Multivalent protein conjugates

Abstract

The present invention relates to multivalent peptide conjugates, and pharmaceutical compositions, methods of preparation, and methods of use thereof.

Description

Attorney Docket No.: 052566-511001 WOMULTIVALENT PROTEIN CONJUGATESCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 722,904, filed on November 20, 2024; U.S. Provisional Application No. 63 / 800,041, filed on May 5, 2025; U.S. Provisional Application No. 63 / 818,964, filed on June 6, 2025; U.S. Provisional Application No. 63 / 874,443, filed on September 2, 2025; and U.S. Provisional Application No. 63 / 899,734, filed on October 15, 2025, each of which is incorporated herein by reference in its entirety for all purposes.SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 18, 2025, is named 052566-511001 WO_ST26. xml and is 39.4 kilobytes in size.BACKGROUND OF THE INVENTION

[0003] The use of biopolymers to modify the properties of biologically active agents is a recurring theme across a wide range of medical and biological applications. A variety of chemical linkers can be used to attach bioactive peptides or proteins to biopolymers to modify the pharmacological properties of the resulting conjugate for use as a drug that can provide optimal treatment of specific diseases. Peptide-polymer conjugate comprising multiple copies of one or more species of peptide conjugated to a single biopolymer chain have been employed to impart specific improvements to the pharmacological properties of the peptides, including: (1) higher binding affinity to the biological target, (2) slower diffusivity through a target tissue, and (3) inhibition of proteases that could deactivate the biological activity of the peptides or proteins.

[0004] These improved pharmacological properties of peptide-polymer conjugates are particularly useful for the delivery of potent drugs that are be delivered directly into the diseased tissue. The dose delivered directly into the tissue can be lower than would be required to achieve the same therapeutic effect after systemic administration because the drug has been administered locally to the target tissue. It is also possible to administer to drugs to tissues that otherwise have poor transport properties from the blood. Specific examples ofAttorney Docket No.: 052566-511001 WO tissues where direct drug administration is common include the posterior eye chamber via intravitreal injection and articular joints via intra-articular injection.

[0005] However, local tissue administration requires a professional to safely provide the required injection, which makes them more burdensome and costly to administer compared to systemic administration. When the peptide drug is administered as part of a peptide-polymer conjugate, it is possible to substantially reduce the frequency of drug administration, thereby reducing the burden on the patient to receive effective treatment. Furthermore, a reduction in the number of local injections reduces the risk of local tissue injury or adverse effects to the injection. Finally, the need for less frequent administrations can reduce the amount of time that the drug concentration in the target tissue is below the therapeutic concentration, thereby improving the overall efficacy of the drug. Based on these advantages, there is a strong motivation to develop protein-polymer drug products for a variety of diseases.

[0006] Many humanized monoclonal antibodies display poor biophysical properties, such as low stability and a propensity to aggregate. These unfavorable tendencies can be even more pronounced for humanized antibody fragments, which often require a considerable degree of modification.

[0007] To appropriately formulate a peptide-polymer conjugate as a drug product, it is necessary to achieve sufficiently high drug concentrations to enable appropriate dosing in the patient. It is also necessary to prepare purified peptide-polymer conjugates that exhibit high bioactivity and shelf-stability, for example, by being able to remain in solution for up to two years from the date of manufacture to the date of clinical use. Interactions between the peptide-polymer conjugates can negatively impact the ability to complete any of these drugenabling properties.

[0008] The degree of humanness of the peptides and the secondary structure of the peptide linkers used in the attachment to the polymer can have a substantial impact on the pharmacological properties of the conjugates, intra-conjugate interactions, as well as conjugate-to-conjugate interactions. Therefore, there is a need to develop humanized peptide-polymer conjugates with specific peptide linkers that will enable them to achieve the preferred pharmacological properties for a given disease as well as to be successfully formulated into a drug product. The present invention meets this and other needs.Attorney Docket No.: 052566-511001 WOBRIEF SUMMARY OF THE INVENTION

[0009] In some embodiments, the conjugate of the present invention is a conjugate that is a random polymer of Formula Illa:(X1-X2-Y-Z1)n-(Z2)p-(Z3)q(Illa), having a molecular weight of about 0.8 MDa; wherein each X1is a peptide having an anti-VEGF amino acid sequence comprising SEQ ID NO: 96; each X2is a peptide linker having an amino acid sequence comprisingAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21); each Y is an organic linker having the structure:each X1-X2-Y-Z1moiety has the structure:each Z2has the structure:each Z3independently has the structure:Attorney Docket No.: 052566-511001 WOeach Z3ais independently OH or Y'; each Y' has the structure:each R1and R2is ethyl or -(CH2)3-NMe2; subscript n is an integer of from 10 to 300 and less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer of from 1 to 15 and less than about 0.5% of the sum of subscripts n, p, and q; and subscript q is an integer of from 1000 to 3000.

[0010] In some embodiments, the pharmaceutical composition of the present invention comprises a conjugate as described herein and a pharmaceutically acceptable excipient.

[0011] In some embodiments, the method of the present invention is a method of treating an ocular disorder in a subject in need thereof, comprising administering to the subject a conjugate as described herein.

[0012] In some embodiments, the method of the present invention is a method of treating a disease or disorder in an articular joint in a subject in need thereof, comprising administering to the subject a conjugate as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 A-1B show purity of exemplary conjugate preparations. FIG. 1 A shows (left) a size exclusion chromatography (SEC) trace and (right) differential light scattering (DLS) traces of Conjugate 5 (“VLTR-559”) compared to the unconjugated antibody component (“Unconj VHH”). FIG. IB shows graph of the hydrodynamic radius (nm) obtained by DLS ofAttorney Docket No.: 052566-511001 WO the unconjugated single domain antibody (“sdAb only”) compared with Conjugate 5 (“VLTR-559”).

[0014] FIG. 2A shows anti-VEGF cell bioassays for Conjugate 1 and bevacizumab (left), and Conjugate 2 and bevacizumab (right). FIG. 2B shows anti-VEGF cell bioassays for Conjugate 6 (VLTR-559), aflibercept, and faricimab-svoa. FIG. 2C shows anti-VEGF cell bioassays for Conjugate 6 (VLTR-559), aflibercept (“Eylea”), faricimab-svoa (“Vabysmo”), and unconjugated single domain antibody (“sdAb only”) of Conjugate 6.

[0015] FIG. 3 A-3B show reduction of the neovascularization area in response to the laser- induced lesion formation in an in vivo mouse model: FIG. 3 A shows effects based on lesion area; FIG. 3B shows effects based on % neovascular inhibition with aflibercept or Conjugate 4 (VLTR-559) compared to vehicle.

[0016] FIG. 4A-4C show the intravitreal concentration of Conjugate 5 in rabbit eyes at various time points after administration via intravitreal injection (n=6 eyes per timpoint; R2=0.8864): FIG. 4A shows effects up to 50 days; FIG. 4B shows effects up to 77 days with individual data points; FIG. 4C shows effects up to 77 days.

[0017] FIG. 5 shows the stability of Conjugate 6 under physiological conditions was assessed over 180 days based on hydrodynamic radius (top) and association rate constant (Kon, bottom).

[0018] FIG. 6 shows the anti-VEGF activity (IC50 in nM) of Conjugate 5 obtained from rabbit eye at 0, 15, 29, 46, and 77 days after intravitreal injection in the in vivo study of Example 4.

[0019] FIG. 7A-7B show the vitreous cell and aqueous flare SPOTS scores from cynomolgus monkey eyes in Groups 1 and 2 in an in vivo tolerability study of Example 8: FIG. 7A: Group 1; FIG. 7B: Group 2.

[0020] FIG. 8A-8B show the intraocular pressure (IOP in mm Hg) from cynomolgus monkey eyes in Groups 1 and 2 in an in vivo tolerability study of Example 8: FIG. 8 A: Group 1; FIG. 8B: Group 2.Attorney Docket No.: 052566-511001 WODETAILED DESCRIPTION OF THE INVENTIONI. GENERAL

[0021] The present invention provides multivalent peptide-hyaluronic acid polymer conjugates, pharmaceutical compositions, and methods of the same. As described in Examples herein, anti-VEGF VHH SEQ ID NO: 96 was synthesized based on mutations from a literature anti-VEGF VHH (SEQ ID NO: 91). See, Example 1. SEQ ID NO: 96 exhibited improved anti-VEGF binding activity compared to similar VHH with only point mutation differences. See, Example 2. The anti-VEGF multivalent protein Conjugate 2 comprising SEQ ID NO: 102 exhibited better anti-VEGF cell activity compared to bevacizumab (FIG. 2A). A similar preparation Conjugate 6 exhibited similar properties (FIG. 2B-2C). In contrast, Conjugate 1 (SEQ ID NO: 101) performed poorly in the same cell bioassay relative to bevacizumab. Conjugate 6 demonstrated high in vitro stability with no or minimal change in hydrodynamic radius or VEGF binding affinity over 180 days (FIG. 5). In an in vivo mouse model, Conjugate 4 (SEQ ID NO: 102) demonstrated similar effects as afibercept in reduction of the neovascularization area in response to laser-induced lesion formation in the eye (FIG. 3A-3B). Conjugate 4 performed better than Conjugate 3 (SEQ ID NO: 101) in the in vivo mouse assay. Conjugate 5, a separate preparation from Conjugate 4 with the same sequence, demonstrated an in vivo half-life of at least 12.2 days in Dutch belted white rabbits after intravitreal administration (FIG. 4A-4C). Conjugate 5 further demonstrated no significant loss of anti-VEGF activity in rabbit vitreous humor up to 77 days after intravitreal administration in vivo (FIG. 6). Illustrative Conjugate 7 demonstrated low levels of ocular toxicity based on SPOTS scoring in an in vivo tolerability study in non-human primates. See, FIG. 7A-7B and 8A-8B.II. DEFINITIONS

[0022] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined.

[0023] “About” when referring to a value includes the stated value + / - 10% of the stated value. For example, about 50% includes a range of from 45% to 55%, while about 20 molarAttomey Docket No.: 052566-511001 WO equivalents includes a range of from 18 to 22 molar equivalents. Accordingly, when referring to a range, “about” refers to each of the stated values + / - 10% of the stated value of each end of the range. For instance, a ratio of from about 1 to about 3 (weight / weight) includes a range of from 0.9 to 3.3.

[0024] “Alkyl” is a linear or branched saturated monovalent or divalent hydrocarbon. For example, an alkyl group can have 1 to 10 carbon atoms (i.e., Ci-io alkyl) or 1 to 8 carbon atoms (i.e., Ci-s alkyl) or 1 to 6 carbon atoms (i.e., Ci-6 alkyl) or 1 to 4 carbon atoms (i.e., (Ci- 4 alkyl). Examples of alkyl groups include, but are not limited to, methyl (Me, -CEE), ethyl (Et, -CH2CH3), 1 -propyl ( / / -Pr, / / -propyl, -CH2CH2CH3), 2-propyl (z-Pr, z-propyl, -CH(CH3)2), 1 -butyl (zz-Bu, / / -butyl, -CH2CH2CH2CH3), 2-methyl-l -propyl (z-Bu, z-butyl, -CH2CH(CH3)2), 2-butyl (.s-Bu, .s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl ( / - Bu, / -butyl, -C(CH3)3), 1 -pentyl ( / / -pentyl, -CH2CH2CH2CH2CH3), 2-pentyl(-CH(CH3)CH2CH2CH3), 3 -pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3 -methyl- 1 -butyl (-CH2CH2CH(CH3)2), 2-methyl-l- butyl (-CH2CH(CH3)CH2CH3), 1 -hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3 -hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3 -methyl-3 -pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3 -pentyl (- CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (- CH(CH3)C(CH3)3, and octyl (-(CH2)7CH3).

[0025] “Cycloalkyl” refers to a single saturated or partially unsaturated all carbon ring having 3 to 20 annular carbon atoms (i.e., C3-20 cycloalkyl), for example from 3 to 12 annular atoms, for example from 3 to 10 annular atoms, or 3 to 8 annular atoms, or 3 to 6 annular atoms, or 3 to 5 annular atoms, or 3 to 4 annular atoms. The term “cycloalkyl” also includes multiple condensed, saturated and partially unsaturated all carbon ring systems (e.g., ring systems comprising 2, 3 or 4 carbocyclic rings). Accordingly, cycloalkyl includes multicyclic carbocycles such as a bicyclic carbocycles (e.g., bicyclic carbocycles having about 6 to 12 annular carbon atoms such as bicyclo[3.1.0]hexane and bicyclo[2.1.1]hexane), and polycyclic carbocycles (e.g. tricyclic and tetracyclic carbocycles with up to about 20 annular carbon atoms). The rings of a multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, 1-Attorney Docket No.: 052566-511001 WO cyclopent- 1-enyl, l-cyclopent-2-enyl, 1 -cyclopent-3 -enyl, cyclohexyl, 1 -cyclohex- 1-enyl, 1- cyclohex-2-enyl and 1 -cyclohex-3 -enyl.

[0026] “Organic linker” as used herein refers to a chemical moiety that directly or indirectly covalently links the peptide to the polymer. Organic linkers useful in the present invention can be about 100 Da to 500 Da. The types of organic linkers of the present invention include, but are not limited to, imides, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonate and thioureas. One of skill in the art will appreciate that other types of organic linkers are useful in the present invention.

[0027] “ Thiol” refers to the -SH functional group.

[0028] “ Thiol reactive group” refers to a group capable of reacting with a thiol to form a covalent bond to the sulfur atom. Representative thiol reactive groups include, but are not limited to, thiol, TNB-thiol, haloacetyl, aziridine, acryloyl, vinylsulfone, APN (3- arylpropiolonitrile), maleimide and pyridyl disulfide. Reaction of the thiol reactive group with a thiol can form a disulfide or a thioether.

[0029] “Coupling agent” as used herein refers to a reagent that effects reaction between a carboxylic acid (-(C=O)-OH) and an amine (-NH2) group to form an amide (-(C=O)-NH-).

[0030] “Peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to naturally occurring and synthetic amino acids of any length, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. The term “peptide” includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like. Peptides further include post-translationally modified peptides.

[0031] “VHH” as used herein refers to a single-domain heavy chain antibody.

[0032] An “alpha-helix” or “a-helix” is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N-H group hydrogen bonds to the backbone C=O group of the amino acid located four residues earlier along the protein sequence. The alpha helix is also known as a classic Pauling-Corey- Branson a-helix, or 3.613-helix, which denotes the average number of residues per helical turn (3.6) with 13 atoms being involved in the ring formed by the hydrogen bond. Peptides thatAttomey Docket No.: 052566-511001 WO contain an alpha-helix is said to be alpha-helical. Such peptides may be partly or entirely alpha-helical. As understood in the art, an alpha-helix has at least four amino acid residues. In some embodiments, an alpha-helix has from 4 to 40 amino acids.

[0033] Provided are also pharmaceutically acceptable salts of the peptides or conjugates described herein. “Pharmaceutically acceptable” or “physiologically acceptable” refer to compounds, salts, compositions, dosage forms and other materials which are useful in preparing a pharmaceutical composition that is suitable for veterinary or human pharmaceutical use.

[0034] “Pharmaceutical composition” as used herein refers to a product comprising the specified ingredients in the specified amounts, as well as any product, which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. The pharmaceutical composition is generally safe for biological use.

[0035] “Pharmaceutically acceptable excipient” as used herein refers to a substance that aids the administration of an active agent to an absorption by a subject. Pharmaceutically acceptable excipients useful in the present invention include, but are not limited to, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors. One of skill in the art will recognize that other pharmaceutically acceptable excipients are useful in the present invention.

[0036] The conjugates described herein may be prepared and / or formulated as pharmaceutically acceptable salts or when appropriate as a free base. Pharmaceutically acceptable salts are non-toxic salts of a free base form of a compound that possess the desired pharmacological activity of the free base. These salts may be derived from inorganic or organic acids or bases. For example, a conjugate that contains a basic nitrogen may be prepared as a pharmaceutically acceptable salt by contacting the compound with an inorganic or organic acid. Non-limiting examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne- 1,4-dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methyl sulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene- 1-Attorney Docket No.: 052566-511001 WO sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, y-hydroxybutyrates, glycolates, tartrates, and mandelates. Lists of other suitable pharmaceutically acceptable salts are found in Remington: The Science and Practice of Pharmacy, 21stEdition, Lippincott Wiliams and Wilkins, Philadelphia, Pa., 2006.

[0037] Examples of “pharmaceutically acceptable salts” of the conjugates disclosed herein also include salts derived from an appropriate base, such as an alkali metal (for example, sodium, potassium), an alkaline earth metal (for example, magnesium), ammonium and NR (wherein R is C1-C4 alkyl). Also included are base addition salts, such as sodium or potassium salts.

[0038] “Therapeutically effective amount” as used herein refers to a dose that produces therapeutic effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, the therapeutically effective dose can be lower than the conventional therapeutically effective dose for non-sensitized cells.

[0039] “Inhibition”, “inhibits” and “inhibitor” as used herein refer to a compound that prohibits or a method of prohibiting, a specific action or function.

[0040] “ Treatment” or “treat” or “treating” as used herein refers to an approach for obtaining beneficial or desired results. For purposes of the present disclosure, beneficial or desired results include, but are not limited to, alleviation of a symptom and / or diminishment of the extent of a symptom and / or preventing a worsening of a symptom associated with a disease or condition. In one embodiment, “treatment” or “treating” includes one or more of the following: a) inhibiting the disease or condition (e.g, decreasing one or more symptoms resulting from the disease or condition, and / or diminishing the extent of the disease or condition); b) slowing or arresting the development of one or more symptoms associated with the disease or condition (e.g, stabilizing the disease or condition, delaying the worsening or progression of the disease or condition); and c) relieving the disease or condition, e.g., causing the regression of clinical symptoms, ameliorating the disease state, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival.Attorney Docket No.: 052566-511001 WO

[0041] “Prophylaxis” refers to preventing or retarding the progression of clinical illness in patients suffering from a disease.

[0042] A “subject” of the present invention is a mammal, which can be a human or a nonhuman mammal, for example a companion animal, such as a dog, cat, rat, or the like, or a farm animal, such as a horse, donkey, mule, goat, sheep, pig, or cow, and the like. In some embodiments, the subject is human.

[0043] “Articular joint” as used herein refers to the fibrous or cartilaginous joints, which is a fibrous or cartilaginous area wherein two or more bones connect to each other.

[0044] “Diffusion half-life” as used herein refers to the time it takes for the initial concentration of the conjugate within a given volume or space to decrease by half, where the decrease in concentration is a function of the concentration gradient.

[0045] “Intra-articular half-life” as used herein refers to the time it takes for the initial concentration of the conjugate within a particular joint to decrease by half, where the transport out of the joint is via convection. Convective transport is the combination of transport via diffusion and advection, where advective transport is the transport of a substance by bulk motion.III. PEPTIDES

[0046] In some embodiments, the peptides of the present invention offer advantages to comparative peptides in the art, for example, greater solubility, greater stability, lower tendency to aggregate in solution, and / or higher expression levels in convenient systems such as A. coli.

[0047] In some embodiments, the peptide of the present invention is an anti-VEGF peptide. In some embodiments, the peptide is a variant peptide of VH3:DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGY KYD AVSLEGRFTISRDNAKNTVYLQINSLRPEDT AVYYC AS SRAYGS SRLRL ADTYE YWGQGTLVTVSS (SEQ ID NO: 91).

[0048] In some embodiments, the peptide has at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% sequence identity to SEQ ID NO:91.Attorney Docket No.: 052566-511001 WO

[0049] In some embodiments, the peptide does not have the amino acid sequence of SEQ ID NO: 91.

[0050] Variations in amino acid sequences of the peptides described herein may be naturally occurring, such as splicing variants or allelic variants. In addition or alternatively, variations in amino acid sequences of the peptides may be introduced by substitution, deletion or insertion of one or more codons into the nucleic acid sequences encoding the antibodies that results in a change in the amino acid sequences of the antibodies. Optionally, the variation may be resulted from substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids with any other amino acid in the peptides. Amino acid substitutions in variants of the peptides may be conservative or non-conservative. Those of skill in the art will understand that a “non-conservative substitution,” when used in reference to a peptide, refers to a substitution of an amino acid in a peptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., serine for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. A non-limiting exemplary non- conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0051] Conservatively modified variant peptides are also contemplated as part of the present disclosure. As discussed above, a “conservatively modified variant” or a “conservative substitution” refers to a variant wherein there is one or more substitutions of amino acids in a peptide with other amino acids having similar characteristics (e.g., charge, side-chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.). Such changes can frequently be made without significantly disrupting the biological activity of the peptide. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a peptide do not substantially alter biological activity. In addition, substitutions of structurally or functionally similar amino acids are less likely to significantly disrupt biological activity.

[0052] Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains:Attorney Docket No.: 052566-511001 WO asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Exemplary conservative amino acids substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagineglutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (1992) Science 256: 1443 45.

[0053] In some embodiments, the peptide has the amino acid sequence:X1VQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAX2GI<EREFVX3AISI<GG YKYX4AVSX5X6GRFTISRDNX7KNTVYLQX8NSLRPEDTAVYYCASSRAYGSSRLRLA DTYEYWGQGTLVTVSS (SEQ ID NO: 92), wherein X1, X2, X3, X4, X5, X6, X7, and X8is independently any amino acid.

[0054] In some embodiments, X1is Q.

[0055] In some embodiments, X2is P. In some embodiments, X2is T.

[0056] In some embodiments, X3is A.

[0057] In some embodiments, X4is Y.

[0058] In some embodiments, X5is V. In some embodiments, X5is L.

[0059] In some embodiments, X6is K. In some embodiments, X6is E.

[0060] In some embodiments, X7is S.

[0061] In some embodiments, X8is M.

[0062] In some embodiments, X1is Q; X3is A; X7is S; and X8is M.

[0063] In some embodiments, X1is Q; X3is A; X5is V; X6is K; X7is S; and X8is M.

[0064] In some embodiments, X1is Q; X3is A; X4is Y; X5is V; X6is K; X7is S; and X8isM.

[0065] In some embodiments, the peptide has the amino acid sequence:Attorney Docket No.: 052566-511001 WOX1VQLVESGGGLVQPGGSLRLSCAASGR.TFSSYSMGWFR.QAX2GI<ER.EFVX3AISI<GG YKYX4AVSX5X6GRFTISRDNX7KNTVYLQX8NSLRPEDTAVYYCASSRAYGSSRLRLA DTYEYWGQGTLVTVSS (SEQ ID NO: 92), wherein X1is Q or D; X2is P or T; X3is A or V; X4is D or Y; X5is L or V; X6is E or K; X7is S or A; and X8is M or I, provided that the peptide does not have the amino acid sequence of SEQ ID NO: 91.

[0066] Illustrative peptides of the present invention are shown below:

[0067] In some embodiments, the peptide has the amino acid sequence according to SEQ ID NO:96. In some embodiments, the peptide has the amino acid sequence according to SEQID NO:97. In some embodiments, the peptide has the amino acid sequence according to SEQID NO: 98. In some embodiments, the peptide has the amino acid sequence according to SEQID NO:99.

[0068] In some embodiments, the peptide of the present invention is linked to a peptide linker. In some embodiments, the peptide linker is from 3 to 100 amino acids in length. In some embodiments, the peptide linker is an alpha-helical peptide.

[0069] In some embodiments, the peptide linker has an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21).

[0070] In some embodiments, the peptide of the present invention has the amino acid sequence:Attorney Docket No.: 052566-511001 WOX1VQLVESGGGLVQPGGSLRLSCAASGR.TFSSYSMGWFR.QAX2GI<ER.EFVX3AISKGG YKYX4AVSX5X6GRFTISRDNX7KNTVYLQX8NSLRPEDTAVYYCASSRAYGSSRLRLA DTYEYWGQGTLVTVSS (SEQ ID NO: 92), wherein X1, X2, X3, X4, X5, X6, X7, and X8are defined anywhere herein, linked at the C-terminus to a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21).

[0071] In some embodiments, the peptide has the amino acid sequence:X1VQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAX2GKEREFVX3AISKGG YKYX4AVSX5X6GRFTISRDNX7KNTVYLQX8NSLRPEDTAVYYCASSRAYGSSRLRLA DTYEYWGQGTLVTVSS AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 100), wherein X1, X2, X3, X4, X5, X6, X7, and X8are defined anywhere herein.

[0072] In some embodiments, the peptide has the amino acid sequence:X1VQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAX2GKEREFVX3AISKGG YKYX4AVSX5X6GRFTISRDNX7KNTVYLQX8NSLRPEDTAVYYCASSRAYGSSRLRLA DTYEYWGQGTLVTVSS AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 100), wherein X1is Q or D; X2is P or T; X3is A or V; X4is D or Y; X5is L or V; X6is E or K; X7is S or A; and X8is M or I.

[0073] Illustrative peptides linked to a peptide linker are shown below:Attorney Docket No.: 052566-511001 WO

[0074] In some embodiments, the peptide has the amino acid sequence according to SEQ ID NO: 102. In some embodiments, the peptide has the amino acid sequence according to SEQ ID NO: 103. In some embodiments, the peptide has the amino acid sequence according to SEQ ID NO: 104. In some embodiments, the peptide has the amino acid sequence according to SEQ ID NO: 105. In some embodiments, the peptide has the amino acid sequence according to SEQ ID NO: 106.

[0075] Anti-VEGF peptides of the present invention with affinity for human VEGF may be desirable for therapeutic and diagnostic uses. Accordingly, the present disclosure contemplates peptides having binding affinity to human VEGF. In specific embodiments, the peptide binds human VEGF with an affinity of at least about 1000 nM, but may exhibit higher affinity, for example, at least about 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 100 pM, 10 pM, 1 pM, 0.1 pM, or even higher. In some embodiments, the peptide binds human VEGF with an affinity in the range of from about 0.1 pM to about 1000 nM, or an affinity ranging between any of the foregoing values.IV. NUCLEOTIDES ENCODING PEPTIDES, EXPRESSION SYSTEMS, AND METHODS OF MAKING

[0076] The present disclosure encompasses nucleic acid molecules encoding genes for the peptides of the present invention, vectors comprising such nucleic acids, and host cells capable of producing the peptides.

[0077] A peptide of the present invention can be prepared by recombinant expression of genes in a host cell. To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the peptide such that the peptide expressed in the host cell and, optionally, secreted into the medium in which the host cells are cultured, from which medium the peptides can be recovered. Standard recombinant DNA methodologies are used to obtain polypeptide genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989), Current Protocols in Molecular Biology (Ausubel, F.M. et al., eds., Greene Publishing Associates, 1989) and in US 4,816,397.Attorney Docket No.: 052566-511001 WO

[0078] To generate nucleic acids encoding such a peptide, DNA fragments encoding the peptides are first obtained. These DNAs can be obtained by amplification and modification of germline DNA or cDNA encoding peptide sequences, for example using the polymerase chain reaction (PCR). Germline DNA sequences for human heavy chain variable region genes are known in the art (See, e.g., the "VBASE" human germline sequence database; see also Kabat, E. A. et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson et al., 1992, J. Mol. Biol. 22T: 116-198; and Cox et al., 1994, Eur. J. Immunol. 24:827-836).

[0079] Once DNA fragments encoding peptide-related VH segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as a flexible linker. The term "operatively linked," as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0080] To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4~Ser)3 (SEQ ID NO: 20), such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker See, e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0081] To express the peptide of the invention, DNAs encoding the peptides, obtained as described above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. As understood in the art, operatively linked is intended to mean that a gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the peptide gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used.

[0082] The genes for the peptide are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the gene fragment and vector, orAttorney Docket No.: 052566-511001 WO blunt end ligation if no restriction sites are present). Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the peptide chain from a host cell. The peptide chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the peptide chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0083] Accordingly, in some embodiments, provided herein is a nucleic acid molecule including a nucleotide sequence encoding a peptide of the present disclosure.

[0084] In addition to the peptide chain genes, the recombinant expression vectors of the disclosure carry regulatory sequences that control the expression of the peptide chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the peptide chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, 1990. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see, e.g., U.S. Patent No.5,168,062, U.S. Patent No. 4,510,245, and U.S. Patent No. 4,968,615.

[0085] In addition to the peptide chain genes and regulatory sequences, the recombinant expression vectors of the disclosure can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (See, e.g., U.S. Patents Nos. 4,399,216 , 4,634,665 and 5,179,017, all by Axel et al .). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductaseAttorney Docket No.: 052566-511001 WO(DHFR) gene (for use in DHFR' host cells with methotrexate selection / amplification) and the neo gene (for G418 selection). The various forms of the term transfection are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calciumphosphate precipitation, DEAE-dextran transfection and the like.

[0086] It is possible to express the peptide of the invention in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of peptides is performed in eukaryotic cells, e.g., mammalian host cells, of optimal secretion of a properly folded and immunologically active peptide. Exemplary mammalian host cells for expressing the recombinant peptides of the invention include Chinese Hamster Ovary (CHO cells) (including DHFR' CHO cells, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding genes are introduced into mammalian host cells, the peptides are produced by culturing the host cells for a period of time sufficient to allow for expression of the peptide in the host cells or secretion of the peptide into the culture medium in which the host cells are grown. Peptides can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact peptides, such as Fab fragments or scFv molecules. It is understood that variations on the above procedure are within the scope of the present disclosure.

[0087] The peptide of the invention can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Variant peptides can also be generated using a cell-free platform (See, e.g., Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals) and Murray et al., 2013, Current Opinion in Chemical Biology, 17:420-426).

[0088] Once a peptide of the invention has been produced by recombinant expression, it can be purified by any method known in the art, for example, by chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of peptides. Further, the peptide of the present invention can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.Attorney Docket No.: 052566-511001 WO

[0089] Once isolated, the peptide can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, eds., Elsevier, 1980), or by gel filtration chromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

[0090] Examples of purification techniques suitable for the purification of the peptide of the present invention include affinity chromatography, anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydroxyapatite chromatography, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), metal affinity chromatography, mixed mode chromatography (MMC), centrifugation, diafiltration, and ultrafiltration.

[0091] Generally, a peptide is “substantially pure,” “substantially homogeneous,” or “substantially purified” when at least about 60 to 75% of a sample exhibits a single species of polypeptide. The peptide may be monomeric or multimeric. A substantially pure peptide generally includes about 50%, 60%, 70%, 80% or 90% W / W of a protein sample, more usually about 95%, 96%, 97%, 98%, or in some embodiments, over 99% pure. Protein purity or homogeneity may be indicated by a number of means available in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single peptide band upon staining the gel with a suitable stain available in the art. For certain purposes, higher resolution may be provided by using HPLC or other means available in the art for purification. Accordingly, in some embodiments, the peptide of the present invention has a purity of greater than 80% such as, for example, a purity of greater than 85%, 90%, 95%, 96%, 97%, 98%, or 99%.V. CONJUGATES

[0092] In some embodiments, the conjugate is a conjugate of Formula Ila:(Xkx^Yjn-Z (Ila), wherein each X1is independently a peptide as described herein; each X2is independently a peptide linker of from 3 to 100 amino acids in length; each Y is independently an organic linker;Attorney Docket No.: 052566-511001 WOZ is a biocompatible polymer having a molecular weight of from about 0.1 MDa to about 3 MDa; and subscript n is an integer of from 1 to 1500.

[0093] In some embodiments, the conjugate is a conjugate of Formula lib :(X1-X2A-Y)n-Z (lib), wherein each X1is independently a peptide having a molecular weight of from about 5 kDa to about 200 kDa; each X2Ais independently a peptide linker that comprises an alpha-helix; each Y is independently an organic linker;Z is a biocompatible polymer having a molecular weight of from about 0.1 MDa to about 3 MDa; and subscript n is an integer of from 1 to 1500.

[0094] In some embodiments, each X1is independently a peptide of the present invention. The peptide of the present invention can be any peptide as described in Section III above.

[0095] In some embodiments, each peptide linker has an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21).

[0096] Each peptide can be linked to the biocompatible polymer by a variety of organic linkers generally known in the art for forming antibody-drug conjugates, such as those provided by Conju-Probe or BroadPharm of San Diego, CA or Creative Biolabs of Shirley, NY. Methods for forming bioconjugate bonds are described in Bioconjugate Techniques, 3rdEdition, Greg T. Hermanson. The organic linkers can be reactive with amines, carbonyls, carboxyl and activated esters, can react via Click-chemistry (with or without copper), or be reactive with thiols.

[0097] Representative organic linkers include an amide or disulfide, or are formed from a reactive group such as succinic anhydride, succinimide, N-hydroxy succinimide, N- chlorosuccinimide, N-bromosuccinimide, maleic anhydride, maleimide, hydantoin, phthalimide, and others. The organic linkers useful in the present invention are small and generally have a molecular weight from about 100 Da to about 500 Da containing two functional groups consisting of a maleimide and either an amine or hydrazide. In some embodiments, the peptide is covalently linked to the polymer via a sulfide bond and anAttorney Docket No.: 052566-511001 WO organic linker having a molecular weight of from about 100 Da to about 500 Da. In some embodiments, the organic linker has a molecular weight of from about 100 Da to about 300 Da. In some embodiments, the organic linker comprises a succinimide. In some embodiments, the organic linker is formed using N-beta-maleimidopropionic acid hydrazide (BMPH), N-epsilon-maleimidocaproic acid hydrazide (EMCH), N-aminoethylmaleimide, N- kappa-maleimidoundecanoic acid hydrazide (KUMH), hydrazide-PEG2-maleimide, amine- PEG2-mal eimide, hydrazide-PEG3-mal eimide, or amine-PEG3-mal eimide.

[0098] Representative organic linkers include, but are not limited to,

[0099] In some embodiments, the organic linker can be N-epsilon-maleimidocaproic acid hydrazide (EMCH):

[0100] In some embodiments, the organic linker has the structure:Attorney Docket No.: 052566-511001 WO wherein subscript m is an integer of from 1 to 300.

[0101] In some embodiments, the organic linker has the structure:

[0102] In some embodiments, preparing the conjugates of the present invention comprises covalently attaching the organic linker to the biocompatible polymer and then covalently attaching the peptide to the organic linker. In some embodiments, after preparing the conjugate of the present invention, unreacted organic linker is present on the biocompatible polymer. The structure of the unreacted organic linker depends on the organic linker and would be understood by a person skilled in the art.

[0103] Representative unreacted organic linkers include, but are not limited to,

[0104] In some embodiments, the unreacted organic linker has the structure:

[0105] In some embodiments, the unreacted organic linker has the structure:Attorney Docket No.: 052566-511001 WOwherein subscript m is an integer of from 1 to 300. In some embodiments, subscript m is an integer from 1 to 100.

[0106] In some embodiments, the unreacted organic linker has the structure:

[0107] In some embodiments, the biocompatible polymer is hyaluronic acid.

[0108] In some embodiments, the biocompatible polymer has a molecular weight of from about 0.4 MDa to about 2 MDa. In some embodiments, the biocompatible polymer has a molecular weight of from about 0.7 MDa to about 1.5 MDa. In some embodiments, the biocompatible polymer has a molecular weight of about 0.8 MDa.

[0109] In some embodiments, subscript n is an integer of from 1 to 1500. In some embodiments, subscript n is an integer of from 5 to 1000. In some embodiments, subscript n is an integer of from 10 to 400. In some embodiments, subscript n is an integer of from 10 to 100.

[0110] In some embodiments, the conjugate is a conjugate of Formula Ila:(Xkx^Yjn-Z (Ila), wherein each X1is independently a peptide as described herein; each X2is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21); each Y is an organic linker having the structure:Attorney Docket No.: 052566-511001 WOZ is a biocompatible polymer that is a hyaluronic acid having a molecular weight of from about 0.1 MDa to about 3 MDa; subscript m is an integer of from 1 to 300; and subscript n is an integer of from 1 to 1500.[OHl] In some embodiments, the conjugate of the present invention is a conjugate that is a random polymer of Formula III:having a molecular weight of from about 0.1 MDa to about 3 MDa; wherein each X is independently a peptide having an amino acid sequence comprising any one of SEQ ID NOS: 101-105; each Y is an organic linker; each X-Y-Z1moiety has the structure:each Z2has the structure:Attorney Docket No.: 052566-511001 WOeach Z3independently has the structure:each R1and R2is independently Ci-Ce alkyl, -(Ci-Ce alkyl)-NR3R4, or Cs-Cs cycloalkyl; each R3and R4is independently H or Ci-Ce alkyl; each Z3ais independently OH or Y'; each Y' is an unreacted organic linker; subscript n is an integer of from 1 to 1500 and less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer of from 0 to 1000 and less than about 10% of the sum of subscripts n, p, and q; and subscript q is an integer of from 100 to 10000.

[0112] In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 101. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 102. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 103. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 104. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 105.

[0113] In some embodiments, the conjugate has the structure of Formula Illa:Attorney Docket No.: 052566-511001 WO(X1-X2-Y-Z1)n-(Z2)p-(Z3)q(Illa), wherein each X1is independently a peptide having an amino acid sequence comprising any one of SEQ ID NOS: 91 and 96-99; and each X2is a peptide linker having the sequence of SEQ ID NO: 21.

[0114] In some embodiments, each X1is a peptide having an amino acid sequence comprising any one of SEQ ID NO: 91. In some embodiments, each X1is a peptide having an amino acid sequence comprising any one of SEQ ID NO: 96. In some embodiments, each X1is a peptide having an amino acid sequence comprising any one of SEQ ID NO: 97. In some embodiments, each X1is a peptide having an amino acid sequence comprising any one of SEQ ID NO: 98. In some embodiments, each X1is a peptide having an amino acid sequence comprising any one of SEQ ID NO: 99.

[0115] In some embodiments, each X2is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21).

[0116] In some embodiments, the organic linker has the structure:

[0117] In some embodiments, the organic linker can be N-epsilon-maleimidocaproic acid hydrazide (EMCH):

[0118] In some embodiments, the organic linker has the structure:Attorney Docket No.: 052566-511001 WOsubscript m is an integer from 1 to 300. In some embodiments, subscript m is an integer from 1 to 100.

[0119] In some embodiments, the organic linker has the structure:The organic linker with the above structure is known as MP2H.

[0120] In some embodiments, the random polymer of Formula III has a molecular weight of from about 0.4 MDa to about 2 MDa. In some embodiments, the random polymer of Formula III has a molecular weight of from about 0.7 MDa to about 1.5 MDa. In some embodiments, the random polymer of Formula III has a molecular weight of about 0.8 MDa.

[0121] In some embodiments, each R1and R2is independently C1-C3 alkyl or -(C1-C3 alkyl)- NR3R4. In some embodiments, each R1and R2is ethyl or -(CH2)3-NMe2. In some embodiments, each R1is ethyl; and each R2is -(CH2)3-NMe2. In some embodiments, each R1is -(CH2)3-NMe2; and each R2is ethyl.

[0122] In some embodiments, each R3and R4is independently C1-C3 alkyl. In some embodiments, each R3and R4is methyl.

[0123] In some embodiments, subscript n is an integer of from 1 to 1500 and less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer of from 1 to 1000 and less than about 10% of the sum of subscripts n, p, and q; and subscript q is an integer of from 100 to 10000. In some embodiments, subscript n is an integer of from 1 to 1000 and less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer of from 1 to 800 and less than about 8% of the sum of subscripts n, p, and q; and subscript q is an integer of fromAttorney Docket No.: 052566-511001 WO100 to 10000. In some embodiments, subscript n is an integer of from 10 to 450 and less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer of from 1 to 300 and less than about 10% of the sum of subscripts n, p, and q; and subscript q is an integer of from 1000 to 3000. In some embodiments, subscript n is an integer of from 10 to 300 and less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer of from 1 to 240 and less than about 8% of the sum of subscripts n, p, and q; and subscript q is an integer of from 1000 to 3000. In some embodiments, subscript n is an integer of from 10 to 300 and less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer of from 1 to 60 and less than about 2% of the sum of subscripts n, p, and q; and subscript q is an integer of from 1000 to 3000. In some embodiments, subscript n is an integer of from 10 to 300 and less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer of from 1 to 30 and less than about 1% of the sum of subscripts n, p, and q; and subscript q is an integer of from 1000 to 3000. In some embodiments, subscript n is an integer of from 10 to 300 and less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer of from 1 to 15 and less than about 0.5% of the sum of subscripts n, p, and q; and subscript q is an integer of from 1000 to 3000.

[0124] In some embodiments, the conjugate of the present invention is a conjugate that is a random polymer of Formula III:having a molecular weight of from about 0.1 MDa to about 3 MDa; wherein each X is independently a peptide having an amino acid sequence comprising any one of SEQ ID NOS: 101-105; each Y is an organic linker; each X-Y-Z1moiety has the structure:each Z2has the structure:Attorney Docket No.: 052566-511001 WOeach Z3independently has the structure:each R1and R2is independently Ci-Ce alkyl, -(Ci-Ce alkyl)-NR3R4, or Cs-Cs cycloalkyl; each R3and R4is independently H or Ci-Ce alkyl; each Z3ais independently OH or Y'; each Y' is an unreacted organic linker; subscript n is an integer of from 1 to 1500 and less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer of from 0 to 1000 and less than about 10% of the sum of subscripts n, p, and q; and subscript q is an integer of from 100 to 10000.

[0125] In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 101. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 102. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 103. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 104. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 105.

[0126] In some embodiments, the conjugate is a conjugate that is a random polymer of Formula Illa:Attorney Docket No.: 052566-511001 WO(X1-X2-Y-Z1)n-(Z2)p-(Z3)q(Illa), having a molecular weight of from about 0.1 MDa to about 3 MDa; wherein each X1is a peptide having an anti-VEGF amino acid sequence comprising SEQ ID NO: 96; each X2is a peptide linker having an amino acid sequence comprisingAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21); each Y is an organic linker having the structure:each X1-X2-Y-Z1moiety has the structure:each Z2has the structure:each Z3independently has the structure:Attorney Docket No.: 052566-511001 WOeach Z3ais independently OH or Y'; each Y' has the structure:each R1and R2is ethyl or -(CH2)3-NMe2; subscript n is an integer of from 10 to 300 and less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer of from 1 to 15 and less than about 0.5% of the sum of subscripts n, p, and q; and subscript q is an integer of from 1000 to 3000.

[0127] In some embodiments, the conjugate is a conjugate that is a random polymer of Formula Illa:(X1-X2-Y-Z1)n-(Z2)p-(Z3)q(Illa), having a molecular weight of about 0.8 MDa; wherein each X1is a peptide having an anti-VEGF amino acid sequence comprising SEQ ID NO: 96; each X2is a peptide linker having an amino acid sequence comprisingAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21); each Y is an organic linker having the structure:each X1-X2-Y-Z1moiety has the structure:Attorney Docket No.: 052566-511001 WOeach Z2has the structure:each Z3independently has the structure:each Z3ais independently OH or Y'; each Y' has the structure:each R1and R2is ethyl or -(CH2)3-NMe2; subscript n is an integer of from 10 to 300 and less than about 10% of the sum of subscripts n, p, and q;Attorney Docket No.: 052566-511001 WO subscript p is an integer of from 1 to 15 and less than about 0.5% of the sum of subscripts n, p, and q; and subscript q is an integer of from 1000 to 3000.

[0128] In some embodiments, a conjugate of the present invention exhibits favorable in vitro stability as measured, e.g., in minimal physicochemical changes over time as compared to its initial synthetic preparation, that is, at time 0. In some embodiments, the in vitro stability is measured after aging, e.g., after from about 1 day to about 360 days or more, such as after about 7, about 14, about 21, about 28, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 210, about 240, about 270, about 300, about 330, or about 360 days. In some embodiments, the in vitro stability is measured after aging, e.g., after from about 1 month, about 3 months, about 6 months, about 9 months, about 1 year, about 1.5 years, or about 2 years or more.

[0129] In some embodiments, a conjugate of the present invention has a hydrodynamic radius (Rh) upon aging that is within about 50%, about 40%, about 30%, about 20%, or within about 10%, of the hydrodynamic radius measured after initial synthetic preparation.

[0130] In some embodiments, a conjugate of the present invention has an association rate constant (Kon) upon aging that is within about 100-fold, about 10-fold, about 5-fold, about 4- fold, about 3-fold, or about 2-fold of the Kon measured after initial synthetic preparation.

[0131] In some embodiments, a conjugate of the present invention exhibits a half-life in vivo of from about 12 hours to about 24 hours, from about 1 day to about 3 days, from about 3 days to about 7 days, from one week to about 2 weeks, from about 2 weeks to about 4 weeks, or from about 1 month to about 6 months.

[0132] In some embodiments, a conjugate of the present invention exhibits a therapeutically efficacious residence time in vivo of from about 12 hours to about 24 hours, from about 1 day to about 3 days, from about 3 days to about 7 days, from one week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 1 month to about 3 months, from about 3 months to about 6 months, from about 6 months to about 9 months, from about 9 months to about 12 months, or longer than 12 months.

[0133] The biological activity of a conjugate is enhanced relative to the activity of the corresponding peptide in soluble form, e.g., compared to the activity of the peptide notAttorney Docket No.: 052566-511001 WO conjugated to the polymer. In some embodiments, the biological activity of the conjugate is at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 5- fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25- fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75- fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about 1000-fold, or more than 1000-fold, greater than the biological activity of the peptide in soluble (unconjugated) form.

[0134] In some embodiments, a conjugate of the present invention exhibits a favorable toxicity profile compatible with in vivo administration. Various in vitro and in vivo toxicity estimations are known in the art. In some embodiments, a conjugate of the present invention is tolerated in a non-human primate in vivo toxicity study, e.g., in a single dose or in multiple doses.

[0135] In some embodiments, a conjugate of the present invention exhibits a low anti -drug antibody (ADA) titer after in vivo administration. In some embodiments, the ADA titer is measured in serum or in aqueous humor. In some embodiments, the ADA titer is less than about 30,000, less than about 20,000, less than about 10,000, less than about 5,000, less than about 4,000, less than about 3,000, less than about 2,000, or less than about 1,000.VI. COMPOSITIONS

[0136] In some embodiments, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising a conjugate as described herein, and a pharmaceutically acceptable excipient.A. Formulation

[0137] For preparing pharmaceutical compositions from the conjugates of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, cachets, and dispersible granules. A solid carrier can be one or more substances, which may also act as diluents, binders, preservatives, disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").Attorney Docket No.: 052566-511001 WO

[0138] In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain from 5% or 10% to 70% of the conjugates of the present invention.

[0139] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

[0140] Aqueous solutions suitable for oral use can be prepared by dissolving the conjugates of the present invention in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolality.

[0141] Also included are solid form preparations, which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

[0142] Oil suspensions can be formulated by suspending the conjugates of the present invention in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. The oil suspensions can contain aAttorney Docket No.: 052566-511001 WO thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto, J. Pharmacol. Exp. Ther. 281 :93-102, 1997. The pharmaceutical formulations of the invention can also be in the form of oil-in- water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent.

[0143] The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be formulated for administration via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). Both transdermal and intradermal routes afford constant delivery for weeks or months.

[0144] In another embodiment, the compositions of the present invention can be formulated for parenteral administration into a body cavity such as intratumoral administration, intravitreal administration into an eye, or the intra-articular space of a joint. The formulations for administration will commonly comprise a solution of the compositions of the present invention dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting andAttorney Docket No.: 052566-511001 WO buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of the compositions of the present invention in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV, intratumoral, or intravitreal administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3- butanediol.

[0145] In another embodiment, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing ligands attached to the liposome, or attached directly to the oligonucleotide, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46: 1576-1587, 1989).

[0146] Lipid-based drug delivery systems include lipid solutions, lipid emulsions, lipid dispersions, self-emulsifying drug delivery systems (SEDDS) and self-microemulsifying drug delivery systems (SMEDDS). In particular, SEDDS and SMEDDS are isotropic mixtures of lipids, surfactants and co-surfactants that can disperse spontaneously in aqueous media and form fine emulsions (SEDDS) or microemulsions (SMEDDS). Lipids useful in the formulations of the present invention include any natural or synthetic lipids including, but not limited to, sesame seed oil, olive oil, castor oil, peanut oil, fatty acid esters, glycerol esters, Labrafil®, Labrasol®, Cremophor®, Solutol®, Tween®, Capryol®, Capmul®, Captex®, and Peceol®.B. Administration

[0147] The conjugates and compositions of the present invention can be delivered by any suitable means, including oral, parenteral and topical methods. In some embodiments, theAttorney Docket No.: 052566-511001 WO delivery method is intra-articular. In some embodiments, the delivery method is intravitreal. In some embodiments, the delivery method is intratumoral.

[0148] The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the conjugates and compositions of the present invention. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.

[0149] The conjugates and compositions of the present invention can be co-administered with other agents. Co-administration includes administering the conjugate or composition of the present invention within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of the other agent. Coadministration also includes administering simultaneously, approximately simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order. Moreover, the conjugates and compositions of the present invention can each be administered once a day, or two, three, or more times per day so as to provide the preferred dosage level per day.

[0150] In some embodiments, co-administration can be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition including the conjugates and compositions of the present invention and any other agent. Alternatively, the various components can be formulated separately.

[0151] The conjugates and compositions of the present invention, and any other agents, can be present in any suitable amount, and can depend on various factors including, but not limited to, weight and age of the subject, state of the disease, etc. Suitable dosage ranges include from about 0.01 mg to about 10,000 mg, or about 0.1 mg to about 10 mg, or about 1 mg to about 1000 mg, or about 10 mg to about 750 mg, or about 25 mg to about 500 mg, or about 50 mg to about 250 mg. Suitable dosages also include about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3.1.4, 1.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg. The composition can also contain other compatible therapeutic agents. The conjugates described herein can be used in combination with one another, with other active agents known to be useful in modulating a glucocorticoid receptor, or with adjunctive agents that may not be effective alone, but may contribute to the efficacy of the active agent.Attorney Docket No.: 052566-511001 WOVII. METHODS OF TREATMENT

[0152] In some embodiments, the present invention relates to a method and / or use comprising a conjugate or a composition as described herein for the treatment of disease or disorder in a subject in need thereof.

[0153] In some embodiments, the method comprises multiple administrations of the conjugate. In some embodiments, the method comprises administering the conjugate every day, every other day, every three days, or every week. In some embodiments, the method comprises administering the conjugate every week, every 2 weeks, every 3 weeks, or every month. In some embodiments, the method comprises administering the conjugate every month, every two months, every three months, every six months, every nine months, or every 12 months. In some embodiments, the method comprises administering the conjugate twice or three times yearly. In some embodiments, the method comprises administering the conjugate yearly.A. Ocular Disorder

[0154] In some embodiments, the method of the present invention is a method of treating an ocular disorder in a subject in need thereof, comprising administering to the subject a conjugate as described herein.

[0155] In some embodiments, the method comprises intravitreally administering the conjugate.

[0156] In some embodiments, the method comprises administering the conjugate every month, every two months, every three months, every six months, every nine months, or every 12 months.

[0157] In some embodiments, the vitreous half-life of the conjugate is at least 2-fold, 3- fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or at least 100-fold greater than the half-life of the unconjugated peptide. In some embodiments, the vitreous half-life of the conjugate is at least 4-fold greater than the half-life of the unconjugated peptide.

[0158] Ocular disorders that can be treated using a method of the present disclosure include, but are not limited to, uveitis, macular degeneration, also known as age-related macular degeneration (AMD), choroidal neovascularization, retinal neovascularization, proliferative vitreoretinopathy, glaucoma, and ocular inflammation. In some embodiments,Attorney Docket No.: 052566-511001 WO the macular degeneration comprises wet macular degeneration. In some embodiments, the macular degeneration comprises dry macular degeneration. In some embodiments, the macular degeneration comprises geographic atrophy.

[0159] Ocular diseases that can be treated using a method of the present disclosure include, but are not limited to, acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, such as acute macular degeneration, non-exudative age related macular degeneration and exudative age related macular degeneration; edema, such as macular edema, cystoid macular edema and diabetic macular edema; multifocal choroiditis; ocular trauma which affects a posterior ocular site or location; ocular tumors; retinal disorders, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal arterial occlusive disease, retinal detachment, uveitic retinal disease; sympathetic ophthalmia; Vogt Koyanagi-Harada (VKH) syndrome; uveal diffusion; a posterior ocular condition caused by or influenced by an ocular laser treatment; posterior ocular conditions caused by or influenced by a photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorders; branch retinal vein occlusion; anterior ischemic optic neuropathy; non-retinopathy diabetic retinal dysfunction; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (fundus flavimaculatus); inherited macular degeneration; chorioretinal degeneration; Leber congenital amaurosis; congenital stationary night blindness; choroideremia; Bardet-Biedl syndrome; macular telangiectasia; Leber's hereditary optic neuropathy; retinopathy of prematurity; and disorders of color vision, including achromatopsia, protanopia, deuteranopia, and tritanopia.

[0160] In some cases, the ocular disease is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber's Congenital Amaurosis, diabetic retinopathy, achromotopsia, or color blindness.

[0161] In some cases, a composition comprising a conjugate is administered by an intravitreal, transcleral, periocular, conjunctival, subtenon, intracameral, subretinal, subconjunctival, retrobulbar, or intracanalicular route of administration. In some cases, a composition comprising a conjugate is administered intravitreally. In some cases, the composition is delivered intravitreally or in close proximity to the posterior segment of theAttorney Docket No.: 052566-511001 WO eye. In some cases, the composition is administered intravitreally by injection. In some cases, a composition comprising a conjugate is administered by intraocular injection.B. Joint Diseases

[0162] In some embodiments, the method of the present invention is a method of treating a disease or disorder in an articular joint in a subject in need thereof, comprising administering to the subject a conjugate as described herein.

[0163] In some embodiments, the method comprises intraarticularly administering the conjugate.

[0164] In some embodiments, the method comprises administering the conjugate every month, every two months, every three months, every six months, every nine months, or every 12 months.

[0165] In some embodiments, the intraarticular half-life of the conjugate is at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or at least 100-fold greater than the half-life of the unconjugated peptide. In some embodiments, the intraarticular half-life of the conjugate is at least 4-fold greater than the half-life of the unconjugated peptide.

[0166] The present invention also provides methods of treating disease and disorders of the joint tissues using the conjugates of the present invention. Examples of diseases and disorders of the joint tissues include, but are not limited to rheumatoid arthritis, wear-related osteoarthritis, age-related osteoarthritis, post-traumatic osteoarthritis, psoriatic arthritis, and aseptic implant loosening, joint effusion, ankylosing spondylitis, bursitis, gout, reactive, arthritis, synovitis, and avascular necrosis. In some embodiments, the disease or disorder is rheumatoid arthritis, wear-related osteoarthritis, age-related osteoarthritis, post-traumatic osteoarthritis, psoriatic arthritis, and aseptic implant loosening, joint effusion, ankylosing spondylitis, bursitis, gout, reactive arthritis, synovitis, or avascular necrosis.

[0167] Many polypeptides are used as drugs to attenuate immune cell function have substantial utility in treating many joint disorders. Joint tissues are particularly susceptible to injury and disease because the typical cellular responses to these assaults, i.e., upregulating of inflammatory mediators, is also a signal to encourage catabolism of articular cartilage and resorption of the underlying bone tissues. Degeneration of the articular surfaces encourages the worsening of damage to the joint tissues and further up regulation of inflammatoryAttorney Docket No.: 052566-511001 WO mediators. Over time, these mechanisms generate a feed-forward loop that results in cumulative damage to the joint tissues.

[0168] Any joint of the human or animal body can be treated using the methods and conjugates of the present invention. Representative joints include, but are not limited to, fibrous joints, cartilaginous joints, synovial joints, facet joints, synarthrosis joints, amphiarthrosis j oints, and diarthrosis j oints. The j oints can be simple j oints having two articulation surfaces, a compound joint having three or more articulation surfaces, or complex joints having two or more articulation surfaces and an articular knee or meniscus. Anatomical joints that can be treated using the conjugates and methods of the present invention include, but are not limited to, hand joints including the fingers, elbow joints, wrist joints, shoulder joints, joints of the sternum and clavicle, vertebral joints, jaw and skull joints, pelvic and hip joints, knee joints, ankle joints and foot joints including the toes. The joints can also be classified as a plane joint, ball and socket joint, hinge joint, pivot joint, condyloid joint and saddle joint. The conjugates and methods of the present invention can be used to treat the tissues of the joint, including, but not limited to, connective tissue, cartilage, articulation surfaces, synovial cavities, meniscus, and others.

[0169] Examples of drugs that are designed to attenuate immune cell function include antibodies that can interfere with Tumor Necrosis Factor-a and IL-ip, IL-6, or interferon-y. Other examples include selective antibody inhibitors of T cell and B cell function. These antibodies may be monoclonal IgG antibodies, IgG antibody fragments, single chain scFv antibodies, single-domain heavy-chain VHH antibodies, or engineered antibody-like scaffolds such as adnectins, affibodies, anticalins, DARPins, and engineered Kunitz-type inhibitors. Other examples also include receptor decoys of immunomodulatory cytokines such as Tumor Necrosis Factor-a and IL-ip, IL-6, or interferon-y.

[0170] One common side effect of using anti-inflammatory drugs such as those listed above is a higher risk of infection. Because they attenuate the body’s immune responses, the immune system becomes impaired to fight bacteria, viruses, and parasites. Therefore, the benefits of systemic use of these drugs needs to be weighed carefully against the risks associated with systemic immune suppression. In the case of diseases where the whole body is affected by a hyperimmune disorder, such as rheumatoid arthritis, systemic use of immune attenuating drugs may be justified. However, for conditions effecting only one or a limitedAttorney Docket No.: 052566-511001 WO number of joints, the system risk of infection often does not justify the systemic use of these drugs.

[0171] As an alternative, intra-articular (IA) administration of immune modulating drugs has been proposed to prevent or inhibit the long-term effects of inflammation that are associated with osteoarthritis. However, these drugs are rapidly cleared out of the joint space and do not provide adequate duration of therapy after IA administration. After IA injection, the half-life of anti-inflammatory proteins in the synovium is short (<1.5 hours). This is evident from clinical studies where inflammation inhibitors, including infliximab and etanercept, have been administered by IA injection in humans for a variety of joint disorders. Some of these studies report a significant reduction in joint inflammation, but acknowledge that frequent (e.g. weekly) administration was required for a successful outcome. Thus, IA antiinflammatory therapy using existing drugs would be limited by high costs and the inconvenience of frequent IA dosing. Clearly, methods to extend anti-inflammatory drug bioactivity within the synovial fluid are needed to enable this therapeutic approach for treating joint disorders.

[0172] The primary symptoms associated with joint disorders are pain, effusion, limited range of motion, and pathological remodeling of the joint anatomy. Efficacy for a treatment to treat joint disorders may include a reduction in pain as measured by a generalized assessment, such as the visual assessment score. Efficacy may also be determined based on an improved score using a system that is specific to a particular joint disorder, such as the WOMAC score for osteoarthritis, the ACR20 for rheumatoid arthritis, the Psoriatic Arthritis Quality of Life for psoriatic arthritis, or the SASSS for ankylosing spondylitis. Efficacy may also be measured using a functional output, such as an increase in pain free walking distance or an increase in the range of joint motion. Efficacy may also be measured based on radiographic evidence showing restoration of normal joint anatomy.

[0173] The conjugate can be administered at any suitable frequency or amount as discussed above. In some embodiments, the conjugate is injected into the articular joint no more than about once a month. In some embodiments, the conjugate is injected into the articular joint from about once a month to once every 6 months. In some embodiments, the conjugate is injected into the articular joint once every 2 months or once every 3 months.Attorney Docket No.: 052566-511001 WO1. Osteoarthritis

[0174] In 2015, an estimated 7.75 million Americans experienced symptoms of osteoarthritis (OA) that could be associated with a known joint injury. Post-traumatic OA (PTOA) accounts for at least 15% of all OA cases, although it is assumed many other OA diagnoses may also be related to a prior joint trauma. Due to a lack of disease modifying therapies, joint replacement surgery is often the only treatment option to eliminate the associated discomfort and restore mobility. However, PTOA is often diagnosed in younger patients, for whom joint replacement is not a viable option. Overall, the cost of treating these PTOA patients exceeds $4B in health care costs each year.

[0175] Short-term inhibition of injury -related inflammation will limit the long-term symptoms of PTOA. Many types of joint injury have been associated with PTOA, including dislocations, ligament tears, meniscal damage, and intra-articular fractures. Although the initial damage may be acute, the injury is sufficient to initiate a cascade of inflammatory mediators. The resulting chronic whole-joint inflammation can encourage catabolism of the articular cartilage, resulting in further tissue damage that accumulates over time and presents as PTOA. TNFa and IL- 10 have well-known roles in mediating joint inflammation. These cytokines interact to promote destruction of cartilage, which occurs by both downregulating the expression of the cartilage matrix components and upregulating the expression of matrix metalloproteinases (MMPs). TNFa also stimulates osteoclast recruitment, and induces apoptosis of bone-forming osteoblasts in inflammatory environments, which contributes to the erosion of articular cartilage tissues. TNFa and IL- 10 are compelling targets for mitigating the inflammatory response to joint injury. Inhibiting these key acute inflammatory cytokines in the joint environment has been proposed for early intervention to stall the progression of PTOA.2. Inflammation due to immune response to intra-articular microparticles

[0176] Wear occurring between the articular surfaces of a joint can generate particles at the micron scale that drive joint inflammation and osteolysis. Wear particles may be generated due to abrasion between endogenous surfaces, such as ossified cartilage lesions, osteophytes (bone spurs), or exposed subchondral bone lesion. This type of wear particle generationAttorney Docket No.: 052566-511001 WO occurs frequently in later stage of OA, resulting in severe joint pain and immobility. This additional inflammatory response accelerates the rate of joint tissue degeneration in OA.

[0177] Wear particles may also be formed between the surfaces of an artificial joint. In 2015, more than 7 million Americans were living with an implanted artificial joint. Nearly 250,000 of these individuals will eventually require a revision surgery due to osteolysis of the bone surrounding the device, eventually resulting in device loosening and failure.

[0178] Wear-related inflammation stems from the foreign body response to otherwise inert microparticles shed from the articulating surfaces. Macrophages inside the synovial lining readily recognize wear microparticles as foreign bodies, release pro-inflammatory factors that recruit other active immune cells to the synovium, and stimulate osteoclast expansion while simultaneously inhibiting bone formation. Thus, sustained inflammation triggers a feedforward cycle where cartilage degeneration and osteolysis leads to more abrasions between articulating surfaces and more movement and physical stress that in turn produces more particles.

[0179] In some embodiments, the peptide modulates the activity of immune cell function. In some embodiments, the peptide inhibits tumor necrosis factor-a, interleukin- ip, interleukin-6, or interferon-y. In some embodiments, the peptide inhibits tumor necrosis factor-a.

[0180] Tumor necrosis factor (TNFa) is a compelling target for controlling the foreign body response. TNFa has a well-known role in mediating joint inflammation. TNFa also stimulates osteoclast recruitment, and induces apoptosis of bone-forming osteoblasts in inflammatory environments, leading to osteolysis of subchondral bone. Inhibition of TNFa using a systemically-administered receptor antagonist (etanercept) has been shown to reduce bone resorption induced by wear particles in mice, although the risks associated with systemic anti-TNFa are not generally regarded as acceptable for localized conditions. As an alternative, IA anti-TNFa therapy has been proposed to prevent or inhibit the osteolytic response to intra-articular wear particle.

[0181] In some embodiments, a use of the present invention is a use of a conjugate as described herein for the preparation of a medicament for a method of treating a disease or disorder in a subject.

[0182] In some embodiments, the subject is a human.Attorney Docket No.: 052566-511001 WO

[0183] In some embodiments, a use of the present invention is a use for treating a disease or disorder comprising a conjugate or pharmaceutical composition as described herein.

[0184] In some embodiments, a pharmaceutical composition of the present invention is a pharmaceutical composition for use in treating a disease or disorder comprising a conjugate as described herein.

[0185] In some embodiments, a conjugate of the present invention is a conjugate for use in treating a disease or disorder as described herein.VIII. EXAMPLES

[0186] Certain abbreviations and acronyms are used in describing the experimental details. Although most of these would be understood by one skilled in the art, the Table below contains a list of many of these abbreviations and acronyms.Table 1. List of abbreviations and acronyms.Example 1. Anti-VEGF Peptide Synthesis and EvaluationAttorney Docket No.: 052566-511001 WO

[0187] Anti-VEGF VH3 peptide (SEQ ID NO: 91) has been reported. See, e.g., US Patent No. 10,765,759. Mutations of VH3 were prepared by methods as previously described in US 2023 / 0406916. The sequences of the mutated peptides are shown in Table 2 below, with the summary of mutations shown in Table 3. The VHH antibody sequences were expressed, purified, and evaluated using methods described in US 2023 / 0406916.Table 2. PeptidesTable 3. Comparative MutationsTable 4. VHH Antibody Characteristics with Peptide Linker (SEQ ID NO: 21)Attorney Docket No.: 052566-511001 WO

[0188] Methods to determine yield and humanization, and biolayer interferometry were as previously described in US 2023 / 0405133.TmAnalysis for VHH’s and VHH Conjugates

[0189] Unconjugated VHH’s were diluted to 0.5mg / mL using materials within Thermo Protein Thermal Shift Dye Kit (catalog #4461146) according to manufacturers specification, and mixtures were subjected to temperature ramping of 0.05°C / s in an Applied Biosystems Quantstudio5 Real-Time PCR machine, from 25°C to 99°C. Melt curves were analyzed in triplicate using Applied Biosystems Real-Time PCR Design and Analysis Software. anti-VEGF ICso

[0190] A Promega cell assay was performed to determine anti-VEGF IC50. The manufacturer’s protocol was used without modification. The Promega VEGF Bioassay is a bioluminescent cell-based assay that measures VEGF stimulation KDR (VEGFR-2) using luciferase as a readout. The VEGF Responsive Cells have been engineered to express the response element (RE) upstream of luc2P, as well as exogenous VEGF receptor. When VEGF binds to VEGF Responsive Cells, the receptor transduces intracellular signals resulting in luminescence. The bioluminescent signal is detected as a reporter for VEGF signaling, and decreases in the bioluminescent signal indicate that the test article is effective to inhibit the VEGF signal. The concentration at which 50% of the VEGF signaling is inhibited (IC50) a measure of anti-VEGF potency for the test article. Lower values for IC50 indicate greater potency to inhibit anti-VEGF.

[0191] The anti-VEGF VHH SEQ ID NO: 96 with mutations at D1Q, V49A, A74S, I82M showed increased stability based on Tmcompared with SEQ ID NO: 91, while retaining the binding affinity to VEGF. In contrast, SEQ ID NO: 97 and 98 each exhibited no binding affinity to VEGF.Attorney Docket No.: 052566-511001 WOExample 2. Multivalent Protein Conjugates from Anti-VEGF Antibodies

[0192] The preparation of multivalent protein conjugates is similar to the method described in US patent publication 20230405133. Sodium hyaluronate (HA, 830 kDa) was hydrated in 0. IM MES pH 5.7 at 3.47 mg / mL in a reaction vessel for 8 to 24 hours. The temperature of vessel was controlled at 20 °C and the stir rate was 100 rpm. After HA had fully dissolved, HOBt hydrated in DMSO at 50mg / mL was added to the HA hydrate mix. 50mg / mL MP2H TFA, neutralized with tri ethylamine (TEA) and DMSO, was added to the mixture of HA and HOBt. EDC dissolved in 0.1M MES pH 5.7 at lOOmg / mL was added to the mixture while maintaining the pH of mixture greater than 3.5 with sodium hydroxide. The molar equivalents for each reactant per mole of HA and per carboxylate, and example methods are described in Table 5 below.Table 5: Relative Ratios of Coupling Agent, Catalyst, and Linkers in Methods

[0193] The final mixture was allowed to react at 20 °C with 100 rpm stir rate for 60 to 70 minutes. Glycine was then added to stop the reaction. The unpurified intermediate was 7x volume buffer exchanged with lx DPBS pH 6.5 using KrosFlo KR2i TFF system equipped with Tangenx SIUS 30 KDa E channel 0.5m2 cassette. The transmembrane pressure (TMP) was monitored at 10 psi. The purified intermediate was sterile filtered using a 0.22 pm SUPOR EKV capsule filter then aliquoted and stored at -80 °C. Alternatively, the intermediate could be purified in smaller batches using 7 kDa MWCO 5-10 mL Zeba desalting spin column. The desired intermediate was eluted into clean conical tube using centrifuge at RT, elution time -25-60 minutes. The mal eimide concentration and number of modifications per polymer was determined using UV absorbance, NMR, or a modified Ellman’s reaction assay.

[0194] Alternatively, reaction pH or equivalents of hydrazide linker, catalyst, and coupling agent (EDC) were altered higher or lower to increase or decrease the number of thiol reactive small molecule linkers covalently linked per biopolymer (valency).

[0195] Alternative coupling reagents can be used in place of EDC and HOBt such as DMTMM or oxyma. Activated biopolymer intermediate can also be purified away fromAttorney Docket No.: 052566-511001 WO reactants using size exclusion chromatography, other desalting columns, tangential flow filtration, ion exchange chromatography, dialysis, or alcohol / acetone precipitation.

[0196] After purification, a UV spectrum (200-324 nm) was taken for intermediates prepared using different methods on a BioTek Synergy plate reader using a Take3 microspot plate. Maleimide concentration can be determined by absorbance at 230 nm, or by comparing spectra to a reference standard intermediate. Maleimide:HA molar ratios are based on the starting concentration of HA in the reaction and assume no HA is lost during purification. Chemical analysis of example reaction products are described in Table 6 below.Table 6. Exemplary Intermediate Prepared Using a Method Described Above

[0197] The method of generating cyclic peptide MVPs using the anti-VEGF VHH antibodies and maleimide-activated hyaluronic acid was similar to the method used to generate protein MVP described in US patent publication 20230405133. Analytical methods for characterizing the protein conjugates are the same as described previously.

[0198] SEC Methods: MVP samples were diluted in mobile phase and filtered using 5, 0.65, and 0.2 pm PVDF spin filters sequentially prior to analysis. The samples were analyzed using the KW-405-4F SEC column (Shodex) equilibrated in pH=6, 50 mM potassium phosphate, 300 mM KC1, 400 mM arginine mobile phase containing 0.025% w / v sodium azide at 0.35 ml / min, using an Agilent 1260 II HPLC with a multi wavelength detector monitoring at 280 nm, vialsampler, and thermostatted column compartment set to 30 °C, injecting between 5 and 15 ug of sample based on VHH mass. Unconjugated VHH diluted in mobile phase was also injected at known masses to quantify recovery of the MVP sample. Sample protein concentration was quantified using UV absorbance at 280 nm.

[0199] Table 7 shows illustrative conjugates prepared by the methods.Table 7. ConjugatesAttorney Docket No.: 052566-511001 WO*Note: all conjugates showed binding affinity of <1 pM by biolayer interferometry (BLI)

[0200] Exemplary data are shown for Conjugate 5 (FIG. 1A-1B).

[0201] Anti-VEGF cell bioassays were performed using methods as described in Example 1. Direct comparisons to other anti-VEGF agents were performed (FIG. 2A-2B).

[0202] FIG. 2A shows relative cell activities of bevacizumab and Conjugate 1 (left), and bevacizumab and Conjugate 2 (right). Conjugate 1 exhibited lower cellular activity against both VEGF-A121 and VEGF-A165 compared to bevacizumab. In contrast, Conjugate 2 exhibited higher cellular activity compared to bevacizumab against both VEGF splice variants. Conjugate 1 showed poor inhibition of the HEK-derived VEGFs, suggesting that it is sensitive to post-translational modifications to its target.

[0203] In an in vitro stability assay, Conjugate 6 remained stable when aged at 37 °C for at least 180 days (FIG. 5).Example 3. In Vivo Pharmacodynamics in Mouse

[0204] Conjugate 3 and Conjugate 4 were assessed in an in vivo pharmacodynamic model. Efficacy of the conjugates was evaluated for inhibiting laser-induced choroidal neovascularization (CNV) in transgenic mice in which the mouse VEGF gene has been replaced with a human VEGF gene. This model eliminates the risk that species-cross reactivity will bias efficacy results for treatments that target human VEGF.

[0205] On the day of the procedure, animals were given buprenorphine (0.05-0.1 mg / kg subcutaneous). Eyes were dilated with a cocktail of 1.0% tropicamide and 2.5% phenylephrine HC1 topically to dilate and proptose the eyes. Animals were then tranquilized for the procedure with a ketamine / xylazine cocktail (80-90 / 10-20 mg / kg IP), and one drop of 0.5% proparacaine HC1 was applied to both eyes. A 532 nm diode laser delivered through a slit-lamp was used to create 4 single laser spots surrounding the optic nerve. Both eyesAttorney Docket No.: 052566-511001 WO received laser treatment as indicated in the experimental design table and eye lubricant was applied topically to the eyes following the laser treatment.

[0206] Following completion of the laser CNV procedures, treatments were delivered in both eyes (OU) via 1-pL intravitreal injections containing the mass indicated in Table 8 below. The conjunctiva was gently grasped with Dumont #4 forceps, and the injection was made using a 33G needle (NF33BV-2, WPI) and 10 pL syringe (NANOFIL, 10 pL, WPI). After dispensing the syringe contents, the syringe needle was slowly withdrawn. Following the injection procedure, 1 drop of 0.3% ofloxacin ophthalmic solution and eye lubricant was applied topically to the ocular surface.Table 8. Treatment Protocols

[0207] Eight days after generating CNV lesions, animals were humanely euthanized. The eyes were enucleated, immediately fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), and stored overnight at approximately 4°C. On the following day, the eyes were transferred to cold immunocytochemistry (ICC) buffer (PBS containing 0.5% BSA and 0.2% Tween 20) until processing. Using a dissecting microscope, the eyes were trimmed of extraneous tissue and the anterior segments and lenses were removed and discarded. The retina was detached and removed from the optic nerve head with fine curved scissors. The remaining eye cups were rinsed with cold ICC buffer, and the eye cups were incubated in ICC buffer as shown in Table 9 below.Table 9. ICC BufferAttorney Docket No.: 052566-511001 WO

[0208] Eye cups were incubated at 4 °C with gentle rotation for 4 hours and washed with cold ICC buffer. Radial cuts were then be made toward the optic nerve head avoiding lesions, and the sclera-choroid / RPE complexes were flat mounted, covered and sealed. Two- dimensional (2D) fluorescent microscopy images will be acquired, digitized, and analyzed using an Olympus Bx63 upright fluorescent microscope and Cell Sens (Olympus) software and post-acquisition analysis was performed with FIJI / ImageJ software. To quantify neoformed vessels, the isolectin IB4 area was measured based on pixel area.

[0209] Both aflibercept (Eylea) and Conjugate 4 resulted in a significant reduction the neovascularization area in response to the laser-induced lesion formation (FIG. 2). There was no significant difference in lesion area for Conjugate 3 and the vehicle control. Statistical analysis was performed using a Kruskal-Wallis test with Dunn’s multiple comparison tests (letters above the error bars indicate groups with significant differences).Example 4. In Vivo Half Life of a Purified Peptide-Polymer Conjugate - Vitreous Humor

[0210] An extended intravitreal retention time of the conjugates was shown in a well- established pharmacokinetics model. Dutch belted rabbits were divided into 3 groups randomized by weight. All animals received 80-pL ITV injections of Conjugate 5 bilaterally using a 31 G insulin syringe. Both eyes received an equivalent molar dose of antibody. At 1 hour, and at 4 days, 7 days, 14 days, 28, days, 45 days, and 75 days post injection, one group of three rabbits were sacrificed, and their eyes enucleated for analysis of intravitreal VHH. Both eyes were flash frozen, and the vitreous, retina, and aqueous humor were isolated from the frozen eye. Each tissue sample was then homogenized with a bead beater and with hyaluronidase digestion. After homogenization, the VHH concentrations were quantified either using ELISA or by digesting the peptide using trypsin and subjecting the samples to LC / mass spectrometry, or a similar method.

[0211] The intravitreal half-life was calculated by plotting VHH concentrations versus time and fitting the data using a one-phase exponential decay equation. Based on the fit for Conjugate 5 (R2=0.8864), its intravitreal half-life was determined to be at least 12.2 days (FIG. 4A-4C). Based on this information, one previously published species-scaling factorAttorney Docket No.: 052566-511001 WO(Crowell, S. R. et al. Translational Vision Science & Technology 2019 Nov 1; 8(6): 1) suggests that the intravitreal half-life of Conjugate 5 in humans would be at least 21-28 days.

[0212] The potency of Conjugate 5 in the vitreous humor samples obtained from the PK analysis was assessed using cell assay measurements of IC50. Vitreous humor homogenate samples from two separate left eyes from the PK study at select timepoints (T=lhr, 15, 29, 46 and 77 days) were thawed and mixed well. The reported concentration of the single domain antibody (SEQ ID NO: 102) in the vitreous humor homogenate from the LC / MS / MS results is shown in Table 10 below. The homogenate was diluted to a final top concentration of 4.5 nM in assay buffer. All dilutions had a final 2% (v / v) concentration of vitreous humor homogenate that supplemented to the final concentration using blank vitreous humor homogenate that was processed in the same manner as samples. The top 4.5 nM concentration sample was serially diluted 9x to a final concentration of 0.012 nM in assay buffer.

[0213] Promega cell assays were performed to determine anti-VEGF IC50 according to the method described in Example 1.Table 10: Tissues Measured and Corresponding sdAb Concentration in Vitrous Humor Homogenate

[0214] The IC50 values for Conjugate 5 versus the number of days the conjugates were in the rabbit eye during the PK study were determined. An anti-VEGF IC50 of approximately 0.1Attorney Docket No.: 052566-511001 WO nM was measured throughout the study (FIG. 6). There was no significant difference in the anti-VEGF potency of Conjugate 5 between day 0 and day 77, indicating that the composition remained stable in vivo while in the vitreous humor during that time.Example 5. In Vivo Half Life of a Purified Peptide-Polymer Conjugate - Retina

[0215] The concentration of Conjugate 5 in the retina samples was estimated by making cell bioassay measurements on tissues obtained from the PK study described in Example 4. Retina samples from two separate left eyes from the PK study at select timepoints (T=15, 29, and 46 days) were thawed and homogenized in a Bioruptor bead beater in IX PBS blocker casein and 0.05% Tween-20 + protease inhibitor cocktail at 5X the tissue weight. Spiked standard samples were prepared in a similar manner as PK study samples except that before homogenization, the retina tissue was spiked with a known concentration of SEQ ID NO: 102, incubated at 37°C for 1 hour and then snap frozen. Samples and standards were centrifuged to pellet debris, and the supernatant was removed and aliquoted. Samples for Days 15-46 were diluted and prepared for a VEGF Bioassay using the same volume of retina homogenate with the top concentration at 1.1% (v / v) homogenate to assay buffer and serially diluted 9x to a final 0.003% of retina homogenate in assay buffer. Samples for day 77 were prepared with top concentration of 3.9% (v / v) homogenate to assay buffer and serially diluted 9x to a final 0.0095% of retina homogenate in assay buffer

[0216] Promega cell assays were performed according to the method described in Example 1, except that in this Example, they were used to estimate concentrations of the anti-VEGF sdAb (SEQ ID NO: 102) in Conjugate 5.

[0217] The output of the Promega cell assay is an inhibition curve showing VEGF activity versus anti-VEGF concentration. Although the exact concentration of the sdAb anti-VEGF in the retina was unknown, it could be estimated based on the position of their inhibition curves relative to those of spiked standards with known concentrations. These concentrations were comparable to what has been observed in previous studies and were similar to the concentrations of SEQ ID NO: 102 in the vitreous humor tissue before homogenization.Attorney Docket No.: 052566-511001 WOExample 6. Intermediate Manufacturing for In Vivo Tolerability Study

[0218] The preparation of INTV 162 was similar to the method described in US patent publication 20230405133. Sodium hyaluronate (HA, 830 kDa) was hydrated in 0.1M MES pH 5.7 at 3.69 mg / mL in a reaction vessel for 12 to 24 hours. The temperature of vessel was controlled at 20 °C and the stir rate was 200 rpm. After HA had fully dissolved, HOBt hydrated in DMSO at 50mg / mL was added to the HA hydrate mix. MP2H HC1 (415mg / mL) was added to the mixture of HA and HOBt. EDC dissolved in 0. IM MES pH 5.7 at lOOmg / mL was added to the mixture while maintaining the pH of mixture greater than 3.5 with sodium hydroxide. The molar equivalents for each reactant per mole of HA and per carboxylate, and example methods are described in Table 11 below.

[0219] The final mixture was allowed to react at 20 °C with 200 rpm stir rate for 60 to 70 minutes. Glycine was then added to stop the reaction. The unpurified intermediate was 7x volume buffer exchanged with lx DPBS pH 6.5 using KrosFlo KR2i TFF system equipped with Tangenx SIUS 30 KDa E channel 0.02m2cassette. The transmembrane pressure (TMP) was monitored at 10 psi. The purified intermediate was sterile filtered using a 0.22 pm SUPOR EKV capsule filter then aliquoted and stored at -80 °C. The mal eimide concentration and number of modifications per polymer was determined using UV absorbance or a modified Ellman’s reaction assay (Table 12).Table 11: Relative Ratios of Coupling Agent, Catalyst, and Linkers in MethodsAttorney Docket No.: 052566-511001 WOTable 12: Exemplary Intermediate Prepared Using a Method Described AboveExample 7. Multivalent Protein Synthesis for In Vivo Tolerability Study

[0220] The anti-VegF VHH (SEQ ID NO: 102) was solubly expressed within the periplasmic compartment of E.coli, and subjected to purification via standard Ion Exchange and Hydrophobic chromatographies. Purified protein was prepared for MVP reaction in 25mM sodium citrate pH 5.5, lOOmM NaCl, 10% glycerol, ImM EDTA at a concentration of ~100mg / mL. Purity was assessed with SEC, thiol reactivity via Ellman’s reagent, potency via BLI and cell bioassay, and host cell impurities via LAL and HCP ELISA.

[0221] The preparation of MVP V552 was similar to the method described in US patent publication 20230405133. To obtain the purified peptide-polymer conjugates, 1.25 equivalents of reactive peptide per mal eimide was combined with INTV162 from Example 5, and allowed to react at 20 °C overnight stirring at 200RPM. 1.0 M pH 7.0 HEPES was added to a final concentration of 0.1 M to adjust reaction pH. During the conjugation reaction, one or more of the following was added to improve the reaction efficiency: 0.5-10 mM EDTA to minimize free thiol oxidation, tween 20, carbohydrate, additional buffer, or glycerol to stabilize protein and / or help reduce non-specific interactions between protein and activated biopolymer, increased or decreased salt concentration to stabilize protein and / or help reduce non-specific interactions between protein and activated biopolymer. Unreacted peptide was removed thru 7x volume buffer exchange with lOmM sodium phosphate, 58mM Sodium Chloride, 159mM trehalose, pH 6.2 from the peptide-polymer conjugates using tangential flow filtration equipped with a hollow-fiber cartridge (500kDa MWCO) . The purified peptide-polymer conjugate was concentrated to a target of 8.0 mg / mL with TFF. The purified peptide-polymer conjugate was characterized by BLI, cell bioassay, SDS-PAGE, DLS, LAL, HCP ELISA, and sterility was determined via direct injection (Table 13).Attorney Docket No.: 052566-511001 WOTable 13: ConjugateExample 8. In Vivo Tolerability

[0222] The ocular tolerability of the conjugates was shown in a well-established cynomolgus non-human primate model. Male cynomolgus monkeys (~3 years old) were divided into two groups (n=3 / group) and given Conjugate 7 through intravitreal injections using a 31 G insulin syringe to both eyes. Group 1 received 0.34 mg / eye in a 40 uL injection and Group 2 received 0.85 mg / eye in two 50 uL injections spaced ~ 15 minutes apart (Table 14).Table 14: Experimental Design

[0223] For ocular examinations (Table 15), animals were sedated and a handheld slit lamp (Kowa SL15) was used to examine the eyelids, conjunctiva, cornea, anterior chamber, iris, lens, and anterior vitreous. The fundus (retina, choroid, and optic nerve head) was examined by indirect ophthalmoscopy. The semi quantitative preclinical ocular toxicology scoring (SPOTS) system was used to score the anterior and posterior segments, as described in Eaton, J. S., Miller, P. E., Bentley, E., Thomasy, S. M., & Murphy, C. J. (2017). The SPOTS System: An Ocular Scoring System Optimized for Use in Modem Preclinical Drug Development and Toxicology. Journal of Ocular Pharmacology and Therapeutics, 33(10), 718-734. Two key ocular exam scores that were indicative of intravitreal anti-VEGF tolerability (aqueous flare and vitreous cells) were averaged per eye at each timepoint for both groups and plotted with the mean + / - SD. For some timepoints, the posterior right eye of animal #0043 was not quantified due to severe anterior inflammation. Vitreous Cell Scores for each eye are shown inTable 16 and Table 17. Aqueous flare scores for each eye are shown in Table 18 and Table 19. Graphical representations of the mean vitreous cell and aqueous flare cell scores are shown in FIG. 7A-7B.Attorney Docket No.: 052566-511001 WOTable 15: In-Life Ocular Examinations and Testing ScheduleTable 16: Group 1 Vitreous Cell ScoresOD, right eye; OS, left eye; W, predominantly white; B, predominantly brown;+, ~2mm white vitreous clump at dorsal anterior vitreous but no other vitreous cellTable 17: Group 2 Vitreous Cell ScoresOD, right eye; OS, left eye; W, predominantly white; B, predominantly brown; M, mixed brown and white; *due to fibrin clots blocking pupillary apertureTable 18: Group 1 Aqueous Flare ScoresAttorney Docket No.: 052566-511001 WOOD, right eye; OS, left eyeTable 19: Group 2 Aqueous Flare ScoresOD, right eye; OS, left eye; F, fibrin

[0224] Intravitreal administration of Conjugate 7 in Group 1 (0.34 mg / eye) was well tolerated through Day 56 of the dosing phase. Mild (0.5+ to 1+) vitreous cell in 5 / 6 eyes were found mostly starting on Day 3 to 7 of the dosing phase. By Day 56, mild (0.5+ to 1+) vitreous cell were found in 3 / 6 eyes. The majority of those vitreous cells were pigmented and predominantly brown in color, which can result from injection.

[0225] Intravitreal administration of Conjugate 7 in Group 2 (0.85 mg / eye) was well tolerated through Day 56 of the dosing phase. Mild vitreous cells (0.5+ to 1+) were observed in 4 / 6 eyes, with onset typically occurring between Day 3 and Day 7. On three occasions, one animal exhibited moderate vitreous cell presence (2+), with the majority of cells being pigmented and brown in color. By Day 56, mild vitreous cells (0.5+ to 1+) were present in 3 / 6 eyes, with the majority of these cells also being pigmented and brown in color.

[0226] An outlier case in Group 2 (0.85 mg / eye), was the right eye of Animal No. 0043. The eye showed elevated IOP after the second dose and the animal presented with anterior uveitis and severe inflammation by Day 7 that progressed throughout the study and requiredAttorney Docket No.: 052566-511001 WO treatment on Day 15. The condition may be indicative of lens trauma or infectious endophthalmitis and was not considered to be test article related.

[0227] During the examination, intraocular pressure was measured by a tonometer (iCare® TONOVET Model TV011) held perpendicular, 4-8 mm from the cornea, and operated according to manufacturer’s instructions. A minimum of six measurements were collected and averaged to obtain the intraocular pressure. The mean of each eye at each timepoint per group was averaged and presented (Table 20, FIG. 8A and FIG. 8B). No statistically significant change in intraocular pressure was observed in either Group 1 or Group 2 over 56 days during the study.Table 20: Intraocular Pressure Measurements

[0228] On the basis of the in-life observations (i.e., IOP measurements and SPOTS scoring of ocular exam observations), Conjugate 7 did not appear to generate substantial toxicity at either dose level. There was no trend of elevated IOP or aqueous cellular flare in most animals over the duration of the study. Vitreous cells were observed, but these were generally scored trace to mild (i.e., SPOTS score <1) and did not appear to be dose dependent. This ocular tolerance response to Conjugate 7 in non-human primates appeared to be consistent with other humanized antibodies that have demonstrated clinical safety in humans (e.g., ranibizumab and faricimab). Accordingly, these results are useful to mitigate the risk of toxicity during clinical translation of Conjugate 7.

[0229] During the study, blood was collected at noted timepoints and processed into serum using standard blood component collection techniques and frozen at -80 °C (Table 21). After euthanasia, the vitreous humor was dissected from the left eye and frozen at -80 °C. The whole right eyes were placed in 50 mL conical tubes and submerged in Davidson’s Fixative for 24-48 hours before storage in 70% EtOH.Attorney Docket No.: 052566-511001 WOTable 21: Tissue Collections

[0230] Anti-drug antibodies (ADA) in the serum and vitreous humor (VH) tissue were detected using a master-mix immunogenicity assay approach. Each tissue sample was first homogenized with a bead beater and with hyaluronidase digestion. After homogenization and digestion, all VH samples, controls, and standards were acid treated, neutralized and concentrated using biotin-labeled VHH (SEQ ID NO: 102) bound to a streptavidin plate in preparation for the second acid-dissociation step. For all tissues, after an acid-dissociation step, controls and cynomolgus monkey samples were neutralized with a master-mix, comprised of biotin-labeled VHH (SEQ ID NO: 102) anti-VEGF sdAb (biotin drug) and sulfo-tag-labeled VHH (SEQ ID NO: 102) anti-VEGF sdAb (sulfo-tag drug).

[0231] Anti-drug antibodies (ADA) in tissues bound these reagents to form antibody-drug complexes, which then bound to a blocked streptavidin-coated plate (MSD GOLDTM 96- Well Streptavidin SECTOR plate* MSD Cat L15SA). The plate was then treated according to the manufacturer’s directions using the MSD Blocker A Kit* (MSD CAT R-93AA-1) to remove unbound material and read on an MSD SQ120 imager, reporting the signal generated by the sulfo-tag drug as relative luminescence units (RLU). The RLU level was proportional to the amount of ADA in the tissue sample.

[0232] The presence of ADA was determined by comparing the signal in the sample or control to a statistically derived threshold, the assay cut point. Screening cut points (SCP) were set to generate a 5% false positive rate (FPR) by testing treatment- naive cynomolgus tissue samples. The SCP was the NormRLU signal level at which a sample was considered positive or negative for ADA in the screening assay at the minimum required dilution (MRD), defined for that assay. All samples that screen positive were used in a subsequent titer assay.Attorney Docket No.: 052566-511001 WO

[0233] Samples slated for titer assays were measured again at the MRD and then subjected to 2-fold serial dilutions. The titer value for each sample was set as the last dilution factor when the mean NormRLU is above the titer cut point (TCP). The TCP was determined similary as the SCP but with a stricter 0.1% FPR. Titer was reported as the MRD multiplied by the highest sample dilution factor below which the mean NormRLU falls below the TCP for the first time.

[0234] Of the 36 total serum samples screened, 11 were positive (Table 22). These 11 samples underwent titration. The antibody titer levels gradually increased over the duration of the study so that all animals had positive signals at euthanasia. The titer levels were generally low, similar for both group 1 and group 2 animals, and did not follow any dose-dependent trends.Table 22: Serum Anti-Drug Antibody Titer ResultsN / T = not tested because screened negative

[0235] Of the 6 total VH samples screened, all 6 were positive. These 6 samples and underwent titration, only one animal was again positive at the MRD when using the stricter TCP.Table 23: Vitreous Humor Anti-Drug Antibody Titer ResultsAttorney Docket No.: 052566-511001 WO

[0236] The observed ADA titer responses to IVT administered human proteins / biologic therapeutics were low, and a common response to a heterologous protein in cynomolgus monkeys. These results did not indicate an adverse immunogenicity signal to Conjugate 7 in NHPs.

[0237] The right eyes from all animals were fixed, trimmed, processed, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). The slides were imaged, evaluated, and microscopic findings were scored using semi -quantitative grading criteria as follows and are presented in Table 24:Minimal (1): A focal, subtle, or trivial change,Mild (2): An easily identifiable change of limited severity and / or distribution,Moderate (3): An obvious change with normal tissue remaining,Marked (4): An extensive change that obliterates much of the normal tissue,Severe (5): A maximal change.Table 24: Incidence and Severity of Microscopic Findings (Day 57 or 58)OD = right eye; - = no finding.Attorney Docket No.: 052566-511001 WO

[0238] In all eyes treated with either 0.34 or 0.85 mg / eye of Conjugate 7, minimal to moderate mononuclear cells were observed. In the trabecular meshwork / ciliary body, sclera / conjunctiva / comea, and iris, cells consisted predominantly of lymphocytes and macrophages. Mononuclear cells were observed within the trabecular meshwork, around the canal of Schlemm, and extended into the ciliary body. Within the (limbal) sclera and associated conjunctiva, and peripheral cornea, mononuclear cells were most often forming perivascular cuffs, and this was also observed occasionally within the iris.

[0239] Minimal or moderate perivascular mononuclear cells were also observed in the inner retina in a single animal each at 0.34 and 0.85 mg / eye. Minimal or mild inflammation in the vitreous was observed at 0.34 mg / kg in one animal and consisted predominantly of macrophages. In the outlier animal # 0043 at 0.85 mg / eye, there was marked inflammation, this condition was determined by the veterinary ophthalmologist to not be related to the test article.

[0240] In summary, intravitreal administration of Conjugate 7 at both doses was determined to be well tolerated in cynomolgus monkeys by a board-certified veterinary ophthalmologist over a 56-day observation period. This was evidenced by:• Inflammation scores from ocular exam observations were generally low (0-1) in the ocular exams for all groups throughout the study• Mean intraocular pressure was in normal range (17-21 mmHg) for all groups throughout the study• ADA titers were generally low (i.e. <10,000) in any of the tissues for all groups throughout the study• Microscopical examination scores of the retina were generally low (0-1) for all groups throughout the study

[0241] No dose-dependent toxicity was identified in any of the tolerability outcomes.

[0242] Although the foregoing invention has been described in some detail by way of illustration and Example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.Attorney Docket No.: 052566-511001 WOWhere a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.Table 25. SequencesAttorney Docket No.: 052566-511001 WO

Claims

Attorney Docket No.: 052566-511001 WOWHAT IS CLAIMED IS:

1. A conjugate that is a random polymer of Formula Illa: (X1-X2-Y-Z1)n-(Z2)p-(Z3)q(Illa), having a molecular weight of about 0.8 MDa; wherein each X1is a peptide having an anti-VEGF amino acid sequence comprising SEQ ID NO: 96; each X2is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO: 21); each Y is an organic linker having the structure:each X1-X2-Y-Z1moiety has the structure:each Z2has the structure:each Z3independently has the structure:Attorney Docket No.: 052566-511001 WOeach Z3ais independently OH or Y'; each Y' has the structure:each R1and R2is ethyl or -(CH2)3-NMe2; subscript n is an integer of from 10 to 300 and less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer of from 1 to 15 and less than about 0.5% of the sum of subscripts n, p, and q; and subscript q is an integer of from 1000 to 3000.

2. A pharmaceutical composition comprising a conjugate of claim 1, and a pharmaceutically acceptable excipient.

3. A method of treating an ocular disorder in a subject in need thereof, comprising administering to the subject a conjugate of claim 1.

4. The method of claim 3, comprising intravitreally administering the conjugate.

5. The method of claim 3 or 4, comprising administering the conjugate every month, every two months, every three months, every six months, every nine months, or every 12 months.

6. The method of any one of claims 3 to 5, wherein the vitreous half-life of the conjugate is at least 4-fold greater than the half-life of the unconjugated peptide.Attorney Docket No.: 052566-511001 WO7. The method of any one of claims 3 to 6, wherein the ocular disorder is uveitis, macular degeneration, choroidal neovascularization, retinal neovascularization, proliferative vitreoretinopathy, glaucoma, or ocular inflammation.

8. A method of treating a disease or disorder in an articular joint in a subject in need thereof, comprising administering to the subject a conjugate of claim 1.

9. The method of claim 8, comprising intraarticularly administering the conjugate.

10. The method of claim 8 or 9, comprising administering the conjugate every month, every two months, every three months, every six months, every nine months, or every 12 months.

11. The method of any one of claims 8 to 10, wherein the intraarticular half-life of the conjugate is at least 4-fold greater than the half-life of the unconjugated peptide.

12. The method of any one of claims 8 to 11, wherein the disease or disorder is rheumatoid arthritis, wear-related osteoarthritis, age-related osteoarthritis, post- traumatic osteoarthritis, psoriatic arthritis, and aseptic implant loosening, joint effusion, ankylosing spondylitis, bursitis, gout, reactive arthritis, synovitis, or avascular necrosis.

13. Use of a conjugate of claim 1 in the manufacture of a medicament for treating an ocular disorder, or a disease or disorder in an articular joint, in a subject in need thereof.

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