaptad pegol.

13. The multivalent C5 binding molecule of claim 12, wherein the multivalent C5 binding molecule inhibits the complement function by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70, at least about 80%, at least about 90%, at least about 100%, or greater than that of the monovalent C5 binding molecule or the avacincaptad pegol.

14. The multivalent C5 binding molecule of any one of claims 1-13, wherein the multivalent C5 binding molecule remains at least about 70%, at least about 80%, or at least about 90% intact after exposure to serum or vitreous for about 48 hours, about 72 hours, or about 96 hours in vitro.

15. The multivalent C5 binding molecule of any one of claims 1 -14, wherein the multivalent C5 binding molecule inhibits at least about 90% of C5 mediated complement activity in human ocular fluid for at least about 90 days, at least about 100 days, at least about 110 days, or at least about 120 days after a single intravitreal injection.

16. The multivalent C5 binding molecule of any one of claims 2-14, wherein each aptamer moiety of the multivalent C 5 binding molecule is covalently attached to the intravitreal half-life extending moiety.

17. The multivalent C5 binding molecule of any one of claims 1 -15, wherein the first aptamer moiety and the second, the third, the fourth, the fifth, the sixth, the seventh, and / or the eighth aptamer moiety are covalently attached via a polyethylene glycol (PEG) linker.

18. The multivalent C5 binding molecule of any one of claims 1 -15, wherein the first aptamer moiety and the second, the third, the fourth, the fifth, the sixth, the seventh and / or the eighth aptamer moiety are attached via a molecular linker.

19. The multivalent C5 binding molecule of any one of claims 2-16, wherein the intravitreal half-life extending moiety comprises a PEG polymer with a molecular weight of about 5 kDa, about 10 kDa, about 20 kDa, about 40 kDa, or about 60 kDa.

20. The multivalent C5 binding molecule of claim 19, wherein the intravitreal half-life extending moiety has hydrodynamic radius that is from about 6 nm to aboutlO nm.

21. The multivalent C5 binding molecule of any one of claims 1 -20, wherein the multivalent C5 binding molecule has an in vivo intravitreal half-life that is at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, or at least about 8 days.WSGR Docket No. 66711-706.60122. The multivalent C5 binding molecule of any one of claims 1 -20, wherein the multivalent C5 binding molecule is a bivalent C5 binding molecule, and wherein the bivalent C5 binding molecule exhibits at least about 90% inhibition of classical complement pathway that persists for at least about 60 days, at least about 90 days, at least about 120 days, or more after a single intravitreal injection.

23. The multivalent C5 binding molecule of claim 21 or 22, wherein the multivalent C5 binding molecule has the in vivo intravitreal half-life or a serum half-life that is greater than that of a control molecule.

24. The multivalent C5 binding molecule of claim 23, wherein the in vivo intravitreal halflife or the serum half-life is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than that of the monovalent C5 binding molecule or the avacincaptad pegol.

25. The multivalent C5 binding molecule of any one of claims 1-24, wherein the multivalent C5 binding molecule comprises or consists of a nucleotide sequence that is at least about 80% identical to a nucleotide sequence of SEQ ID NOs: 10-12.

26. The multivalent C5 binding molecule of any one of claims 1 -25, wherein the multivalent C5 binding molecule comprises a linking moiety of about 1 kDa to about 10 kDa which links the first aptamer moiety and the second, third, fourth, fifth, sixth, seventh, or eighth aptamer moiety .

27. An aptamer comprising a nucleotide sequence that is at least about 80% identical to a nucleotide sequence of SEQ ID NOs: 2-9.

28. A pharmaceutical formulation comprising the multivalent C5 binding molecule of any one of claims 1-26, or the aptamer of claim 27, and a pharmaceutically acceptable excipient, carrier, and / or diluent.

29. The pharmaceutical formulation of claim 28, wherein the pharmaceutical formulation is for an intravitreal administration.

30. The pharmaceutical formulation of claim 28 or 29, wherein the pharmaceutical formulation is administer via a 32 gauge (G), a 31G, a 30G, or a 28G needle.

31. The pharmaceutical formulation of any one of claims 27-30, wherein the pharmaceutical formulation is administered at a concentration of about 1 mg / 100 pL, about2 mg / 100 pL, about 3 mg / 100 pL, or about 4 mg / 100 pL.

32. The pharmaceutical formulation of any one of claims 27-31, wherein the pharmaceutical formulation is administered with a break force of less than 12 N.

33. The pharmaceutical formulation of any one of claims 28-32, wherein the pharmaceutical formulation is formulated at about 0.1 mg, about 0.25 mg, about 0.5 mg, about 1 mg, about 2WSGR Docket No. 66711-706.60138.mg, about 3 mg, about 4 mg, about 8 mg per 100 uL injection volume, calculated as an acid form of the first aptamer moiety of the multivalent C5 binding molecule of any one of claims 1 to 25.

34. A method of inhibiting complement activity in a subject in need thereof, the method comprising:40.administering to the subject in need thereof a therapeutically effective amount of the multivalent C5 binding molecule of any one of claims 1 -26, the aptamer of claim 27, or the pharmaceutical formulation of any one of claims 28-33.

35. A method of treating a subject in need thereof, the method comprising:42.administering to the subject in need thereof a therapeutically effective amount of the multivalent C5 binding molecule of any one of claims 1 -26, the aptamer of claim 27, or the pharmaceutical formulation of any one of claims 28-33.

36. The method of claim 34 or 35, wherein the therapeutically effective amount of the multivalent C5 binding molecule comprises a dose and frequency that does not exceed an average mass of PEG present in the multivalent C5 binding molecule or the pharmaceutical formulation.

37. The method of any one of claims 34-36, wherein the therapeutically effective amount of the multivalent C5 binding molecule comprises a dose and frequency that does not exceed about 5 mg per eye per month, about 4 mg per eye per month, about 3 mg per eye per month, or 2 mg per eye per month.

38. The method of any one of claims 34-37, wherein the subject in need thereof has, is suspected of having, or is at risk of having a retinal disease or disorder, an ocular disease or disorder, or one or more symptoms associated with the retinal disease or disorder or the ocular disease or disorder.

39. The method of claim 38, wherein the retinal disease or disorder or the ocular disease or disorder is selected from the group consisting of : wet age-related macular degeneration, dry age-related macular degeneration, geographic atrophy, diabetic retinopathy, diabetic macular edema, proliferative diabetic retinopathy, retinal vein occlusion, Stargardt disease, retinitis pigmentosa, retinal detachment, and non-arteritic anterior ischemic optic neuropathy (NAION).

40. The method of any one of claims 34-39, wherein the administering comprises an intravitreal administration.

41. The method of any one of claims 34-40, wherein the therapeutically effective amount of the multivalent C5 binding molecule, the aptamer, or the pharmaceutical formulation is administered at every 4 weeks (q4w), every 8 weeks (q8w), every 12 weeks (ql2w), every 16WSGR Docket No. 66711-706.60149.weeks (ql6w), every 20 weeks (q20w), every 24 weeks (q24w), every 36 weeks (q36w), or every 48 weeks (q48w) intervals.

42. A method of reducing a growth rate of Geographic Atrophy (GA) lesion or area, the method comprising:51.(a) identifying or having identified a subject as having or at risk of having GA;52.(b) identifying or having identified the subject not having hypoactive (ocular) complement pathway activity; and53.(c) administering to the subject, or recommending that the subject be treated with, a C5 inhibitor;54.(d) optionally, measuring the Geographic Atrophy lesion or area at least two different time points; and55.(e) optionally, calculating the growth rate of the Geographic Atrophy lesion or area growth between the at least two different time points.

43. The method of claim 42, wherein the identifying of having identified of (b) comprises measuring the complement pathway activity by proteomic assay.

44. The method of claim 42 or claim 43, wherein the hypoactive (ocular) complement pathway activity comprises having a Ba level that is about 10 ng / mL or less.

45. The method of any one of claims 42-44, wherein the hypoactive (ocular) complement pathway activity comprises having a Bb level that is about 40 ng / mL or less.

46. The method of claim 44 or 45, wherein the Ba level or the Bb level is measured in a sample collected from the subject.

47. The method of claim 46, wherein the sample comprises a sample from or processed from serum, aqueous humor, or vitreous humor.

48. The method of any one of claims 42-47, wherein the multivalent C5 binding molecule of any one of claims 1 -26, the aptamer of claim 27, or the pharmaceutical formulation of any one of claims 28-33.

49. The method of any one of claims 42-48, wherein the administration to the subject the C5 inhibitor results in an average reduction in the growth rate of the GA lesion or area by at least about 30% as compared to that of a patient received no treatment.

50. The method of any one of claims 42-49, wherein the administration to the subject the C5 inhibitor results in significantly increase in visual function outcomes, when compared to that of a patient received no treatment.

51. The method of claim 50, wherein the visual functional outcomes are measured by Best Corrected Visual Acuity (BCVA) or Low Luminance Visual Acuity (LLVA).WSGR Docket No. 66711-706.60152. The method of any one of claims 42-51, wherein the C5 inhibitor is administered, or is recommended to administer, every 8 weeks (q8w), every 12 weeks (ql2w), every 16 weeks (ql6w), every 20 weeks (q20w), every 24 weeks (q24w), every 36 weeks (q36w), or every 48 weeks (q48w) intervals.

53. A method of reducing the growth rate of Geographic Atrophy area or lesion, the method comprising:67.(a) identifying or having identified a subject as having Geographic Atrophy;68.(b) identifying or having identified a subject as having pathologic complement pathway activity relevant to C5 ;69.(c) administering to the subject, or recommending that the subject be treated with, a C5 inhibitor; and70.(d) optionally, measuring the Geographic Atrophy area or lesion at least two different time points and calculating the rate of Geographic Atrophy between time points.

54. The method of claim 53, wherein the having pathologic complement pathway activity relevant to C5 comprise having at least one of:72.(a) above a threshold concentration of at least one ocular complement protein;73.(b) elevated retina flavoprotein fluorescence intensity; or74.(c) presence of at least one genetic polymorphism in a complement pathway protein.

55. The method of claim 53 or 54, wherein the having pathologic complement pathway activity relevant to C5 comprises measuring complement pathway activity via a proteomic assay.

56. The method of claim 54 or 55, wherein the complement pathway protein is measured in a sample collected from the subject.

57. The method of claim 56, wherein the sample comprises a sample from or processed from serum, aqueous humor, or vitreous humor.

58. The method of any one of claims 53-57, wherein the having pathologic complement pathway activity relevant to C5 comprises having a Ba level that is at least 10 ng / mL, at least 15 ng / mL, at least 20 ng / mL, at least 25 ng / mL, or at least 30 ng / mL.

59. The method of any one of claims 53-58, wherein the having pathologic complement pathway activity relevant to C5 comprises having a Bb level that is at least 40 ng / mL, at least 80 ng / mL, at least 100 ng / mL, at least 120 ng / mL, or at least 150 ng / mL.

60. The method of claim 53-59, wherein the having pathologic complement pathway activity relevant to C5 comprises having a C5a level that is at least 20 pg / mL, at least 25 pg / mL, at least 30 pg / mL, at least 35 pg / mL, or at least 40 pg / mL.WSGR Docket No. 66711-706.60161. The method of any one of claims 53-60, wherein the having elevated retina flavoprotein fluorescence intensity comprises having an intensity value that is at least about 1-fold, at least about 1.25-fold at least about 1.5-fold, at least about 1.75-fold, or at least about 2-fold greater than that of an age-matched healthy patient group.

62. The method of any one of claims 53-61, wherein the having pathologic complement activity relevant to C5 comprises having a genetic test.

63. The method of claim 62, wherein the genetic test comprises testing for a mutation of the Tyr at amino position 402 of the human complement factor H (CFH) protein.

64. The method of claim 63, wherein the mutation is present in both alleles.

65. The method of claims 62, wherein the genetic test comprises testing for a mutation in CD46, CD55, or CD59.

66. The method of claim 65, wherein the mutation in the CD59 comprises having c.146delA (p.Asp49Valfs*31), c,123delC (p.Val42Serfs*38), c.266G>A (p.Cys89Tyr), c.238A>G (p.Arg80Gly), and / or c,146A>T (p.Asp49Val) mutation in a RPE cell.

67. The method of claim 65 or 66, wherein the genetic test comprises measuring CD59 expression level in the RPE cell of the subject.

68. The method of claim 67, wherein the CD59 expression level of the subjectis reduced by at least about 20%, at least about 30%, at least about 40%, or at least about 50% as compared to that of a healthy subject.

69. The method of any one of claims 53-68, wherein the C5 inhibitor comprises the multivalent C5 binding molecule of any one of claims 1 -26, the aptamer of claim 27, or the pharmaceutical formulation of any one of claims 28-33.

70. The method of any one of claims 53-69, wherein the method results in an average reduction in the growth rate of the Geographic Atrophy area or lesion by at least about 40% or more after 1 year of treatment, when compared to no treatment.

71. The method of any one of claims 53-70, wherein the method results in a significantly increase in visual function outcomes when compared to that of a patient received no treatment.

72. The method of claim 71, wherein the visual function outcomes are measured by Best Corrected Visual Acuity (BCVA) or Low Luminance Visual Acuity (LLVA).

73. The method of any one of claims 53-72, wherein the method results in at least about 50% likelihood that an individual eye has at least about 40% slower growth rate of the Geographic Atrophy area or lesion as compared to that of an individual eye prior to the administering.

74. The method of any of claim 53-73 wherein the C5 inhibitor is administered, or is recommended for administration, beginning at treatment initiation, at q8w, ql2w, ql6w, or q24w intervals.

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