Methods of Treating Uveitis with Multivalent Protein-Hyaluronic Acid Polymer Conjugates

Abstract

The present invention relates to multivalent protein-polymer conjugates, compositions and methods for treating uveitis, such as chronic non-infectious uveitis.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 331,554, filed April 15, 2022, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing The material in the attached Sequence Listing is hereby incorporated by reference in its entirety. 2023-04-11 Sequence_Listing_ST26 The attached file named 052566-508001WO.xml was created on April 11, 2023 and is 117,702 bytes in size. [Background technology]

[0003] The use of biopolymers to alter the properties of bioactive substances is a recurring theme across a wide range of medical and biological applications. By attaching bioactive peptides or proteins to biopolymers using various chemical linkers, the pharmacological properties of the resulting conjugates can be modified and used as drugs to provide optimal treatment for specific diseases. Peptide-polymer conjugates, including multiple copies of one or more peptides conjugated to a single biopolymer chain, have been employed to provide specific improvements in the pharmacological properties of the peptides, including (1) increased binding affinity to biological targets, (2) reduced diffusibility in target tissues, and (3) inhibition of proteases that may inactivate the biological activity of the peptide or protein.

[0004] These improved pharmacological properties of peptide-polymer conjugates are particularly useful for delivering potent drugs directly to affected tissues. The dose delivered directly to tissues can be lower than would be required to achieve the same therapeutic effect after systemic administration because the drug is administered locally to the target tissue. It also allows the administration of drugs to tissues that are otherwise poorly transported from the blood. Specific examples of tissues where direct drug administration is common include the posterior chamber of the eye via intravitreal injection and the joint via intra-articular injection.

[0005] However, local tissue administration requires a specialist to safely provide the necessary injections, which makes local tissue administration more cumbersome and costly compared to systemic administration. When peptide drugs are administered as part of a peptide-polymer conjugate, the frequency of drug administration can be substantially reduced, thereby reducing the burden on patients to receive effective treatment. Furthermore, the reduced number of local injections reduces the risk of local tissue injury or adverse effects to the injection. And, the less frequent administration can reduce the time that the drug concentration in the target tissue falls below the therapeutic concentration, thereby improving the overall efficacy of the drug. These advantages have increased the motivation for the development of protein-polymer formulations for various diseases.

[0006] Uveitis is a group of sight-threatening intraocular inflammatory diseases that are responsible for approximately 5-10% of cases of blindness worldwide. Chronic non-infectious uveitis can result in nerve damage and vision loss. Most patients are treated with corticosteroids, which can have serious side effects. Intravitreal administration of biologic TNFα inhibitors can substantially reduce the need for steroids. However, these products have not been designed or validated for intravitreal use, and off-label intravitreal treatment with existing TNFα inhibitors is not recommended.

[0007] Thus, there is a need to develop purified peptide-polymer conjugates and methods for treating uveitis, such as chronic non-infectious uveitis. The present invention addresses these and other needs. Summary of the Invention

[0008] In some embodiments, the method of the present invention provides a method for treating uveitis in a subject in need thereof, comprising administering to the subject a conjugate of Formula V: (XY) n -Z (V), (In the formula, each X is, independently, an anti-inflammatory peptide having a molecular weight of about 5 kDa to about 200 kDa; each Y is an organic linker; Z is a hyaluronic acid polymer having a molecular weight of about 0.1 MDa to about 3 MDa; The subscript n is an integer from 1 to 1000. The method comprises administering a therapeutically effective amount of

[0009] In some embodiments, the conjugate of the present invention is a random polymer of formula VI having a molecular weight of about 0.1 MDa to about 3 MDa: (XYZ 1 ) n -(Z 2 ) p -(Z 3 ) q (VI), During the ceremony, Each X is, independently of the other, an anti-TNF-α peptide or an anti-IL-1β peptide, including: QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSPSTPPTPSPSTPPGGCDDDDK (SEQ ID NO: 101), QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 102), QVQLQDSGGGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGTTVYADSVLGRFEISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 103), QVQLQESGGGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGTTVYADSVKGRFTISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 104), CGGGVDNKFNKEVGWAFGEIGALPNLNALQFRAFIISLWDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 105), or EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSSSPSTPPTPSPSTPPGGC (SEQ ID NO: 106); Each Y is an organic linker having the structure: [ka] Each XYZ 1The moiety has the structure: [ka] Each Z 2 has the following structure: [ka] Each Z 3 have, independently of each other, the structure: [ka] Each R 1 and R 2 are each independently C1-C6 alkyl, -(C1-C6 alkyl)-NR 3 R 4 or C5-C8 cycloalkyl; Each R 3 and R 4 are, independently of each other, H or C1-C6 alkyl; Each Z 3a are, independently of each other, OH or Y'; each Y' is an unreacted organic linker; subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of subscripts n, p, and q; The subscript q is an integer from 100 to 10,000. It is a random polymer. [Brief description of the drawings]

[0010] [Figure 1A] Figure 1 shows SDS-PAGE images of representative anti-inflammatory peptide polymer conjugates by SEQ ID NO: Figure 1A: (A) SDS-PAGE image of (SEQ ID NO: 101) + HyA (850 kDa) conjugate #2 with valency 55 compared to unconjugated VHH. [Figure 1B]Figure 1 shows SDS-PAGE images of representative anti-inflammatory peptide polymer conjugates by SEQ ID NO: (B) SDS-PAGE image of (SEQ ID NO: 102) + HyA (850 kDa) conjugate #3 of valency 65 compared to unconjugated VHH. [Figure 1C] Figure 1 shows SDS-PAGE images of representative anti-inflammatory peptide polymer conjugates by SEQ ID NO: (C) SDS-PAGE image of (SEQ ID NO: 103) + HyA (850 kDa) conjugate #5 of valency 121 compared to unconjugated VHH. [Figure 1D] Figure 1 shows SDS-PAGE images of representative anti-inflammatory peptide polymer conjugates by SEQ ID NO: (D) SDS-PAGE image of (SEQ ID NO: 104) + HyA (850 kDa) conjugate #6 of valency 51 compared to unconjugated VHH. [Figure 1E] Figure 1 shows SDS-PAGE images of representative anti-inflammatory peptide polymer conjugates by SEQ ID NO: (E) SDS-PAGE image of valency 21 (SEQ ID NO: 105) + HyA (850 kDa) conjugate #8 compared to unconjugated affibody. [Figure 1F] Figure 1 shows SDS-PAGE images of representative anti-inflammatory peptide polymer conjugates by SEQ ID NO: 1. Figure 1F shows an SDS-PAGE image of (F) valency 15 (SEQ ID NO: 106) + HyA (850 kDa) conjugate #9. [Figure 2A] Figure 2A shows that (A) the TNFα binding affinity of the valency 120 (SEQ ID NO: 102) + HyA (850 kDa) conjugate #4 is greater than that of unconjugated TNFα as determined by biolayer interferometry (***p<0.001, Student's t-test, n=3). The dashed line indicates the detection limit of the instrument, below which the binding affinity of the conjugate is lower. [Figure 2B]FIG. 2B shows (B) the bioactivity of valency 9 (SEQ ID NO: 101)+HyA (850 kDa) conjugate #1 in inhibiting TNFα-induced apoptosis in L929 fibroblasts, approximately 10-fold greater than unconjugated VHH. [Diagram 3] FIG. 3 shows that the hydrodynamic radius of conjugate #3 consisting of (SEQ ID NO: 102) + HyA (850 kDa) with a valency of 65 is greater than that of the unconjugated VHH (***p<0.001, Student's t-test, n=3). [Figure 4A] Figure 4A shows (A) normalized absorbance at 280 nm ("A280") of the unconjugated protein (SEQ ID NO: 102) with increasing temperature. The oxidized version of the VHH showed minimal absorbance change when the temperature was increased from 37°C to 50°C, while the reduced construct showed an increase in absorbance starting at 50°C, indicating that the reduced construct was unfolded and less thermostable. Error bars represent SD. [Figure 4B] FIG. 4B shows (B) the change in binding constant of TNFα binding affinity for mu_anti-TNFα_aH_CYS conjugates with or without the 3Mut stability enhancing mutation after incubation at 37° C. for the indicated number of days, as measured by biolayer interference. The samples used were either (SEQ ID NO: 103)+HyA(850 kDa) conjugate #5 with valency 121 ("mu_anti-TNFα_aH_CYS MVP") or (SEQ ID NO: 104)+HyA(850 kDa) conjugate #6 with valency 51 ("mu_anti-TNFα_3Mut_aH_CYS MVP"). After 4 days at 37° C., there was little change in the binding constant of conjugate #6 up to 35 days at 37° C. In contrast, the binding constant of the non-mutated conjugate #5 was dramatically decreased after 5 days at 37° C., indicating a decreased stability. [Figure 4C]FIG. 4C shows (C) representative DLS data of conjugate size in vitreous mimicking buffer after incubation at 37° C. for up to 35 days. Each conjugate was made with (SEQ ID NO: 104) + HyA (850 kDa) at valence 51 (conjugate #6, “mu_anti-TNFα_3Mut_aH_CYS MVP 1:51”) or (SEQ ID NO: 104) + HyA (850 kDa) at valence 98 (conjugate #7, “mu_anti-TNFα_3Mut_aH_CYS MVP 1:98”). There was no significant difference in MVP size based on the valence range of about 50 to about 100 antibodies per polymer. Each conjugate slowly decreased in size to about 75% of the original radius after 35 days at 37° C. Error bars represent SD. [Diagram 5] Figure 5 shows that conjugation can extend the intravitreal half-life of anti-inflammatory therapeutics in a rabbit intravitreal pharmacokinetic model. Each rabbit received an equimolar 50μL intravitreal injection of either unconjugated SEQ ID NO: 102 or valency 120 (SEQ ID NO: 102) + HyA (850kDa) conjugate #4. Intravitreal half-life was determined using nonlinear fitting of VHH concentrations at each time point. Multivalent conjugation extended half-life by at least 2-fold compared to unconjugated VHH. [Figure 6A] FIG. 6A shows that anti-TNFα conjugates significantly suppressed ocular inflammation in a rat experimental autoimmune uveitis model. Conjugate #7 was made with (SEQ ID NO: 104) + HyA (850 kDa) and valency 98. (A) Mean inflammation scores (0=none to 4=severe) observed in rat eyes after EAU induction in rats (n=8) intravitreally injected with vehicle, dexamethasone (5 μg), or conjugate #7 ("mu_anti-TNFα_3Mut_aH_CYS MVP") (12.5 μg). One day after ITV treatment, both dexamethasone- and conjugate-treated eyes showed reduced inflammation compared to vehicle controls. [Figure 6B]FIG. 6B shows (B) the mean histological inflammation scores (0=none to 4=severe) in the same rat cohort sacrificed 14 days after model induction. Left bar: vehicle, middle bar: after dexamethasone treatment, right bar: after conjugate #7 treatment. Conjugate-treated eyes (right bar) were less inflamed than vehicle-treated eyes (left bar) (*p<0.05, Student's t-test). [Figure 7A] FIG. 7A shows that anti-TNFα conjugates significantly suppressed ocular inflammation in a rat experimental autoimmune uveitis model. Conjugate #10 was made with (SEQ ID NO: 104) + HyA (850 kDa) and a valency of 96.5. (A) Mean inflammation scores (0=none to 4=severe) observed in rat eyes after EAU induction in rats intravitreally injected with vehicle (n=24), triamcinolone (40 μg) (n=22), or conjugate #10 ("Conjugate ID: 10") (19 μg) (n=20). One day after ITV treatment, both triamcinolone- and conjugate-treated eyes showed reduced inflammation compared to vehicle controls. There were no statistically significant differences in measurements between triamcinolone and conjugate #10 at any time point. There was a significant difference between conjugate #10 and vehicle on days 10 (p=0.002), 12 (p<0.001), and 14 (p=0.007). [Figure 7B] FIG. 7B shows (B) cytokine analysis of vitreous samples prepared from rat eyes intravitreally injected with vehicle (n=24), triamcinolone (40 μg) (n=22), or conjugate #10 ("Conjugate ID:10") (19 μg) (n=20). Bar graphs show concentrations of key pro-inflammatory cytokines and inflammatory regulators. [Figure 8A]FIG. 8A shows that anti-TNFα conjugates significantly suppressed ocular inflammation in a rabbit TNFα-induced ocular inflammation model. Conjugate #11 was made with (SEQ ID NO: 102) + HyA (850 kDa) and a valency of 132. (A) Mean inflammation score over time (0=none to 4=severe) observed in rabbit eyes following inflammation induction with 7.5 μg TNFα in rabbits intravitreally injected with vehicle, or conjugate #11 ("Conjugate ID11") (0.26 mg) or not (n=26). In rabbits treated with 7.5 μg TNFα, there was statistical significance between conjugate #11-treated eyes and vehicle-treated eyes at 24 and 48 hours. [Figure 8B] Figure 8B shows that (B) after 48 hours, at all TNFα dose concentrations, conjugate #11 treated eyes showed reduced inflammation compared to vehicle controls 48 hours after inflammation induction, with statistically significant differences observed at TNFα doses of 5.0 μg (p=0.03) and 7.5 μg (p<0.001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] I. Definition Unless otherwise specifically indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any methods or materials similar or equivalent to those described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined:

[0012] When "about" refers to a numerical value, it includes the stated value plus or minus 10% of the stated value. For example, about 50% includes the range of 45% to 55%, and about 20 molar equivalents includes the range of 18 molar equivalents to 22 molar equivalents. Thus, when referring to a range, "about" refers to the stated value plus or minus 10% of the stated value at each endpoint of the range. For example, a ratio (weight / weight) of about 1 to about 3 includes the range of 0.9 to 3.3.

[0013] "Alkyl" refers to a straight or branched, saturated, monovalent or divalent hydrocarbon. For example, an alkyl group can have 1 to 10 carbon atoms (i.e., C 1~10 alkyl), having 1 to 8 carbon atoms (i.e., C 1~8 alkyl), having 1 to 6 carbon atoms (i.e., C 1~6 alkyl), or may have 1 to 4 carbon atoms (i.e., (C 1~4Examples of alkyl groups include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)C H2CH3), 2-Methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-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 (-CH2CH2C H(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4- These include, but are not limited to, 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).

[0014] "Cycloalkyl" refers to a single, saturated or partially unsaturated, all-carbon ring (i.e., C 3~20The term "cycloalkyl" refers to a cycloalkyl group having, for example, 3 to 12 ring atoms, such as 3 to 10 ring atoms, or 3 to 8 ring atoms, or 3 to 6 ring atoms, or 3 to 5 ring atoms, or 3 to 4 ring atoms, etc. The term "cycloalkyl" also includes all polycyclic, fused, saturated and partially unsaturated carbocyclic ring systems (e.g., ring systems containing 2, 3, or 4 carbocyclic rings). Thus, cycloalkyl includes multicyclic carbocyclic rings such as bicyclic carbocyclic rings (e.g., bicyclic carbocyclic rings having about 6 to 12 ring carbon atoms, such as bicyclo[3.1.0]hexane and bicyclo[2.1.1]hexane), and polycyclic carbocyclic rings (e.g., tricyclic and tetracyclic carbocyclic rings having up to about 20 ring carbon atoms). The rings of a polycyclic fused ring system may be linked to each other via fused bonds, spiro bonds, and bridged bonds, where valency requirements permit. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3-enyl.

[0015] "Organic linker," as used herein, refers to a chemical moiety that covalently links, directly or indirectly, a peptide to a polymer. Organic linkers useful in the present invention can be from about 100 Da to about 500 Da. Types of organic linkers of the present invention include, but are not limited to, imides, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas. It will be appreciated by those of skill in the art that other types of organic linkers are also useful in the present invention.

[0016] "Thiol" refers to the -SH functional group.

[0017] "Thiol-reactive group" refers to a group capable of reacting with a thiol to form a covalent bond to a sulfur atom. Exemplary thiol-reactive groups include, but are not limited to, thiol, TNB-thiol, haloacetyl, aziridine, acryloyl, vinylsulfone, APN (3-arylpropiolonitrile), maleimide, and pyridyl disulfide. The reaction of a thiol-reactive group with a thiol can form a disulfide or a thioether.

[0018] "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 natural amino acids. The term "peptide" encompasses fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, with or without an N-terminal methionine residue, fusions with heterologous and homologous leader sequences; immunologically tagged proteins; and the like. Peptides also encompass post-translationally modified peptides.

[0019] "VHH" as used herein refers to a single domain heavy chain antibody.

[0020] "DARPin" refers to designed ankyrin repeat proteins, which are genetically engineered antibody-mimetic proteins capable of exhibiting highly specific, high-affinity target protein binding.

[0021] An "alpha helix" or "α helix" is a common motif in protein secondary structure, a right-handed conformation in which all backbone NH groups are hydrogen bonded to the backbone C=O group of the amino acid located four residues earlier along the protein sequence. An α helix is ​​classically referred to as the Pauling-Corey-Branson α-helix or 3.6 13 -Also known as Helix, this 3.6 13represents the average number of residues per helix turn (3.6) and that the ring formed by hydrogen bonds contains 13 atoms. Peptides containing an α-helix are said to be α-helical. Such peptides may be partially α-helical or entirely α-helical. As understood in the art, an α-helix has at least four amino acid residues. In some embodiments, an α-helix has between 4 and 40 amino acids.

[0022] Also provided are pharma- ceutically acceptable salts of the compounds or peptides described herein. "Pharmaceutically acceptable" or "physiologically acceptable" refers to compounds, salts, compositions, dosage forms, and other materials that are useful in the preparation of medicinal compositions suitable for pharmaceutical use in animals or humans.

[0023] "Pharmaceutical composition" as used herein refers to a product that contains specific ingredients in specific amounts, and any product that is the result of combining, directly or indirectly, specific ingredients in specific amounts. Pharmaceutical compositions are generally safe for biological use.

[0024] "Pharmaceutically acceptable excipients" as used herein refers to substances that aid in the administration and absorption of active agents by a subject. Pharmaceutically acceptable excipients useful in the present invention include, but are not limited to, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, and coloring agents. Those skilled in the art will recognize that other pharma-ceutically acceptable excipients are also useful in the present invention.

[0025] The conjugates described herein can be prepared and / or formulated as pharma- ceutically acceptable salts, or as free bases, if appropriate. Pharmaceutically acceptable salts are non-toxic salts of the compounds in free base form, which have the desired pharmacological activity of the free base. These salts can be derived from inorganic or organic acids or bases. For example, conjugates containing a basic nitrogen can be prepared as pharma- ceutically acceptable salts by contacting the compound with an inorganic or organic acid. Non-limiting examples of pharma- ceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioxide, hex ... Examples of suitable pharma- ceutically acceptable salts include 1,6-diacidote, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, besylate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, and mandelate. A list of other suitable pharma-ceutically acceptable salts can be found in Remington: The Science and Practice of Pharmacy, 21 st Edition, Lippincott William and Wilkins, Philadelphia, Pa., 2006.

[0026] Examples of "pharmaceutically acceptable salts" of the conjugates disclosed herein include alkali metal (e.g., sodium, potassium), alkaline earth metal (e.g., magnesium), ammonium and NR4 +(wherein R is C1-C4 alkyl), as well as base addition salts such as sodium or potassium salts.

[0027] "Therapeutically effective amount" as used herein refers to a dosage that produces the therapeutic effect that is intended for administration. The exact dosage will depend on the therapeutic purpose and can be ascertained by those skilled in the art using known methods (see, for example, 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 dosage may be lower than the conventional therapeutically effective dosage for non-sensitized cells.

[0028] "Treatment" or "treat" or "treating" as used herein refers to an approach to obtain a beneficial or desired result. For purposes of this disclosure, beneficial or desired results include, but are not limited to, alleviating symptoms and / or reducing the severity of symptoms and / or preventing the worsening of symptoms associated with a disease or condition. In one embodiment, "treatment" or "treating" includes one or more of the following: a) inhibiting a disease or condition (e.g., reducing one or more symptoms caused by a disease or condition and / or reducing the severity of a disease or condition); b) slowing or arresting the development of one or more symptoms associated with a disease or condition (e.g., stabilizing a disease or condition, slowing the worsening or progression of a disease or condition); and c) relieving a disease or condition, e.g., causing regression of clinical symptoms, ameliorating the condition, slowing the progression of a disease, increasing quality of life, and / or prolonging survival.

[0029] "Prophylaxis" refers to preventing or slowing the progression of a clinically morbid state in a patient suffering from a disease.

[0030] A "subject" of the present invention is a mammal, which may be a human or a non-human 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. In some embodiments, the subject is a human.

[0031] II. Conjugates In some embodiments, the conjugate of the invention is a conjugate of Formula V: (XY) n -Z (V), During the ceremony, each X is, independently, an anti-inflammatory peptide having a molecular weight of about 5 kDa to about 200 kDa; each Y is an organic linker; Z is a hyaluronic acid polymer having a molecular weight of about 0.1 MDa to about 3 MDa; The subscript n is an integer from 1 to 1000. It is a conjugate.

[0032] In some embodiments, each X is, independently of the other, an anti-TNF-α peptide or an anti-interleukin-1β peptide.

[0033] In some embodiments, each X is a monoclonal IgG, an IgG fragment, a single chain scFv, a single domain heavy chain VHH, an adnectin, an affibody, an anticalin, a DARPin, or an engineered Kunitz-type inhibitor. In some embodiments, each X is a monoclonal IgG. In some embodiments, each X is an IgG fragment. In some embodiments, each X is a single domain heavy chain VHH. In some embodiments, each X is a DARPin.

[0034] In some embodiments, each X is a peptide having an amino acid sequence including any one of SEQ ID NOs: 61-73, 81-85, 91-98, 101-109, 111-118, and 145-151. In some embodiments, each X is a peptide having an amino acid sequence including any one of SEQ ID NOs: 101-109 and 148-154.

[0035] In some embodiments, each X is a peptide having an amino acid sequence including any one of SEQ ID NOs: 61-73, 81-85, 91-95, 101-106, and 111-118.

[0036] In some embodiments, each X is a peptide having an amino acid sequence comprising: QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSPSTPPTPSPSTPPGGCDDDDK (SEQ ID NO: 101), QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 102), QVQLQDSGGGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGTTVYADSVLGRFEISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 103), QVQLQESGGGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGTTVYADSVKGRFTISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 104), CGGGVDNKFNKEVGWAFGEIGALPNLNALQFRAFIISLWDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 105), or EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSSSPSTPPTPSPSTPPGGC (sequence number 106).

[0037] 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. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 106.

[0038] Each peptide may be linked to a biocompatible polymer by a variety of organic linkers commonly known in the art for forming antibody-drug conjugates, such as those provided by BroadPharm, San Diego, Calif. Methods for forming bioconjugate bonds are described in Bioconjugate Techniques, 3 (2003) and elsewhere in this publication. rd Edition, Greg T. Hermanson. The organic linkers are reactive with amines, carbonyls, carboxyls, and activated esters, or react via click chemistry (in the presence or absence of copper), or are reactive with thiols.

[0039] Representative organic linkers include amides or disulfides, or are formed from reactive groups such as succinic anhydride, succinimide, N-hydroxysuccinimide, N-chlorosuccinimide, N-bromosuccinimide, maleic anhydride, maleimide, hydantoin, phthalimide, and the like. Organic linkers useful in the present invention are small molecules, typically having a molecular weight of about 100 Da to about 500 Da, and containing two functional groups consisting of maleimide and amine or hydrazide. In some embodiments, the peptide is covalently linked to the polymer via a sulfide bond and an organic linker having a molecular weight of about 100 Da to about 500 Da. In some embodiments, the organic linker has a molecular weight of about 100 Da to about 300 Da. In some embodiments, the organic linker comprises succinimide. In some embodiments, the organic linker is formed using N-β-maleimidopropionic acid hydrazide (BMPH), N-ε-maleimidocaproic acid hydrazide (EMCH), N-aminoethylmaleimide, N-κ-maleimidoundecanoic acid hydrazide (KUMH), hydrazide-PEG2-maleimide, amine-PEG2-maleimide, hydrazide-PEG3-maleimide, or amine-PEG3-maleimide.

[0040] In some embodiments, the organic linker has the structure: [ka]

[0041] In some embodiments, the organic linker can be N-ε-maleimidocaproic acid hydrazide (EMCH). [ka]

[0042] In some embodiments, the organic linker has the structure: [ka] In the above formula, the subscript m is an integer from 1 to 300. In some embodiments, the subscript m is an integer from 1 to 100.

[0043] In some embodiments, the organic linker has the structure: [ka] The organic linker having the above structure is known as MP2H.

[0044] In some embodiments, each Y is an organic linker having the structure: [ka] In the above formula, the subscript m is an integer from 1 to 300.

[0045] In some embodiments, Z has a molecular weight of about 0.4 MDa to about 2 MDa. In some embodiments, Z has a molecular weight of about 0.7 MDa to about 1.5 MDa. In some embodiments, Z has a molecular weight of about 0.8 MDa.

[0046] In some embodiments, the conjugate of formula V has the structure of formula Va: (X 1 -X 2 -Y) n -Z (Va), (In the formula, Each X 1 is an anti-TNF-α peptide or an anti-interleukin-1β peptide having a molecular weight of about 5 kDa to about 200 kDa; Each X 2 is an α-helix-containing peptide linker; Each Y is an organic linker having the structure: [ka] In the above formula, Z is a hyaluronic acid polymer having a molecular weight of about 0.1 MDa to about 3 MDa; The subscript m is an integer from 1 to 300. has.

[0047] In some embodiments, each X 1 is a peptide having an amino acid sequence including any one of SEQ ID NOs: 61 to 73, 81 to 85, 91 to 98, 101 to 109, and 111 to 118.

[0048] In some embodiments, each X 2 is a peptide linker having an amino acid sequence comprising: AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21), AEEEKRKAEEEKRKAEEEAGC (SEQ ID NO:22), AEEEKRKAEEEKRKAEEEKRKAEEEAGC (SEQ ID NO:23), AEEEEKKKKEEEEKKKKAGC (SEQ ID NO:24), AEAAAKEAAAKAGC (SEQ ID NO:25), PSRLEEELRRRLTEGC (SEQ ID NO:26), or AEEEEKKKQQEEEAERLRRIQEEMEKERKRREEDEERRRKEEEERRMKLEMEAKRKQEEEEERKKREDDEKRKKKAGC (sequence number 27).

[0049] In some embodiments, each X 2 is a peptide linker having an amino acid sequence comprising AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21).

[0050] In some embodiments, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO: 107, 2 is a peptide linker having an amino acid sequence comprising SEQ ID NO: 21. In some embodiments, each X 1is a peptide having an amino acid sequence comprising SEQ ID NO: 108, 2 is a peptide linker having an amino acid sequence comprising SEQ ID NO: 21. In some embodiments, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO: 109, 2 is a peptide linker having an amino acid sequence comprising SEQ ID NO:21.

[0051] In some embodiments, the conjugate of formula V is a random polymer of formula VI having a molecular weight of about 0.1 MDa to about 3 MDa: (XYZ 1 ) n -(Z 2 ) p -(Z 3 ) q (VI), During the ceremony, each X is, independently, an anti-inflammatory peptide having a molecular weight of about 5 kDa to about 200 kDa; each Y is an organic linker; Each XYZ 1 The moiety has the structure: [ka] Each Z 2 has the following structure: [ka] Each Z 3 have, independently of each other, the structure: [ka] Each R 1 and R 2 are each independently C1-C6 alkyl, -(C1-C6 alkyl)-NR 3 R 4 or C5-C8 cycloalkyl; Each R 3 and R 4are, independently of each other, H or C1-C6 alkyl; Each Z 3a are, independently of each other, OH or Y'; each Y' is an unreacted organic linker; subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of subscripts n, p, and q; The subscript q is an integer from 100 to 10,000.

[0052] In some embodiments, each X is a peptide having an amino acid sequence including any one of SEQ ID NOs: 61-73, 81-85, 91-98, 101-109, 111-118, and 145-154. In some embodiments, each X is a peptide having an amino acid sequence including any one of SEQ ID NOs: 101-109 and 148-154.

[0053] In some embodiments, each R 1 and R 2 are each independently C1-C3 alkyl or -(C1-C3 alkyl)-NR 3 R 4 In some embodiments, each R 1 and R 2 is ethyl or -(CH)-NMe. In some embodiments, each R 1 is ethyl; each R 2 is -(CH2)3-NMe2. In some embodiments, each R 1 is -(CH2)3-NMe2; 2 is ethyl.

[0054] In some embodiments, each R 3 and R 4 are each independently C1 to C3 alkyl.

[0055] In some embodiments, the preparation of the conjugate of the present invention includes covalently attaching an organic linker to a biocompatible polymer, and then covalently attaching a peptide to the organic linker. In some embodiments, after the preparation of the conjugate of the present invention, an unreacted organic linker is present on the biocompatible polymer. The structure of the unreacted organic linker depends on the organic linker, as will be understood by those skilled in the art.

[0056] Representative unreacted organic linkers include, but are not limited to, the following: [ka]

[0057] In some embodiments, the unreacted organic linker has the structure: [ka]

[0058] In some embodiments, the unreacted organic linker has the structure: [ka] wherein the subscript m is an integer from 1 to 300. In some embodiments, the subscript m is an integer from 1 to 100.

[0059] In some embodiments, the unreacted organic linker has the structure: [ka]

[0060] In some embodiments, the organic linker has the structure: [ka] The unreacted organic linker has the structure: [ka]

[0061] In some embodiments, the conjugate is a random polymer of formula VI having a molecular weight of about 0.1 MDa to about 3 MDa: (XYZ 1 ) n -(Z 2 ) p -(Z 3 ) q (VI), During the ceremony, Each X is, independently of the other, an anti-TNF-α peptide or an anti-IL-1β peptide, including: QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSPSTPPTPSPSTPPGGCDDDDK (SEQ ID NO: 101), QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 102), QVQLQDSGGGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGTTVYADSVLGRFEISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 103), QVQLQESGGGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGTTVYADSVKGRFTISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 104), CGGGVDNKFNKEVGWAFGEIGALPNLNALQFRAFIISLWDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 105), or EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSSSPSTPPTPSPSTPPGGC (SEQ ID NO: 106); Each Y is an organic linker having the structure: [ka] Each XYZ 1 The moiety has the structure: [ka] Each Z 2 has the following structure: [ka] Each Z 3 have, independently of each other, the structure: [ka] Each R 1 and R 2are each independently C1-C6 alkyl, -(C1-C6 alkyl)-NR 3 R 4 or C5-C8 cycloalkyl; Each R 3 and R 4 are, independently of each other, H or C1-C6 alkyl; Each Z 3a are, independently of each other, OH or Y'; each Y' is an unreacted organic linker; subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of subscripts n, p, and q; The subscript q is an integer from 100 to 10,000.

[0062] In some embodiments, subscript n is an integer between 1 and 1500 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 1000 and is less than about 10% of the sum of subscripts n, p, and q; and subscript q is an integer between 100 and 10000. In some embodiments, subscript n is an integer between 1 and 1000 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 800 and is less than about 8% of the sum of subscripts n, p, and q; and subscript q is an integer between 100 and 10000. In some embodiments, subscript n is an integer between 10 and 450 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 300 and is less than about 10% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000. In some embodiments, subscript n is an integer between 10 and 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 240 and is less than about 8% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000. In some embodiments, subscript n is an integer between 10 and 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 60 and is less than about 2% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000. In some embodiments, subscript n is an integer between 10 and 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 30 and is less than about 1% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000. In some embodiments, subscript n is an integer between 10 and 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 15 and is less than about 0.5% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000.

[0063] In some embodiments, the conjugates of the invention are for use in methods of treating uveitis as described herein.

[0064] III. Composition In some embodiments, the invention relates to a pharmaceutical composition as described herein. In some embodiments, the pharmaceutical composition is a pharmaceutical composition comprising a conjugate as described herein and a pharma- ceutical acceptable excipient.

[0065] A. Formulation When formulating pharmaceutical compositions from the conjugates of the present invention, pharma- ceutically acceptable carriers can be solid or liquid. Solid formulations include powders, cachets, and dispersible granules. A solid carrier can be one or more substances that may act as diluents, binders, preservatives, disintegrants, or encapsulating materials. Details regarding formulation and administration techniques are fully explained in the scientific and patent literature, see, for example, the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. ("Remington's").

[0066] In powders, the carrier is a finely divided solid, in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compressed into the desired shape and size. Powders and tablets preferably contain 5% or 10% to 70% of the conjugate of the present invention.

[0067] Liquid preparations include solutions, suspensions, and emulsions, such as, for example, water or water / propylene glycol solutions.For parenteral injection, liquid preparations can be formulated in aqueous polyethylene glycol solution.

[0068] Aqueous solutions suitable for oral use can be prepared by dissolving the conjugate of the invention in water and adding suitable colorants, flavors, stabilizing, and thickening agents, as desired. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active ingredient in water containing viscous substances such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic, and dispersing or wetting agents such as natural phosphatides (e.g., lecithin), condensations of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensations of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensations of ethylene oxide with fatty acids and partial esters derived from hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensations of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspensions may 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. The preparations may be adjusted for osmolality.

[0069] Also included are solid preparations, which are intended to be converted into liquid preparations for oral administration immediately before use. Such liquid preparations include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active ingredient, colorants, flavorings, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, etc.

[0070] Oil suspensions can be formulated by suspending the conjugate of the present invention in a vegetable oil, such as peanut oil, olive oil, sesame oil, or coconut oil, or a mineral oil, such as liquid paraffin; or a mixture thereof. Oil suspensions can contain thickening agents, such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners can be added to provide a palatable oral preparation, such as glycerol, sorbitol, or sucrose. These preparations can be preserved by the addition of an antioxidant, such as ascorbic acid. For examples of oil vehicles for injection, see Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997. The pharmaceutical preparation of the present invention can also be in the form of an oil-in-water emulsion. The oily phase can be a vegetable oil or a mineral oil, as described above, or a mixture thereof. Suitable emulsifying agents include natural gums such as gum arabic and gum tragacanth, natural 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 monooleate. Emulsions can also contain sweetening and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain demulcents, preservatives, or coloring agents.

[0071] The compositions of the present invention can also be delivered as microspheres for sustained release in the body. For example, microspheres can be formulated for administration via intradermal injection of drug-containing microspheres that slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995); for administration as a biodegradable injectable gel formulation (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or for administration as oral microspheres (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). Both transdermal and intradermal routes allow constant delivery over a period of weeks or months.

[0072] In another embodiment, the composition of the present invention can be formulated for parenteral administration into a body cavity, such as intravitreal administration into the eye or into the articular cavity of a joint. The formulation for administration will usually comprise a solution of the composition of the present invention dissolved in a pharma- ceutically acceptable carrier. Among the acceptable vehicles and solvents that can be used are water and Ringer's solution, which is an isotonic saline solution. In addition, sterile, fixed oils can be conventionally used as a solvent or suspending medium. For this purpose, any bland, fixed oil can be used, for example, synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can also be used in the formulation of injections. These solutions are sterile and usually free of undesirable substances. These formulations can be sterilized by conventional, well-known sterilization methods. The formulations may contain pharma-ceutically acceptable auxiliary substances as necessary to approximate physiological conditions, for example, pH adjusting agents such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, pH buffers, toxicity adjusting agents, and the like. The concentration of the composition of the present invention in these preparations can vary widely and will be selected according to the specific mode of administration selected and the patient's requirements, mainly based on the volume of fluid, viscosity, body weight, etc. For IV or intravitreal administration, the preparation can be a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. This suspension can be formulated according to known technology using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a 1,3-butanediol solution.

[0073] In another embodiment, the formulation of the composition of the present invention can be delivered by using liposomes that fuse with cell membranes, i.e., undergo endocytosis, i.e., by using a ligand that binds to a cell surface membrane protein receptor that causes endocytosis, either attached to the liposome or directly attached to the oligonucleotide. The use of liposomes can focus the delivery of the composition of the present invention to target cells in vivo, especially when the liposome surface carries a ligand specific to the target cell or is otherwise preferentially targeted to a particular organ. (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).

[0074] 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 cosurfactants that can spontaneously disperse 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 oil, olive oil, castor oil, peanut oil, fatty acid esters, glycerol esters, Labrafil®, Labrasol®, Cremophor®, Solutol®, Tween®, Capryol®, Capmul®, Captex®, and Peceol®.

[0075] B. Administration The conjugates and compositions of the invention can be delivered by any suitable means, including oral, parenteral, and topical, hi some embodiments, the delivery method is intravitreal.

[0076] The pharmaceutical preparation is preferably in unit dose form. In such form, the preparation is divided into unit doses containing appropriate amounts of the conjugates and compositions of the present invention. The unit dose form can be a packaged preparation, the package containing individual quantities of the preparation, such as tablets, capsules, and powders packaged in vials or ampoules.

[0077] The conjugates and compositions of the present invention can be co-administered with other agents. Co-administration includes administration of the conjugates and compositions of the present invention within 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, or 24 hours of the other agent. Co-administration also includes administration at the same time, approximately the same time (e.g., within about 1 minute, within about 5 minutes, within about 10 minutes, within about 15 minutes, within about 20 minutes, or within about 30 minutes of each other), or sequentially in any order. Furthermore, the conjugates and compositions of the present invention can each be administered once a day, or two, three or more times a day to achieve a preferred daily dosage level.

[0078] In some embodiments, simultaneous administration can be achieved by combination, i.e., by preparing a single pharmaceutical composition containing the conjugates and compositions of the invention and any other agent, or alternatively, the various components can be formulated separately.

[0079] The conjugates and compositions of the invention, and any other agents, may be present in any suitable amount, which may depend on a variety of factors, including, but not limited to, the weight and age of the subject, the disease state, etc. Suitable dosage ranges include about 0.1 mg to about 10,000 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 1 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg. The compositions may also contain other compatible therapeutic agents. The conjugates described herein may be administered in combination with each other, with other active agents known to be useful in modulating the glucocorticoid receptor, or with adjuvants that are not effective alone but may contribute to the efficacy of the active agent.

[0080] In some embodiments, the compositions of the invention are for use in methods of treating uveitis, as described herein.

[0081] IV.Treatment In some embodiments, the present invention relates to a method and / or use comprising a conjugate or composition as described herein for the treatment of uveitis in a subject in need of such treatment. Uveitis is an eye disease occurring when the middle layer of the eyeball is inflamed, red, and / or enlarged. This layer, called the uvea, contains many blood vessels that nourish the eye. Uveitis can damage important eye tissues and result in permanent vision loss. Uveitis can be anterior uveitis, intermediate uveitis, and / or posterior uveitis.

[0082] In some embodiments, the method of the present invention provides a method for treating uveitis in a subject in need thereof, comprising administering to the subject a conjugate of Formula V: (XY) n -Z (V), (In the formula, each X is, independently, an anti-inflammatory peptide having a molecular weight of about 5 kDa to about 200 kDa; each Y is an organic linker; Z is a hyaluronic acid polymer having a molecular weight of about 0.1 MDa to about 3 MDa; The subscript n is an integer from 1 to 1000. The method comprises administering a therapeutically effective amount of

[0083] In some embodiments, each X is, independently of the other, an anti-TNF-α peptide or an anti-interleukin-1β peptide.

[0084] In some embodiments, each X is a monoclonal IgG, an IgG fragment, a single chain scFv, a single domain heavy chain VHH, an adnectin, an affibody, an anticalin, a DARPin, or an engineered Kunitz-type inhibitor. In some embodiments, each X is a monoclonal IgG. In some embodiments, each X is an IgG fragment. In some embodiments, each X is a single domain heavy chain VHH. In some embodiments, each X is a DARPin.

[0085] In some embodiments, each X is a peptide having an amino acid sequence including any one of SEQ ID NOs: 61-73, 81-85, 91-98, 101-109, 111-118, and 145-154.

[0086] In some embodiments, each X is a peptide having an amino acid sequence comprising: QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSPSTPPTPSPSTPPGGCDDDDK (SEQ ID NO: 101), QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 102), QVQLQDSGGGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGTTVYADSVLGRFEISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 103), QVQLQESGGGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGTTVYADSVKGRFTISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 104), CGGGVDNKFNKEVGWAFGEIGALPNLNALQFRAFIISLWDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 105), or EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSSSPSTPPTPSPSTPPGGC (sequence number 106).

[0087] Each peptide may be linked to a biocompatible polymer by a variety of organic linkers commonly known in the art for forming antibody-drug conjugates, such as those provided by Broad Farm, San Diego, Calif., or Creative Biolabs, Shirley, New York. Methods for forming bioconjugate bonds are described in Bioconjugate Techniques, 3rd Edition, Greg T. Hermanson. Organic linkers may react with amines, carbonyls, carboxyls, and activated esters, react via click chemistry (with or without copper), or be reactive with thiols.

[0088] Representative organic linkers include amides or disulfides, or are formed from reactive groups such as succinic anhydride, succinimide, N-hydroxysuccinimide, N-chlorosuccinimide, N-bromosuccinimide, maleic anhydride, maleimide, hydantoin, phthalimide, and the like. Organic linkers useful in the present invention are small molecules, typically having a molecular weight of about 100 Da to about 500 Da, and containing two functional groups consisting of maleimide and amine or hydrazide. In some embodiments, the peptide is covalently linked to the polymer via a sulfide bond and an organic linker having a molecular weight of about 100 Da to about 500 Da. In some embodiments, the organic linker has a molecular weight of about 100 Da to about 300 Da. In some embodiments, the organic linker comprises succinimide. In some embodiments, the organic linker is formed using N-β-maleimidopropionic acid hydrazide (BMPH), N-ε-maleimidocaproic acid hydrazide (EMCH), N-aminoethylmaleimide, N-κ-maleimidoundecanoic acid hydrazide (KUMH), hydrazide-PEG2-maleimide, amine-PEG2-maleimide, hydrazide-PEG3-maleimide, or amine-PEG3-maleimide.

[0089] In some embodiments, the organic linker has the structure: [ka]

[0090] In some embodiments, the organic linker can be N-ε-maleimidocaproic acid hydrazide (EMCH). [ka]

[0091] In some embodiments, the organic linker has the structure: [ka] In the above formula, the subscript m is an integer from 1 to 300. In some embodiments, the subscript m is an integer from 1 to 100.

[0092] In some embodiments, the organic linker has the structure: [ka] The organic linker having the above structure is known as MP2H.

[0093] In some embodiments, each Y is an organic linker having the structure: [ka] In the above formula, the subscript m is an integer from 1 to 300.

[0094] In some embodiments, Z has a molecular weight of about 0.4 MDa to about 2 MDa. In some embodiments, Z has a molecular weight of about 0.7 MDa to about 1.5 MDa. In some embodiments, Z has a molecular weight of about 0.8 MDa.

[0095] In some embodiments, the conjugate of formula V has the structure of formula Va: (X 1 -X 2 -Y) n -Z (Va), (In the formula, Each X 1 is an anti-inflammatory peptide having a molecular weight of about 5 kDa to about 200 kDa; Each X 2 is an α-helix-containing peptide linker; Each Y is an organic linker having the structure: [ka] In the above formula, Z is a hyaluronic acid polymer having a molecular weight of about 0.1 MDa to about 3 MDa; The subscript m is an integer from 1 to 300. has.

[0096] In some embodiments, each X 1 are, independently of each other, anti-TNF-α peptides or anti-interleukin-1β peptides.

[0097] In some embodiments, each X 2 is a peptide linker having an amino acid sequence comprising: AEAAAKEAAAKEAAAKAGC (SEQ ID NO:21), AEEEKRKAEEEKRKAEEEAGC (SEQ ID NO:22), AEEEKRKAEEEKRKAEEEKRKAEEEAGC (SEQ ID NO:23), AEEEEKKKKEEEEKKKKAGC (SEQ ID NO:24), AEAAAKEAAAKAGC (SEQ ID NO:25), PSRLEEELRRRLTEGC (SEQ ID NO:26), or AEEEEKKKQQEEEAERLRRIQEEMEKERKRREEDEERRRKEEEERRMKLEMEAKRKQEEEEERKKREDDEKRKKKAGC (sequence number 27).

[0098] In some embodiments, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO: 107, 2 is a peptide linker having an amino acid sequence comprising SEQ ID NO: 21. In some embodiments, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO: 108, 2 is a peptide linker having an amino acid sequence comprising SEQ ID NO: 21. In some embodiments, each X 1 is a peptide having an amino acid sequence comprising SEQ ID NO: 109, 2 is a peptide linker having an amino acid sequence comprising SEQ ID NO:21.

[0099] In some embodiments, the conjugate of formula V is a random polymer of formula VI having a molecular weight of about 0.1 MDa to about 3 MDa: (XYZ 1 ) n -(Z 2 ) p -(Z 3 ) q (VI), During the ceremony, each X is, independently, an anti-inflammatory peptide having a molecular weight of about 5 kDa to about 200 kDa; each Y is an organic linker; Each XYZ 1 The moiety has the structure: [ka] Each Z 2 has the following structure: [ka] Each Z 3 have, independently of each other, the structure: [ka] Each R 1 and R 2 are each independently C1-C6 alkyl, -(C1-C6 alkyl)-NR 3 R 4 or C5-C8 cycloalkyl; Each R 3 and R 4 are, independently of each other, H or C1-C6 alkyl; Each Z 3a are, independently of each other, OH or Y'; each Y' is an unreacted organic linker; subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of subscripts n, p, and q; The subscript q is an integer from 100 to 10,000.

[0100] In some embodiments, each R 1 and R 2 are each independently C1-C3 alkyl or -(C1-C3 alkyl)-NR 3 R 4 In some embodiments, each R 1 and R 2 is ethyl or -(CH)-NMe. In some embodiments, each R 1 is ethyl; each R 2 is -(CH2)3-NMe2. In some embodiments, each R 1 is -(CH2)3-NMe2; 2 is ethyl.

[0101] In some embodiments, each R 3 and R 4 are each independently C1 to C3 alkyl.

[0102] In some embodiments, the preparation of the conjugate of the present invention includes covalently attaching an organic linker to a biocompatible polymer, and then covalently attaching a peptide to the organic linker. In some embodiments, after the preparation of the conjugate of the present invention, an unreacted organic linker is present on the biocompatible polymer. The structure of the unreacted organic linker depends on the organic linker, as will be understood by those skilled in the art.

[0103] Representative unreacted organic linkers include, but are not limited to, the following: [ka]

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

[0105] In some embodiments, the unreacted organic linker has the structure: [ka] wherein the subscript m is an integer from 1 to 300. In some embodiments, the subscript m is an integer from 1 to 100.

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

[0107] In some embodiments, subscript n is an integer between 1 and 1500 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 1000 and is less than about 10% of the sum of subscripts n, p, and q; and subscript q is an integer between 100 and 10000. In some embodiments, subscript n is an integer between 1 and 1000 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 800 and is less than about 8% of the sum of subscripts n, p, and q; and subscript q is an integer between 100 and 10000. In some embodiments, subscript n is an integer between 10 and 450 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 300 and is less than about 10% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000. In some embodiments, subscript n is an integer between 10 and 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 240 and is less than about 8% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000. In some embodiments, subscript n is an integer between 10 and 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 60 and is less than about 2% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000. In some embodiments, subscript n is an integer between 10 and 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 30 and is less than about 1% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000. In some embodiments, subscript n is an integer between 10 and 300 and is less than about 10% of the sum of subscripts n, p, and q; subscript p is an integer between 1 and 15 and is less than about 0.5% of the sum of subscripts n, p, and q; and subscript q is an integer between 1000 and 3000.

[0108] In some embodiments, the uveitis is chronic uveitis. In some embodiments, the uveitis is chronic non-infectious uveitis.

[0109] In some embodiments, the method comprises intravitreal administration. In some embodiments, the method comprises repeated administration of the conjugate. In some embodiments, the method comprises administering the conjugate monthly, every two months, or every three months. In some embodiments, the method comprises administering the conjugate two or three times a year. In some embodiments, the method comprises administering the conjugate once a year.

[0110] In some embodiments, the method of the present invention is a method for treating chronic non-infectious uveitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate that is a random polymer of Formula VI having a molecular weight of about 0.1 MDa to about 3 MDa: (XYZ 1 ) n -(Z 2 ) p -(Z 3 ) q (VI), During the ceremony, Each X is, independently of the other, an anti-TNF-α peptide or an anti-IL-1β peptide, including: QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSPSTPPTPSPSTPPGGCDDDDK (SEQ ID NO: 101), QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 102), QVQLQDSGGGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGTTVYADSVLGRFEISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 103), QVQLQESGGGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGTTVYADSVKGRFTISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSAEAAAKEAAAKEAAAKAGC (SEQ ID NO: 104), CGGGVDNKFNKEVGWAFGEIGALPNLNALQFRAFIISLWDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 105), or EIVMTQSPSTLSASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSRLSCTASGFSLSSAAMAWVRQAPGKGLEWVGIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSSSPSTPPTPSPSTPPGGC (SEQ ID NO: 106); Each Y is an organic linker having the structure: [ka] Each XYZ 1The moiety has the structure: [ka] Each Z 2 has the following structure: [ka] Each Z 3 have, independently of each other, the structure: [ka] Each R 1 and R 2 are each independently C1-C6 alkyl, -(C1-C6 alkyl)-NR 3 R 4 or C5-C8 cycloalkyl; Each R 3 and R 4 are, independently of each other, H or C1-C6 alkyl; Each Z 3a are, independently of each other, OH or Y'; each Y' is an unreacted organic linker; subscript n is an integer from 1 to 1500 and is less than about 15% of the sum of subscripts n, p, and q; subscript p is an integer from 0 to 1000 and is less than about 10% of the sum of subscripts n, p, and q; The subscript q is an integer between 100 and 10,000. This is the method.

[0111] In some embodiments, the random polymer of formula VI has a molecular weight of about 0.4 MDa to about 2 MDa. In some embodiments, the random polymer of formula III has a molecular weight of 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.

[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. In some embodiments, each X is a peptide having an amino acid sequence comprising SEQ ID NO: 106.

[0113] In some embodiments, uses of the present invention include formulating a medicament for a method of treating uveitis as described herein.

[0114] In some embodiments, the subject is a human.

[0115] In some embodiments, the application of the present invention is the use of a conjugate or pharmaceutical composition as described herein for treating uveitis.

[0116] In some embodiments, a pharmaceutical composition of the invention is a pharmaceutical composition for use in treating uveitis, comprising a conjugate as described herein.

[0117] In some embodiments, the conjugate of the invention is a conjugate for use in treating uveitis as described herein. EXAMPLES

[0118] V. Working Examples In describing the experimental details, certain abbreviations and acronyms have been used, and while most of these will be understood by those of skill in the art, the following table contains a list of many of these abbreviations and acronyms.

[0119] [Table 1]

[0120] Example 1. Preparation of peptides Bioactive peptides were prepared with a C-terminal peptide linker for attachment to a polymer, if desired.

[0121] [Table 2]

[0122] Example 2. Preparation of purified thiol-reactive hyaluronic acid conjugate intermediate Hyaluronic acid (HA, 830 kDa) was suspended at 4 mg / mL in water or 0.1 M 2-(N-morpholino)ethanesulfonic acid buffer (pH 5.7) overnight at room temperature with gentle rotation or nutation mixing. To a solution of 3 mg (3.6 nmol, amount varies based on polymer composition and MW) of HA is added hydroxybenzotriazole (HOBt) hydrate as a stock solution of about 5-100 mg / mL in DMSO, a thiol-reactive linker agent (e.g., hydrazide-X-thiol reactive group or amine-X-thiol reactive group, e.g., MP2H or EMCH) in 10-100% DMSO (10-100 mg / mL stock), and a coupling agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) as a stock of about 1-0.05 g / mL in water or 0.1 M MES buffer, pH 5.7. The molar equivalents of each reactant per mole of HA and per mole of carboxylate for the different and exemplary methods of carrying out the above reaction are listed in the table below:

[0123] [Table 3]

[0124] The solution was mixed by gentle pipetting after each addition of reagent, and the final reaction volume was made up to 1 mL with buffer. The final mixture was allowed to react at room temperature for 45 min to 2 h using a nutating mixer, depending on the method. After reaction, the thiol-reactive biopolymer was purified using a 5-10 mL Zeba desalting spin column with a 7 kDa MWCO equilibrated with 10% v / v glycerol (pH 6.5) DPBS, and 0.01% v / v polysorbate 20 (optional), with 20% of the crude reaction mass (by volume) of resin loaded onto the column. The desired intermediate was eluted into a clean conical tube using a centrifuge at room temperature for an elution time of approximately 25-60 min. The intermediate was either used immediately for reaction with thiols or aliquoted and flash frozen on dry ice. Maleimide concentration and number of modifications per polymer were determined using UV absorbance, NMR, or a modified Ellman reaction assay.

[0125] Alternatively, the number of thiol-reactive small molecule linkers covalently attached per biopolymer (valency) was increased or decreased by varying the reaction pH or the equivalents of hydrazide linker, catalyst, and coupling agent (EDC).

[0126] Instead of EDC and HOBt, other coupling reagents such as DMTMM or Oxyma can be used. The activated biopolymer intermediate can also be purified from the reaction using molecular sieve chromatography, other desalting columns, tangential flow filtration, ion exchange chromatography, dialysis, or alcohol / acetone precipitation.

[0127] Example 3. Conjugate preparation A fixed concentration of peptide was mixed with the polymer at various predefined feed ratios in PBS and allowed to react with mixing on a rotating or orbital mixer for at least 4 hours and at least 2 hours, respectively, at either 4°C or ambient temperature (most reactions are performed at room temperature to improve solubility). Prior to the conjugation reaction, a reducing agent such as DTT or TCEP HCl was added at 10-100 equivalents per protein, corresponding to the reduction of disulfide bridges between the peptides. The reducing agent was either removed from the protein solution prior to conjugation by desalting columns or buffer exchange, or added directly to the conjugation reaction in the form of TCEP immobilized on polymer beads. During the conjugation reaction, one or more of the following were added to improve reaction efficiency: 0.5-10 mM EDTA to minimize oxidation of free thiols, Tween® 20, carbohydrates, or glycerol to stabilize the protein and / or help reduce non-specific interactions between the protein and the activated biopolymer, increasing or decreasing the salt concentration to stabilize the protein and / or help reduce non-specific interactions between the protein and the activated biopolymer. Unreacted peptide was removed from the peptide-polymer conjugate by one or more of the following methods: dialysis against an appropriate buffer (pH should be >1 unit higher or lower than the pI of the peptide) with a MWCO of 50-1000 kDa, twice for 4 hours each and once for at least 4 hours at 4° C. to room temperature, tangential flow filtration against DPBS (pH 6-8) or 50 mM Tris 150 mM NaCl (pH 8-8.5) containing EDTA and other additives such as tween® or trehalose depending on the peptide, FPLC polishing using a size exclusion column, FPLC polishing using an affinity chromatography column designed to bind the polymer component of the conjugate, or selective precipitation of the conjugate. If the reaction efficiency is high enough (less than 4% unreacted protein present), purification may not be necessary.

[0128] Alternatively, peptide was added to each solution of activated polymer at the appropriate peptide:polymer molar feed ratio, and Tween®-20 was added to a final concentration of 0.01%-0.03% (optional). The solutions were allowed to react at ambient temperature with stirring on a rotary mixer (approximately 5 rpm) or orbital mixer for 2 hours to overnight. Unreacted peptide was removed by dialysis against phosphate buffered saline or equivalent citrate or succinate buffered saline (pH and buffer salt used dependent on peptide) containing 0.01-0.03% Tween®-20 (optional) using 100-1000 kDa MWCO membranes for 3-5 cycles, each for 4-18 hours, at 4°C to room temperature. Alternative methods include tangential flow filtration against appropriate buffers or FPLC polishing using size exclusion columns. Optionally, depending on the peptide, additives such as tween® 20, EDTA, and carbohydrates were added to enhance protein stability.

[0129] [Table 4]

[0130] The conjugates in the table below were made with hyaluronic acid (830 kDa or 850 kDa lots). After purification, the products of the conjugation reactions were analyzed by SDS-PAGE separation, which confirmed that less than 20% of the peptide monomers entered the separating gel and more than 90% of the peptide was present at the top of the stacking gel as macromolecular conjugates (Figures 1A-1F). Additionally, protein concentration, percent unconjugated peptide, percent conjugated peptide, valency (molar ratio of conjugated peptide to polymer), and hydrodynamic radius (R hThe reaction products were analyzed for: Protein concentration was determined spectrophotometrically at A280, the percentage of unconjugated protein was determined by densitometric analysis of SDS-PAGE gels, and hydrodynamic radius was measured using dynamic light scattering (DLS).

[0131] [Table 5]

[0132] Example 4. Efficacy of MVPs containing anti-inflammatory proteins As an example, anti-inflammatory proteins were designed with peptide and thiol linkers for conjugation. These antibodies were conjugated to HyA to generate a range of valencies, multivalent conjugates with different polymer backbones and sizes. Biolayer interferometry (BLI) was performed to quantify the binding kinetics of purified MVPs for bioactivity assessment using a GatorPrime (Gator Bio) or similar device and a streptavidin-coated probe (Cat. No. 160002) for AVI-tagged ligands or an anti-human Fc (Cat. No. 160003)-coated probe for Fc-tagged ligands. All analytes and ligands were diluted in BLI buffer (1x dPBS, 0.1% w / v BSA and 0.1% v / v polysorbate 20, 0.2 μm filter sterilized). The appropriate ligands for each analyte as listed in Table 6 were first resuspended according to the manufacturer's instructions and stored for long-term use. Unconjugated analytes were diluted to a maximum concentration ranging from 5 μM to 1 nM. Multivalent conjugates were diluted to a maximum concentration of 50 to 1.0 nM based on the molecular weight of the entire multivalent conjugate ((protein MW x valency) + polymer mw). The concentration range for each ligand-analyte pair is one that shows dose-dependent binding affinity in preliminary range-finding experiments titrating over a wide concentration range from 10 μM to 1 nM.

[0133] All reagents were equilibrated to room temperature for at least 30 min before use. Two probes per sample (one for the kinetic assay and one for a no ligand control) were equilibrated in 250 μL of BLI buffer (PBS pH 7.4, 0.2% Tween® and 0.2% BSA, 0.2 μm filter sterilized) in a Gator Bio Max plate for a minimum of 10 min. Ligands were diluted in BLI buffer to fixed concentrations of 25–100 nM based on their performance in preliminary reactions. Analytes were prepared in BLI buffer to their highest concentration determined in preliminary reactions and serially diluted 1:3 two to five times in BLI buffer (Table 6). 200 μL of ligand and analyte dilutions were dispensed in columns into a black flat-bottom uncoated 96-well plate (Greiner Bio-One, catalog no. 655209, or equivalent), with one column of BLI buffer dispensed for each ligand and analyte column. One well in each analyte column was filled with BLI buffer to be used as a blank for reference subtraction. The sample plate was placed on the Gator's tilted platform set at 25°C. The loading and kinetic steps for the Gator K assay were set using the double reference and step times shown in Table 7. Ligand was loaded until the signal reached 0.4-0.6 nm, then returned to the buffer column and a baseline measurement was taken for 60-90 seconds. A kinetic measurement was then started using the step parameters described above. Once the kinetic measurement was completed with the ligand-loaded probe, a no-ligand control was run using a new probe with no ligand loaded. The same kinetic assay timing and sample wells were used as analyzed with the ligand-loaded probe. This data was used to correct for nonspecific interactions between the sample and the probe.

[0134] Once the kinetic assays were completed, the data was analyzed using Gator software. Raw data was corrected to include binding times from 1 second to 180 seconds. The Y-axis was aligned to the start of the binding step, and inter-step correction was used. Savitzky-Golay filtering of the data was used. Samples were set to double reference by specifying in the software which probe and well were buffer references. The reference subtraction equation for each assay was then edited so that each assay was double referenced with the formula (kinetic assay wells - no ligand assay wells) - (kinetic assay buffer reference wells - no ligand assay buffer wells). All titrations of the same MVP were grouped by color, and parameters were adjusted to a 1:1 binding model, including both binding and dissociation, with a global, Rmax uncoupled fit. The time window of interest was shifted to include only the 100 seconds of dissociation. Binding curves were fitted and the residuals were checked to see if they did not vary more than 10% from the actual curve, and the overall R 2 exceeds 0.98, and the overall X 2 was confirmed to be less than 3.0. Kinetics and the variable K D , K on , and the responses were recorded.

[0135] [Table 6]

[0136] [Table 7]

[0137] Example 5. Hydrodynamic radius of MVP Anti-inflammatory agents were designed containing peptide and thiol linkers for conjugation. These agents were conjugated to HyA to generate a range of multivalent conjugates with different valencies, polymer backbones, and sizes. The hydrodynamic radius (R) of purified unconjugated proteins or MVPs was used to assess size. h To quantify the amount of , dynamic light scattering (DLS) was performed using either a Wyatt Dynapro single cuvette Nanostar, a plate reader, or similar equipment.

[0138] Samples were allowed to equilibrate to room temperature for at least 30 minutes. The solution was diluted with 0.1 μm filter-sterilized formulation buffer without polysorbate 20 to a final concentration of 100 nM in 100 μL (typically a 1:10 dilution) and mixed by gentle trituration in a 1.5 mL centrifuge tube or by placing on an orbital mixer (neutator) for up to 30 minutes. Large aggregates and dust particles could be removed by spinning the tube at 5000 g for 5 minutes in a centrifuge. For single cuvette measurements on the NanoStar, a 40 μL sample of the sample solution was loaded into a Wyatt Technology disposable microcuvette with lid (Wyatt catalog number WNDMC), tapped to remove bubbles, placed in the instrument and analyzed. For multiple readings on a plate reader, 25-35 μL of sample was added to a clear bottom black well 384-well plate (Corning, catalog number P8802-384 or equivalent). Bubbles in the sample wells were removed. The instrument settings for analysis of this and other samples by DLS herein are shown in Table 8. DLS acquisition parameters are shown in Table 8 and result specifications in Table 9. A representative DLS intensity plot of purified, filtered MVP is shown in Figure 3.

[0139] [Table 8]

[0140] [Table 9]

[0141] Example 6. Stability of MVP in vitreous mimetic buffer Thermal stability was used as a proxy to evaluate anti-inflammatory MVPs, which may act as long-term treatments, and to compare the relative stability of different constructs. Unconjugated antibodies were diluted to 1.0 mg / mL and anti-inflammatory MVPs to 0.5 mg peptide / mL in formulation buffer. 3 x 30 μL of each sample was transferred into a UV-VIS compatible 384-well plate (Greiner Bio-One, Cat. No. 781801, or equivalent), bubbles were removed, and the plate was sealed with UV-transparent sealing tape (Greiner Bio-One, Cat. No. 676070, or equivalent). The temperature was increased from 25°C to 37°C using a temperature-controlled plate reader (Biotek Synergy HTX plate reader with UV / VIS capability, or equivalent). The plate was incubated for 15 min and the absorbance at 280 nm was measured in each well at each step. The program was continued until the instrument reached 50° C. and the samples were held for a total of 60 minutes, measuring absorbance at 280 nm every 15 minutes. At each temperature, the reference absorbance at 280 nm (A280) value of the formulation buffer was subtracted from the sample measured A280 value, then normalized to the measurement at 37° C. and plotted. These thermal stability plots were compared between the different antibodies, variants, and peptide linkers to determine the anti-inflammatory constructs that would be most resistant to thermal changes and therefore more likely to be stable enough for long-term intraocular therapy.

[0142] Top performers from the thermal stability experiments were then used for long-term 37°C stability studies. MVP was synthesized under sterile conditions and diluted to approximately 0.4 mg / mL in filter-sterilized human vitreous mimetic buffer (see Table 10) or maintained in formulation buffer. These samples were either filter-sterilized using a sterile 0.2 μm or 5 μm spin filter prior to use or mixed with 0.01% sodium azide as an antimicrobial agent. Several 100-150 μL aliquots of each sample were then added to wells of a sterile 96-well plate, and one day 0 aliquot was stored at 4°C. The remaining wells were filled with filter-sterilized human vitreous buffer + 0.01% sodium azide to minimize evaporation. The plates were incubated in a standard tissue culture incubator at 37°C and 5% CO2. At discrete time points, one aliquot of each sample was removed from the plate under sterile conditions and analyzed. First, UV-VIS spectra of the samples were acquired from 200 to 600 nm in 10 nm increments to monitor any dramatic changes in sample composition. Second, protein concentrations were measured to adjust for possible volume differences. Binding affinities to appropriate ligands were measured using the BLI method described above using 5–10 nM MVP as the highest concentration. To assess relative stability over time, K on (binding constant) over time was used. To monitor the change in radius over time, samples were spun at 5000 g for 5 min to remove large aggregates and dust particles, and Rh was measured using the DLS method described above, but with the instrument at 37° C. and no sample dilution.

[0143] [Table 10]

[0144] Example 7. Peptide-polymer conjugates exhibited intravitreal retention In an established pharmacokinetic model, the conjugate showed an extended intravitreal residence time. New Zealand White rabbits (n=9) were divided into three groups randomized by weight. All animals received hu_anti-TNFα_aH MVP in the left eye and unconjugated VHH in the right eye in a 50 μL ITV injection using a 31G insulin syringe. Both eyes received equimolar antibody doses. At 1 hour, 5 days, and 10 days after injection, a group of three rabbits was sacrificed and their eyes enucleated for analysis of intravitreal VHH. Both eyes were flash frozen and vitreous, retina, and aqueous humor were isolated from the frozen eyes. Each tissue sample was then homogenized with a bead beater. After homogenization, VHH concentrations were quantified using ELISA or by digesting peptides with trypsin and subjecting samples to LC / mass spectrometry or similar methods. Representative results of intravitreal half-life extension in rabbit eyes after bioconjugation are shown in Figure 5.

[0145] Example 8. MVP efficacy in a rat model of uveitis The efficacy of mu_anti-TNFα_aH_CYS MVP was examined to determine whether it could sufficiently reduce the symptoms of uveitis. An experimental autoimmune uveitis (EAU) rat model was used as a human model of chronic posterior uveitis. This model was induced by systemic immunization with uveitis-inducing interphotoreceptor retinoid-binding protein (IRBP), and symptoms of uveitis appeared after 9–11 days. Rats were treated with 12.5 μg of mu_anti-TNFα_aH_CYS MVP (conjugate #7) intravitreally. Dexamethasone was used as a positive control.

[0146] Male Lewis rats were randomized into 4 groups (n=8) based on body weight. Each group received 12.5 μg mu_anti-TNFα_aH_CYS MVP(conjugate #7) (using anti-mouse TNFα VHH), dexamethasone (40 μg), or vehicle control. On day 1, rats were immunized with 30 μg bovine IRBP peptide R16 in 0.2 mL Freund's adjuvant by subcutaneous injection at the base of the tail and each thigh. On days 8 and 10, rats were treated with 5 μL ITV injections of either the left or right eye to administer either assigned treatment. Ocular inflammation was assessed by slit lamp examination before the start of the study and on days 7, 9, 11, and 14, and a clinical EAU score of 0 to 4 was assigned depending on the appearance of inflammation. On day 14, the animals were euthanized and one eye from each animal was subjected to a histopathology examination process and assigned a score of 0 to 4 based on the appearance of inflammation and cellular infiltration. EAU and histopathological characteristics were scored based on published standard scoring methods. The study results are summarized in Figure 6.

[0147] Example 9. MVP Efficacy in a Second Uveitis Rat Model The efficacy of conjugate #10 was examined for successful treatment to sufficiently reduce the symptoms of uveitis. Male Lewis rats were randomized into 4 groups (n=12 for EAU induction and n=8 for uninduced controls) based on body weight. Each group received 19 μg of conjugate #10, triamcinolone (40 μg), or vehicle control. On day 1, rats in the induction group were immunized subcutaneously with 25 μg of interphotoreceptor retinoid binding protein (IRBP) peptide R16 in 0.1 mL of Freund's complete adjuvant for a total of 50 μg in each flank. On days 4 and 8, both left and right eyes were treated with 5 μL intravitreal (ITV) injections of conjugate #10 or vehicle control, or 1 μL ITV injections of triamcinolone, based on the assigned treatment. Ocular inflammation was assessed by slit lamp examination at baseline and on days 3, 6, 10, 12, and 14, and a clinical EAU score of 0 to 4 was assigned depending on the appearance of inflammation. Figures 7A-B show that the efficacy of conjugate #10 was comparable to triamcinolone in reducing ocular inflammation as measured by slit lamp and by inflammatory cytokine or inflammatory regulator levels.

[0148] On day 14, animals were euthanized and one eye from each animal was dissected into vitreous and aqueous humor for cytokine analysis. After dissection, aqueous humor from each group was pooled and all tissues were flash frozen. Relative cytokine concentrations in tissues were assessed using a Multiplex Rat Cytokine / Chemokine Magnetic Bead Panel (Millipore, Cat. No. RECYMAG65K27PMX) according to the manufacturer's protocol. Briefly, ocular tissues were thawed on ice and sample volumes were measured. Assay buffer was then added to samples to achieve a final volume of 55 μL for duplicate readings and mixed well. 25 μL of sample, standard, or control was then added to the assay plate, mixed with 25 μL of beads, and incubated at room temperature for 2 hours. Wells were washed with a magnetic plate washer and incubated with 25 μL of detection antibody for 1 hour, followed by incubation with 25 μL of Streptavadin-Phycoerythrin for 30 minutes. Wells were washed with a magnetic plate washer and 125 μL of Sheath Fluid Plus was added per well, followed by a Luminex read. The amount of cytokines recovered from each sample was normalized to the volume of tissue recovered and plotted. Graphical analysis of key pro-inflammatory cytokines and inflammatory regulators is shown in FIG. 7B. Selected cytokine concentrations are shown in Table 11. No statistical significance was observed in cytokine levels between triamcinolone-treated and conjugate #10-treated rats.

[0149] [Table 11]

[0150] Example 10. MVP efficacy in a rabbit TNF-α-induced uveitis model Anti-TNFα MVP was evaluated in a rabbit model of TNF-α-induced uveitis (EIU) involving ITV injection of human TNF-α, which increases other inflammatory cytokines and induces the ocular inflammatory properties of noninfectious uveitis (NIU) in humans.

[0151] Male New Zealand White (NZW) rabbits were randomized into 9 groups based on body weight (n=3). On day 0, each group received 0.26 mg of hu_anti-TNFα_aH MVP (conjugate #11) (4 groups) or vehicle control (4 groups) in a bilateral 50 μL ITV injection, or no injection (1 group). One day after ITV drug delivery, ocular inflammation was induced by delivering 7.5 μg, 5.0 μg, or 2.5 μg of human TNFα, or PBS vehicle control, in a unilateral 50 μL ITV injection to the left eye. One uninduced group did not receive an intravitreal injection. Severity of inflammation was assessed by ocular examination and intraocular pressure was measured using a rebound tonometer before TNFα injection and 6, 24, and 48 hours after TNFα administration. A clinical score was assigned to each eye based on a published scale. The rabbits were euthanized 48 hours after TNF-α injection, and the results are shown in Figures 8A-8B.

[0152] The left eye is dissected and separated into aqueous and vitreous humor, and the vitreous humor may be processed for inflammatory cytokine analysis. Briefly, the dissected vitreous humor is thawed on ice and weighed. The vitreous is gently mixed in homogenization buffer: PBS 0.05% Tween®-20, 1% casein w / v, and 0.01% protease inhibitor cocktail set III (Sigma, Catalog No. 535140) to a concentration of 500 mg vitreous / mL. A solution of bovine testicular hyaluronidase (MP Biochemicals, Catalog No. 37326-33-3) in PBS containing 50 μM MgCl2 and 100 μM CaCl2 is then added to the vitreous tissue in homogenization buffer to a final concentration of 0.04 mg hyaluronidase / g vitreous tissue. The tissue homogenization reaction is incubated for 1 h at room temperature and then overnight at 4 °C. Finally, the homogenized vitreous tissue is spun at 5000 g for 5 min to pellet debris and used for cytokine analysis.

[0153] Relative cytokine concentrations in tissues may be assessed using a Multiplex Bovine Cytokine / Chemokine Magnetic Bead Panel (Millipore, Cat. No. BCYT1-33K-12) following the manufacturer's protocol. Post-mortem aqueous humor is thawed and used directly, and vitreous humor is homogenized in hyaluronidase as described above. 25 μL of sample, standard, or control is added to the assay plate, mixed with 25 μL of beads, and incubated for 2 hours at room temperature. Wells are washed with a magnetic plate washer, incubated with 25 μL of detection antibody for 1 hour, and then incubated with 25 μL of Streptavadin-Phycoerythrin for 30 minutes. Wells are washed with a magnetic plate washer, and 125 μL of Sheath Fluid Plus is added per well, followed by a Luminex read. The amount of cytokine recovered from each sample is normalized to the volume of tissue recovered and plotted.

[0154] Example 11. MVP efficacy in a rabbit endotoxin-induced uveitis model The hu_anti-TNFα_aH MVP MVP is evaluated in an endotoxin-induced uveitis (EIU) rabbit model involving ITV injection of lipopolysaccharide (LPS), which elevates TNFα levels and induces the ocular inflammatory properties of NIU in humans.

[0155] NZW rabbits were divided into 4 groups randomized based on weight (n=7, 3 males / 3 females, 1 random). Each group received hu_anti-TNFα_aH MVP, adalimumab or triamcinolone positive control, or vehicle control, via bilateral 50 μL ITV injections. Two groups received anti-TNFα MVP and one group received adalimumab equimolar to the dose of antigen-binding epitope in each eye: 225 μg total VHH antibody, 1 mg adalimumab, or 1 mg triamcinolone.

[0156] Fifteen days after ITV drug delivery, EIU is induced with 10 μg LPS in 50 μL of ITV injection solution into the left eye of each animal except one of the anti-TNFα MVP groups (durability cohort). Sixty days after ITV drug delivery, EIU is induced in the durability cohort using the same method. Severity of inflammation and EIU is assessed by ocular examination before LPS injection, and at 6 and 24 hours after LPS administration. An EIU clinical score is assigned to each eye based on a published scale. Rabbits are euthanized 24 hours after LPS injection. Eyes after LPS induction are processed for aqueous humor cellular infiltration, inflammatory cytokine analysis, and histopathology to quantify the cellular infiltrate. Uninduced right eyes are snap frozen and anti-TNFα concentrations are measured in the vitreous and aqueous humor.

[0157] Example 12. Persistence of MVP in a TNFα uveitis rabbit model NZW rabbits were divided into 4 randomized groups based on weight (n=7, 3 males / 3 females, 1 random). Groups received hu_anti-TNFα_aH MVP (0.25 mg), triamcinolone (1 mg), or vehicle control via bilateral 50 μL ITV injections. Two groups received anti-TNFα MVP and one group received triamcinolone.

[0158] One to 30 days after ITV drug delivery, ocular inflammation is induced by delivering 7.5 μg of human TNFα, or PBS vehicle control, in a unilateral 50 μL ITV injection into the left eye. One uninduced group received no intravitreal injection. Sixty days after ITV drug delivery, EIU is induced in the durability cohort using the same method. The severity of inflammation is assessed by ocular examination before TNFα injection and at 6, 24, and 48 hours after TNFα administration. Intraocular pressure measurements are also taken daily using a rebound tonometer. A clinical score is assigned to each eye based on a published scale. Rabbits are euthanized 48 hours after TNFα injection.

[0159] The left eye is dissected and separated into aqueous and vitreous humor, and the vitreous humor is processed for inflammatory cytokine analysis. Briefly, the dissected vitreous humor is thawed on ice and weighed. The vitreous is gently mixed in homogenization buffer: PBS 0.05% v / v Tween®-20, 1% w / v casein, and 0.01% v / v protease inhibitor cocktail set III (Sigma, Catalog No. 535140) to a concentration of 500 mg vitreous / mL. A solution of bovine testicular hyaluronidase (MP Biochemicals, Catalog No. 37326-33-3) in PBS containing 50 μM MgCl2 and 100 μM CaCl2 is then added to the vitreous tissue in homogenization buffer to a final concentration of 0.04 mg hyaluronidase / g vitreous tissue. The tissue homogenization reaction is incubated at room temperature for 1 h and then overnight at 4 C. Finally, the homogenized vitreous tissue is spun at 5000 g for 5 min to pellet debris and used for cytokine analysis.

[0160] Relative cytokine concentrations in tissues are assessed using a Multiplex Bovine Cytokine / Chemokine Magnetic Bead Panel (Millipore, Cat. No. BCYT1-33K-12) following the manufacturer's protocol. Post-mortem aqueous humor is thawed and used, and vitreous humor is homogenized in hyaluronidase as described above. 25 μL of sample, standard, or control is added to the assay plate, mixed with 25 μL of beads, and incubated for 2 hours at room temperature. Wells are washed with a magnetic plate washer, incubated with 25 μL of detection antibody for 1 hour, and then incubated with 25 μL of Streptavadin-Phycoerythrin for 30 minutes. Wells are washed with a magnetic plate washer, and 125 μL of Sheath Fluid Plus is added per well, followed by a Luminex read. The amount of cytokine recovered from each sample is normalized to the volume of tissue recovered and plotted.

[0161] [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7]

[0162] Although the above invention has been described in some detail by way of illustration and example for clarity of understanding, it will be understood by those skilled in the art that certain variations and modifications may be made without departing from the scope of the appended claims. Furthermore, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. In the event of any conflict between this application and the references provided herein, this application shall control.

Claims

1. The following conjugate of formula V: (X-Y) ｎ -Z (V)、 (In the formula, Each X is an anti-inflammatory peptide having a molecular weight of approximately 5 kDa to approximately 200 kDa, independently of the others; Each Y is an organic linker; Z is a hyaluronic acid polymer having a molecular weight of approximately 0.1 MDa to approximately 3 MDa; (The subscript n is an integer between 1 and 1000.) A pharmaceutical composition for use in the treatment of uveitis in subjects requiring treatment for uveitis, comprising [a specific ingredient / method].

2. The pharmaceutical composition according to claim 1, wherein each X is independently an anti-TNF-α peptide or an anti-interleukin-1β peptide.

3. The pharmaceutical composition according to claim 1, wherein each X is a peptide having an amino acid sequence containing one of sequence numbers 61-73, 81-85, 91-98, 101-109, 111-118, and 145-154.

4. The pharmaceutical composition according to claim 1, wherein each X is a peptide having an amino acid sequence comprising the following: QVQLQESGGGLVQPGGSLRLSCAASGRRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSSPSTPPPTPSSPSTPPGGCDDDDDK (Sequence ID 101), QVQLQESGGGLVQPGGSLRLSCAASGRRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSSAEAAAAAKEAAAKAGC (Sequence ID 102), QVQLQDSGGGLVQAGGSLRLSCAASGGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGGTTVYADSVLGRFEISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSSAEAAAAAKEAAAKAGC (Sequence ID 103), QVQLQESGGGLVQAGGSLRLSCAASGGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGGTTVYADSVKGRFTISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSSAEAAAAAKEAAAKAGC (Sequence ID 104), CGGGVGDNKFNKEVGWAFGEIGALPNLNALQFRAFIISLWDPSQSANLLLAEAKKLNDAQAPK (Sequence ID 105), or EIVMTQSPSTLSASASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSG AEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGGSGGGGSGGGGSGGGGSEVQLVESG GGLVQPGGSLRLSCTASGFSLSSAAAMAWVRQAPGKGLEWVGIIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSSSSPSPTPPPSPSPTPPGGGC (Sequence No. 106).

5. Each Y is an organic linker having the following structure: 【Chemistry 1】 In the above equation, the subscript m is an integer between 1 and 300. The pharmaceutical composition according to claim 1.

6. The pharmaceutical composition according to claim 1, wherein Z has a molecular weight of approximately 0.8 MDa.

7. The conjugate of equation V has the following structure: (8) １ -8 ２ -9) ｎ -Z (Va)、 (In the formula, Each X １ It is an anti-inflammatory peptide having a molecular weight of approximately 5 kDa to approximately 200 kDa; Each X ２ It is a peptide linker containing an α-helix; Each Y is an organic linker having the following structure: 【Chemistry 2】 In the above formula, Z is a hyaluronic acid polymer having a molecular weight of approximately 0.1 MDa to approximately 3 MDa; (The subscript 'm' is an integer between 1 and 300.) A pharmaceutical composition according to claim 1, having the following characteristics.

8. Each X ２ The pharmaceutical composition according to claim 7, wherein the peptide linker has an amino acid sequence comprising the following: AEAAAAKEAAAAKAGC (Sequence ID 21), AEEEKRKAEEEKRKAEEEAGC (Sequence ID 22), AEEEKRKAEEEEKRKAEEEEKRKAEEEEAGC (Sequence ID 23), AEEEEKKKKEEEEKKKAKAGC (Sequence ID 24), AEAAAAKEAAAAKAGC (Sequence ID 25), PSRLEEELRRRRLTEGC (Sequence ID 26), or AEEEEKKKQQEEEEEAERLRRIQEEMEKERKRRREEDEEERRRRKEEEEERRMKLEMEAKRKQEEEEERKKREDDEKRKKKAGC (Sequence No. 27).

9. The conjugate of formula V is a random polymer of the following formula VI having a molecular weight of about 0.1 MDa to about 3 MDa: (X-Y-Z １ ) ｎ -(Z ２ ) ｐ -(Z ３ ) ｑ (VI)、 During the ceremony, Each X is an anti-inflammatory peptide having a molecular weight of approximately 5 kDa to approximately 200 kDa, independently of the others; Each Y is an organic linker; Each X-Y-Z １ The part has the following structure: 【Transformation 3】 Each Z ２ It has the following structure: 【Chemistry 4】 Each Z ３ They are independent of each other and have the following structure: 【Transformation 5】 Each R １ and R ２ They are independent of each other, C １ ~C ６ Alkyl, -(C １ ~C ６ Alkyl)-NR ３ R ４ , or C ５ ~C ８ It is a cycloalkyl; Each R ３ and R ４ These are H or C, independently of each other. １ ~C ６ It is alkyl; Each Z ３ａ These are OH or Y', independently of each other; Each Y' is an unreacted organic linker; The subscript n is an integer between 1 and 1500, and is less than approximately 15% of the sum of the subscripts n, p, and q; The subscript p is an integer between 0 and 1000, and is less than approximately 10% of the sum of the subscripts n, p, and q; The subscript q is an integer between 100 and 10000. The pharmaceutical composition according to claim 1.

10. Each R １ and R ２ However, C １ ~C ３ Alkyl or -(C １ ~C ３ Alkyl)-NR ３ R ４ The pharmaceutical composition according to claim 9.

11. Each R ３ and R ４ However, C １ ~C ３ The pharmaceutical composition according to claim 9, wherein the component is alkyl.

12. The subscript n is an integer between 10 and 300, and is less than approximately 10% of the sum of the subscripts n, p, and q; The subscript p is an integer between 1 and 60, and is less than approximately 2% of the sum of the subscripts n, p, and q; The subscript q is an integer between 1000 and 3000. The pharmaceutical composition according to claim 9.

13. The pharmaceutical composition according to claim 1, wherein the uveitis is chronic uveitis.

14. The pharmaceutical composition according to claim 13, wherein the uveitis is chronic non-infectious uveitis.

15. The pharmaceutical composition according to claim 14, wherein the composition is for intravitreal administration.

16. The pharmaceutical composition according to claim 15, wherein the use includes repeated administration of the conjugate.

17. The pharmaceutical composition according to claim 16, wherein the use comprises administering the conjugate every one month, every two months, or every three months.

18. The pharmaceutical composition according to claim 16, wherein the use comprises administering the conjugate two or three times a year.

19. The pharmaceutical composition according to claim 16, wherein the use comprises administering the conjugate once a year.

20. A conjugate which is a random polymer of the following formula VI having a molecular weight of approximately 0.1 MDa to approximately 3 MDa: (X-Y-Z １ ) ｎ -(Z ２ ) ｐ -(Z ３ ) ｑ (VI)、 During the ceremony, Each X is independently an anti-TNF-α peptide or anti-IL-1β peptide containing the following: QVQLQESGGGLVQPGGSLRLSCAASGRRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSSPSTPPPTPSSPSTPPGGCDDDDDK (Sequence ID 101), QVQLQESGGGLVQPGGSLRLSCAASGRRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSSSAEAAAAAKEAAAKAGC (Sequence ID 102), QVQLQDSGGGLVQAGGSLRLSCAASGGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGGTTVYADSVLGRFEISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSSAEAAAAAKEAAAKAGC (Sequence ID 103), QVQLQESGGGLVQAGGSLRLSCAASGGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGGTTVYADSVKGRFTISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQVTVSSSAEAAAAAKEAAAKAGC (Sequence ID 104), CGGGVGDNKFNKEVGWAFGEIGALPNLNALQFRAFIISLWDPSQSANLLLAEAKKLNDAQAPK (Sequence ID 105), or EIVMTQSPSTLSASASVGDRVIITCQASQSIDNWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSG AEFTLTISSLQPDDFATYYCQNTGGGVSIAFGQGTKLTVLGGGGGGSGGGGSGGGGSGGGGSEVQLVESG GGLVQPGGSLRLSCTASGFSLSSAAAMAWVRQAPGKGLEWVGIIIYDSASTYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARERAIFSGDFVLWGQGTLVTVSSSSPSPTPPPSPSPTPPGGGC (Sequence ID 106); Each Y is an organic linker having the following structure: 【Transformation 6】 Each X-Y-Z １ The part has the following structure: 【Transformation 7】 Each Z ２ It has the following structure: 【Transformation 8】 Each Z ３ They are independent of each other and have the following structure: 【Chemistry 9】 Each R １ and R ２ They are independent of each other, C １ ~C ６ Alkyl, -(C １ ~C ６ Alkyl)-NR ３ R ４ , or C ５ ~C ８ It is a cycloalkyl; Each R ３ and R ４ These are H or C, independently of each other. １ ~C ６ It is alkyl; Each Z ３ａ These are OH or Y', independently of each other; Each Y' is an unreacted organic linker; The subscript n is an integer between 1 and 1500, and is less than approximately 15% of the sum of the subscripts n, p, and q; The subscript p is an integer between 0 and 1000, and is less than approximately 10% of the sum of the subscripts n, p, and q; The subscript q is an integer between 100 and 10000. Conjugate.

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